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農業生物技術學報

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Journal of Agricultural Biotechnology

《農業生物技術學報》創刊于1993年，由中華人民共和國教育部主管，中國農業大學與中國農業生物技術學會共同主辦，是一份為農業生物技術研究與發展提供服務的科學期刊。現任主編武維華院士，著名的植物生理學與生物化學家。主要刊登與農業科學有關的植物、動物、微生物及林業、海洋等學科在組織、器官、細胞、染色體、蛋白質、基因、酶、發酵工程等不同水平上的農業生物技術研究成果；刊登與農業有關的遺傳與育種、生理、生化與分子生物學、環境與生態、醫學、病理學等與基礎與應用基礎研究成果。

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主管單位：中華人民共和國教育部

主辦單位：中國農業大學中國農業生物技術學會

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農業生物技術學報 >2021年第012期

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共4384條結果

1.大白菜硫代葡萄糖甙合成基因SSR標記的開發
- 鞏紅影;羅雙霞;顧愛俠;王彥華;趙建軍;申書興;
- 《農業生物技術學報》 | 2014年第010期
摘要：硫代葡萄糖甙(glucosinolates,GS)是一種含氮、硫的重要植物次生代謝產物,是十字花科蔬菜風味的主要來源.本研究根據擬南芥(Arabidopsis thaliana)和大白菜(Brassica rapa ssp.pekinensis)基因組序列信息,比對GS生物合成相關基因的序列信息,發現大白菜中與GS含量相關的候選基因102個,分布于10條染色體上.擬南芥GS合成基因在大白菜中的同源基因個數分別為:零拷貝基因8個、單拷貝基因13個、2拷貝基因17個、2個以上多拷貝基因14個.通過FASTPCR6.0軟件分析目標基因上下游序列各5kb,共開發出237個簡單重復序列(SSR)位點,16個基因沒有發現SSR位點,72個基因存在1～4個SSR位點,5個基因存在5個SSR位點,4個基因存在6個SSR位點,4個基因存在7個SSR位點,1個基因存在8個SSR位點.所開發的SSR位點中,共發現4種重復類型:單核苷酸重復最多,共122個,占SSR總數的51.4％;其次是二核苷酸重復類型,共48個,占SSR總數的20.3％;三核苷酸重復類型最少,共7個,占SSR總數的3.0％,另外60個SSR無明顯的重復類型.具有SSR位點的86個基因中,77個基因可以設計SSR特異引物,其中49對特異引物在供試的75份大白菜高代自交系中得到有效擴增.其中16對引物具有多態性,占全部引物的20.8％; 18對引物無多態性,占全部引物的23.4％; 15對引物雜帶多,占全部引物的19.5％;另外28對引物無擴增產物,占全部引物的36.4％.最終11對獲得了清晰多態性擴增產物,共檢測出26個等位基因.本研究為分子標記輔助選擇培育高有益GS成分的大白菜新品種提供了基礎資料,進而加快大白菜營養品質育種的進程.
2.山羊FAM134B基因克隆、序列分析及其mRNA表達與肌內脂肪含量的相關性
- 朱武政;江明鋒;唐璐;王永;
- 《農業生物技術學報》 | 2014年第010期
摘要：FAM134B基因對肌內脂肪沉積有重要的調控作用,為了進一步了解FAM134B基因的結構與功能及其對山羊(Capra hirus)肉質的影響,本研究以川中黑山羊為實驗動物,采用RT-PCR技術克隆了山羊FAM134B基因,并對核苷酸序列和氨基酸序列進行生物信息學分析,運用實時熒光定量PCR技術檢測了FAM134B mRNA的表達情況,對肌肉組織中FAM134B mRNA表達與肌內脂肪(intramuscular fat,IMF)含量進行了關聯分析.結果表明,山羊FAM134B基因(GenBank登錄號:KF684947.1)cDNA全長1 071 bp,編碼356個氨基酸,預測蛋白序列含有4個N-連接的糖基化修飾位點,20多個磷酸化位點;系統進化樹分析顯示,山羊FAM134B核苷酸序列與牛(Bos taurus)的相似性最高;熒光定量PCR分析表明,FAM134B在山羊的心、肝、脾、肺、腎、背最長肌和腿肌中均有表達,其中在肝臟的表達量最高,脾的表達量最低;相關分析顯示:山羊背最長肌、腿肌中FAM134B mRNA表達與肌內脂肪含量呈顯著正相關.研究結果顯示,FAM134B基因可能對山羊肌內脂肪沉積起著重要的調控作用.本研究為進一步了解FAM134B基因的功能及其在反芻動物IMF代謝中的作用和營養調控機制提供一定基礎資料.
- 山羊;
- FAM134B;
- 克隆;
- mRNA;
- 肌內脂肪(IMF);
3.干擾水稻瘤矮病毒（RGDV）非結構蛋白（Pns12）的表達抑制病毒在介體昆蟲培養細胞內的復制
- 鄭立敏;劉華敏;陳紅燕;賈東升;謝聯輝;魏太云;
- 《農業生物技術學報》 | 2014年第011期
摘要：水稻瘤矮病毒(Rice gall dwarf virus, RGDV)屬呼腸孤病毒科(Reoviridae)植物呼腸孤病毒屬(Phytoreovirus)，由介體電光葉蟬(Recilia dorsalis)以持久增殖型方式傳播，其基因組第12條片段編碼的非結構蛋白(nonstructural protein, Pns12)是提供病毒復制和子代病毒粒體裝配場所——病毒原質(viroplasm)的組分之一。為了明確Pns12在RGDV侵染介體電光葉蟬培養細胞中的功能，本研究通過原核表達的Pns12蛋白免疫注射兔(Oryctolagus cuniculus)，制備Pns12抗體，并應用免疫熒光標記和RNA干擾(RNA interference, RNAi)技術研究Pns12在介體培養細胞內的定位和參與病毒原質形成的過程。共聚焦顯微鏡觀察到，病毒侵染的細胞中，與熒光素交聯的Pns12抗體特異地標記在病毒原質上。干擾Pns12蛋白表達后，可有效地阻礙病毒原質的形成、子代病毒粒體的組裝和病毒非結構蛋白Pns12和外殼蛋白P8蛋白的表達，表明Pns12作為病毒原質的組分參與了RGDV在介體培養細胞內的復制。也表明，Pns12可作為理想的靶標用于阻斷電光葉蟬攜帶和傳播RGDV。%Rice gall dwarf virus(RGDV), the genus Phytoreovirus in the family Reoviridae, is transmitted by the leafhopper vector(Recilia dorsalis) in a persistent-propagative manner. Nonstructural protein(Pns12), encoded by segment 12 of RGDV, is one of the components of viroplasm which is the site for viral replication and assembly of progeny virons during viral infection in its insect vector cells. In this study, to investigate the functional role of Pns12 in the formation of viroplasm in its insect vector cells, the polyclonal antibody against Pns12 was prepared and purified, then conjugated to fluorescein isothiocyanate. The immunoglobulin G(IgG) of P8 was purified and conjugated to rhadamine. Immunofluorescence microscopy demonstrated that Pns12 antibodies specifically distributed in the viroplasm matrix during RGDV infection in the vector cells in monolayers (VCMs), while outer capsid protein P8 were accumulated at the periphery of the viroplasm. The viroplasm increased in size over time. Knockdown of Pns12 by RNA interference (RNAi) induced by dsRNAs, targeting Pns12 gene of RGDV, significantly inhibited the formation of viroplasm compared with dsGFP, suggesting that RGDV replication was inhibited. Western blot showed that viral Pns12 and P8 expression reduced in the VCMs treated with dsPns12. Thus, the present study indicated that Pns12 of RGDV played an important role in viroplasm formation and viral replication in its insect vector cells. RNAi induced by dsRNAs derived from the viral genes of viroplasm matrix protein may be an ideal tool for inhibiting the infection and transmission of RGDV by leafhopper vectors.
4.刺糖多孢菌高產菌株和野生型菌株多殺菌素生物合成基因簇（spn）在發酵過程中的表達分析
- 黃穎;趙晨;楊博磊;陳園;關雄;宋淵;張曉琳;
- 《農業生物技術學報》 | 2014年第011期
摘要：Spinosad is a kind of bio-insecticide showing advantages such as high-efficiency and low-toxicity, which is wildly used in pesticide, veterinary drug, sanitary field, etc. However, industrial production of spinosad still has not been implement in China, because of relatively late and undeveloped study on spinosad high-yield strain breeding and relative fermentation process. Previously, a spinosad high-yield strain, Saccharopolyspora spinosa ASAGF73, was obtained through multiple rounds mutagenesis. In this study, we focused on analysing spinosyn biosynthetic gene cluster(spn) expression profile and comparing the difference between wide- type strain ATCC49460 and high- yield ASAGF73 during fermentation process, and preliminarily elucidated the rate-limiting step for spinosad synthesis. Bioinformatic analysis revealed that spn gene cluster contained 9 transcripts, and their expressions were detected using RT-PCR with primers designed inside each transcript. The results showed that the high production of ASAGF73 strain could be due to the high expression of some transcripts in spn cluster, the expression of genes coding for polyketide synthase (PKS) and cross- bridging in ASAGF73 was higher than those in ATCC49460. spnG, which coded rhamnosyltransferase, showed high expression in ASAGF73 strain at the beginning of the fermentation but decreased to a quite low level later, which was not ideal for spinosad high production and might be the rate-limiting step for spinosad synthesis. The present study was the comprehensive and comparative expression analysis of spinosad biosynthetic gene cluster and genes for rhamnose biosynthesis in the 2 S. spinosa strains during fermentation, provided some clues to understand the molecular mechanisms which can lead to the increasing of spinosad production yield.%多殺菌素(spinosad)作為一種高效、低毒生物殺蟲劑，已在農藥、獸藥、衛生用藥等領域得到應用。中國關于多殺菌素的研究起步較晚，進展緩慢，目前仍無法實現產業化生產。本研究旨在分析刺糖多孢菌(Saccharopolyspora spinosa)高產菌株ASAGF73與野生型菌株ATCC49460在發酵過程中多殺菌素合成基因簇(spinosyn biosynthetic gene cluster, spn)的表達變化及差異，初步判斷影響多殺菌素合成的關鍵限速步驟。生物信息學分析表明，spn基因簇含有9個轉錄子，分別在各轉錄子中設計引物，進行RT-PCR檢測其表達情況。結果顯示，突變株ASAGF73中多殺菌素的產量提高可能與spn基因簇中部分基因的高表達有關，其中編碼聚酮合酶(polyketide synthase, PKS)的基因和參與大環內聯橋形成的基因表達量遠高于野生型菌株的。在ASAGF73中編碼鼠李糖轉移酶的spnG基因在發酵前期過量表達，而在后期大量合成多殺菌素時表達減弱，有可能成為多殺菌素合成的限速步驟。本研究初步闡明能夠提高多殺菌素產量的分子機制，為構建多殺菌素高產基因工程菌提供參考。
5.蘇云金芽胞桿菌WB5中營養期殺蟲蛋白3Aa基因（vip3Aa）的克隆、表達與純化
- 陳凡冰;宋飛飛;石鵬;黃志鵬;關雄;
- 《農業生物技術學報》 | 2014年第011期
摘要：營養期殺蟲蛋白(vegetative insectcidal protein, Vip)是由蘇云金芽胞桿菌(Bacillus thuringiensis, Bt)在營養生長指數中期至穩定期期間分泌產生的一類新型殺蟲因子，分為Vip1、Vip2和Vip3三種，以Vip3的研究最為深入。Vip3A對鱗翅目(Lepidoptera)害蟲具有廣譜殺蟲活性，具有重要研究意義。本研究以BtWB5菌株總DNA為模板，采用PCR方法擴增vip3Aa(GenBank登錄號：AAM22456)全長，將該片段純化回收后與pMD18-T連接并轉入大腸桿菌(Escherichia coli)DH5α，進行酶切驗證和序列測定。將vip3Aa基因片段與經同樣酶切的表達載體pCzn1連接，構建重組表達載體pCzn1-vip3Aa，轉入大腸桿菌ArcticExpressTM (DE3)，異丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-1-thiogalactopyranoside, IPTG)進行誘導表達。SDS-PAGE結果表明，在沉淀和上清中均有約88 kD VIP3Aa蛋白質表達產物；采用Ni親和樹脂對上清中的VIP3Aa融合表達蛋白進行了純化，獲得了純化蛋白。本研究成功亞克隆了vip3Aa基因，并表達純化了VIP3Aa蛋白，為后續研究VIP3Aa蛋白在昆蟲中腸的作用受體提供了基礎資料。%Vegetative insecticidal proteins(Vips) are one kind of novel toxic proteins, which are generated and excreted by Bacillus thuringiensis(Bt) cells during growth from mid-exponential phase to the stationary phase. Vips may generally be classified into 3 groups of Vip1, Vip2 and Vip3, of which Vip3 is the most intensively studied. It has important research significance because of broad-spectrum insecticidal activities to Lepidoptera pests. The entire coding region of vip3Aa(GenBank No. AAM22456) gene was amplified by PCR with total DNA extracted from BtWB5 as template. PCR product was purified and ligated with pMD18-T to form the recombinant pMD18-T-vip3Aa, and then transformed into Escherichia coli DH5. The recombinant plasmid DNA extracted from the selected positive clone was used for nucleotide sequencing and NdeⅠ/XbaⅠdigested analysis. Using Universal DNA Purification Kit, an approximate 2.4 kb DNA product was purified from NdeⅠ/XbaⅠdigested pMD18-T-vip3Aa, and then inserted into multiple cloning sites of the expression vector pCzn1 to generate the recombinant expression vector pCzn1-vip3Aa. The inserted fragment and its reading frame were confirmed by NdeⅠ/XbaⅠdigestion and nucleotide sequence analysis. pCzn1-vip3Aa was transformed into E. coli ArcticExpressTM (DE3) and induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). SDS-PAGE showed that the relative molecular weight of the expressed protein was about 88 kD in both supernatant and precipitated. In addition, the expressed protein was purified by Ni-NTA affinity chromatography. The success of the subcloned vip3Aa gene and the expression and purification of Vip3Aa provide basic data for furthur research on its action receptor in insect mid gut.
6.金線蓮根部內生菌多樣性宏基因組的分析
- 陳倩倩;劉波;關雄;唐建陽;
- 《農業生物技術學報》 | 2014年第011期
摘要：Anoectochilus roxburghii (Wall.) Lindl is a traditional Chinese medicine, which has been used for diabetes and tumor treatment. The wild A. roxburghii and its endophytes live together to form a balanced symbiotic system. The endophytes produce various secondary metabolites to protect the host from being attacked by fungi and pests. Endophytes are rich bio-resources. Because of a major limitation of isolation methods and cultivation media, the diversity endophytes still remain undiscovered. Metagenomics simultaneous study of endophytes genomes from environmental samples may avoid the limitations of culture-dependent methods. We enriched the microbiota from the root of A. roxburghii and extracted and purified the metagenomic DNA.16S rDNA fragments were amplified by PCR and cloned to Escherichia coli DH5α to sequence. The endophytes were mainly uncultured bacterium, uncultured compost bacterium, Enterobacteriaceae, Paenibacillus sp., Bacillus sp. and Brevibacillus sp., according to 16S rDNA information. This research provided the basis for mining endophytes resources of A. roxburghii. Analyzed the groups of endophytes by partial sequence of 16S rDNA. This work opens further insight into the great potential of A. roxburghii microbiota.%金線蓮(Anoectochilus roxburghii (Wall.) Lindl)是中國傳統中藥，具有重要的藥用價值，現代醫學用來治療糖尿病和癌癥。金線蓮作為蘭科植物，與內生菌在長期的生長過程中建立起和諧的共生關系，內生菌產生多種次生代謝產物，幫助宿主植物抗菌防蟲。現有實驗條件下，環境中絕大多數的微生物不能培養，而宏基因組學是認識環境中不可培養微生物的重要手段。本研究對福建金線蓮根部內生菌富集進行探索，提取混合內生菌樣品宏基因組DNA，并對宏基因組DNA純化，PCR擴增微生物16S rDNA片段，克隆至大腸桿菌(Escherichia coli)并測序。根據16S rDNA信息初步對富集的內生菌群落分析，內生菌主要是不可培養細菌(uncultured bacterium)、不可培養堆肥細菌(uncultured compost bacterium)、腸桿菌科(enterobacteriaceae)、類芽胞桿菌屬(Paenibacillus sp.)、芽胞桿菌屬(Bacillus sp.)和短芽胞桿菌屬(Brevibacillus sp.)的微生物。本研究為挖掘金線蓮內生菌資源提供基礎資料。
7.蘇云金芽胞桿菌N-酰基高絲氨酸內酯酶基因（aiiA）在畢赤酵母中的優化表達
- 簡思美;何晶晶;毛惠民;楊梅;
- 《農業生物技術學報》 | 2014年第011期
摘要：蘇云金芽胞桿菌(Bacillus thuringiensis)N-酰基高絲氨酸內酯酶基因(auto inducer inactivation A, aiiA)編碼的N-酰基高絲氨酸內酯酶(N-acylhomoserine lactonase, AiiA)能夠水解植物病原菌群體效應的信號分子N-酰基高絲氨酸內酯(N-acylhomoserine lactone, AHL)，進而使革蘭氏陰性細菌群體感應受到抑制，減弱病原菌的致病性。本研究將aiiA基因連接至分泌型穿梭表達載體pPICZαB，獲得重組表達質粒pPICZαB-aiiA，線性化后電擊轉化畢赤酵母(Pichia pastoris)GS115，獲得重組工程菌GS115-pPICZαB-aiiA。利用定點突變技術，對aiiA基因進行密碼子優化，獲得重組工程菌GS115-pPICZαB-MaiiA。以終濃度為1%的甲醇、28℃條件下誘導表達，重組工程菌GS115-pPICZαB-aiiA和GS115-pPICZαB-MaiiA均成功表達并分泌出AiiA蛋白。抗病性分析表明，分泌表達的AiiA蛋白能有效抑制胡蘿卜軟腐歐文氏桿菌(Erwinia carotovora)的致病性。AiiA蛋白在畢赤酵母中的分泌表達，拓寬了AiiA蛋白的獲取途徑，為AiiA蛋白的產業化提供理論依據。密碼子優化為今后改造AiiA蛋白，提高AiiA蛋白的表達效率提供了新的思路。%Many phytopathogens use N-acylhomoserine lactone (AHL) as quorum sensing (QS) signals, inducing the expression of virulence factors to destroy plant. N-acylhomoserine lactonase (AiiA) can degrade AHL, resulting in the disruption of AHL-mediated QS system in gram-negative bacteria. In this study, auto inducer inactivation A(aiiA) gene was firstly inserted into the shuttle expression vector of pPICZαB and introduced to GS115 strain of Pichia pastoris, resulting in a recombinant yeast strain GS115-pPICZαB-aiiA. On the other hand, codon optimized aiiA gene, named as MaiiA, was carried out by using site-directed mutagenesis to adapt to P. pastoris expression system betterly, and was also inserted into the expression vector and introduced into yeast, and constructed GS115-pPICZαB-MaiiA. Both GS115-pPICZαB-aiiA and GS115-pPICZαB-MaiiA were successfully induced to express secreted AiiA proteins by the addition of 1%methanol at 28 ℃. Antimicrobial activity analysis showed that the AiiA protein inhibited the virulence of Erwinia carotovora. The expression of secreted AiiA protein in P. pastoris, broadening the acquisition route of AiiA protein, provides a theoretical basis and new idea for AiiA industrialization. Codon optimization provides a new way of thinking for the future reconstruction and the efficient expression of AiiA protein.
8.鉛鋅礦尾礦壩分離節桿菌12-1對Pb2+的耐受和吸附性能研究
- 陳志;鄒情雅;潘曉鴻;林璋;關雄;
- 《農業生物技術學報》 | 2014年第011期
摘要：The extensive exploitation and usage of lead resources have caused serious environmental pollution problems currently. Bioremediation of Pb2 + contaminated water and soil environments using microbes is regarded as a promising technology due to the advantages of its cost-effective, easy operation and environmental-friendly properties. In order to understand Pb2+ biosorption characterization of microbes, an indigenous lead-resistant bacterium-Arthrobacter sp. 12-1(GenBank No. KM362724) was isolated from lead-zinc mine tailing dam, and the process and mechanism of Pb2+ biosorption by Arthrobacter sp. 12-1 were furtherly investigated in this study. Study on the growth of Arthrobacter sp. 12-1 in LB medium containing different concentration of Pb2+suggested that the highest Pb2+tolerant concentration of Arthrobacter sp. 12-1 was 800 mg/L. In water solution, 105 mg/L of Pb2+could be reduced to 2.17 mg/L by Arthrobacter sp. 12-1 within 24 h with biosorption rate of 97.93%. Microscopic investigation (atomic force microscopy and scanning electronic microscopy) combined with energy dispersive X-ray spectroscopy analysis showed that lead containing mineral was formed on the surface of cell after Pb2+sorption by Arthrobacter sp. 12-1. Further fourier transform infrared (FT- IR) analysis revealed that carboxyl, amide and phosphate groups of Arthrobacter sp. 12-1 might be involved in Pb2 + biosorption process. The results demonstrated that the Arthrobacter sp. 12-1 isolated from lead-zinc mine tailing dam had strong ability of Pb2 + resistance and biosorption, indicating an attractive prospect of practical applications in bioremediation Pb2+ contaminated water and soil environments. The present work provides much fundamental information for help in constructing feasible strategies for Pb2+bioremediation in the environment.%當前鉛資源的粗放式開采和使用對環境造成了嚴重的污染。利用微生物修復鉛污染具有費用低、易操作、環境友好等優點，在水體和土壤鉛污染治理中具有很好的應用前景。為了了解微生物對鉛的吸附特性，本研究從鉛鋅礦尾礦壩分離到的一株耐鉛節桿菌(Arthrobacter sp.)12-1(GenBank登錄號：KM362724)，并研究其對鉛的吸附過程和作用機制。研究耐鉛節桿菌12-1在含不同Pb2+濃度LB培養基中的生長情況表明，其最高可耐受800 mg/L Pb2+。在水溶液中，經24 h吸附，耐鉛節桿菌12-1可將Pb2+濃度從105 mg/L降至2.17 mg/L，吸附率為97.93%。顯微成像(原子力顯微鏡,掃描電鏡)觀察和能譜分析表明，耐鉛節桿菌12-1吸附鉛后在細胞表面形成含鉛的礦物。傅立葉變換紅外光譜(FT-IR)分析表明，耐鉛節桿菌12-1細胞上的羧基、酰胺和磷酸基團可能參與了鉛的吸附和固定過程。以上結果表明，從鉛鋅礦尾礦壩分離到的耐鉛節桿菌12-1對鉛具有較好的耐受和吸附能力，顯示其在鉛污染環境修復中具有潛在的應用前景。本研究為細菌修復鉛污染環境的實踐提供了理論基礎。
9.橘小實蠅蛋白餌劑中氨基酸的HPLC分析及生物測定
- 韓英;王波;許晨;許建中;黃居昌;季清娥;楊建全;陳家驊;
- 《農業生物技術學報》 | 2014年第011期
摘要：采用生物測定的方法比較Prima蛋白餌劑和自行研制蛋白餌劑的引誘效果。結果表明，兩者對橘小實蠅(Bactrocera dorsalis (Hendel))引誘效果相當，但水解后的同種蛋白餌劑引誘效果高于水解前。采用柱前衍生高效液相色譜法(AccQ·Tag法)測定以上兩種蛋白餌劑中氨基酸的種類和含量，結果表明，這兩種蛋白餌劑中均含有游離氨基酸17種，總氨基酸18種。其中Prima蛋白餌劑總氨基酸含量為52.8476 mg/mL，游離氨基酸含量為5.7364 mg/mL，多肽含量為4.71112 mg/mL；自行研制的橘小實蠅蛋白餌劑總氨基酸含量為65.6243 mg/mL，游離氨基酸含量為6.3010 mg/mL，多肽含量為59.3233 mg/mL。通過比較兩者氨基酸的差別，為蛋白餌劑的工業化生產質量監控提供方法依據。%This article mainly compares the similarities and differences between Prima produced by Malaysia Pupuk Alam company and protein bait for Bactrocera dorsalis (Hendel) developed by our research institute. The biological test was used to compare attractive effects, and High Performance Liquid Chromatography with Precolumn Derivatization (AccQ · Tag) was used to analyse the kinds and contents of amino acids. The results showed the attractive effects of Prima and protein bait were similar, and the two protein baits all contained 17 kinds of free amino acids and 18 total amino acids. Prima contained 52.847 6 mg/mL of total amino acids, 5.736 4 mg/mL of free amino acid, and 47.111 2 mg/mL of peptides. The protein bait was consisted of 65.624 3 mg of total amino acids, 6.301 0 mg of free amino acid, 59.323 3 mg of peptides in one milliliter. Besides, the attractive effect of hydrolyzed protein was stronger than before. The results can provide a basis for monitoring protein bait quality on the industrial production.
10.蘇云金芽胞桿菌Cry1Ab毒素domainⅡ內3個loop對其毒性作用的影響
- 邵恩斯;林莉;石鵬;劉斯軍;關雄;黃志鵬;
- 《農業生物技術學報》 | 2014年第011期
摘要：蘇云金芽胞桿菌(Bacillus thuringiensis, Bt)Cry毒素domainⅡβ-折疊頂端的3個loop在靶標昆蟲中腸受體識別中有重要作用，這3個loop在影響Cry毒性中起到的重要性有所不同。本研究利用能夠與Cry毒素非靶標的豌豆蚜(Acyrthosiphon pisum)中腸氨肽酶N(amino peptidase N, APN)受體結合的短肽GBP3.1對Cry1Ab蛋白domainⅡ內3個loop分別進行替換，通過檢測loop替換后3株改造蛋白對小菜蛾(Plutella xylostella)幼蟲的毒性變化來分析3個loop在Cry1Ab毒性功能中的重要性差異。生物測定結果顯示，與野生型Cry1Ab的LC50=0.88μg/mL相比，loop 1、2和3分別被替換后的Cry1Ab的LC50為12.17、32.25和23.00μg/mL。由此可見，loop 2在Cry1Ab殺蟲活性中起到的作用最大，loop 3次之，loop 1對Cry1Ab毒素的毒性作用影響最小。該結論與Cry1A蛋白的三維空間結構分析相吻合，并為進一步了解Bt Cry1A毒素作用機制及其三維結構中功能域的研究提供了依據。%Receptor binding is a key step in the mechanism of Bacillus thuringiensis Cry toxins and mainly determined by 3 loops locate on the apex ofβ-sheet in domainⅡ. The important role of these 3 loops, function in binding with specific receptors in the midgut of target insect, has currently been widely reported. However, the importance of 3 loops to influence the toxicity of Cry is different. To determine the important role of these 3 loops in insecticidal activity of Cry1A toxins, in this research, overlap PCR was conducted by using a pea aphid (Acyrthosiphon pisum) gut APN (amino peptidase N) receptor binding peptide GBP3.1 to replace 3 loops in domainⅡ. Then 3 plasmids carrying each modified cry1Ab gene were constructed. After that, prokaryotic expression and purification of 3 modified Cry toxins were conducted. Leaf residue bioassay was conducted by feeding each modified Cry toxin to Plutella xylotella lavae to test the impact of loop substitution on insecticidal activity. Bioassay results indicated that substitution of each loop decreased the toxicity against P. xylotella larvae at different degree, with the LC50 of 12.17, 32.25 and 23.00 μg/mL for the substitution of loop 1, 2 and 3, respectively, compared with wild-type toxin (LC50=0.88 μg/mL). It suggested that compared with loop 1 and 3, loop 2 played the most important role in exhibition of insecticidal activity of Cry1Ab toxin. Prediction of 3-dimensional structure of Cry1Ab toxin and 3 modified Cry toxins showed that replacement of each loop by GBP3.1 did not change the main structure of Cry1Ab protoxin, which meaned that the variety of toxicity against P. xylatella was mainly attributed to the substitution of each receptor binding loop. Moreover, analysis of 3-dimensional structure of Cry1Ab protoxin indicated that loop 2 was the most exposed receptor binding loop compared with loop 1 and 3. These results will be helpful for the understanding of mechanism of Cry1A toxins in the midgut of target insects and benefit further research on gene modification in receptor binding regions of Bt Cry toxins.
11.煤礦中蘇云金芽胞桿菌（Bt）菌株的分離及其鑒定
- 林白容;章秋萍;王毅斌;關雄;
- 《農業生物技術學報》 | 2014年第011期
摘要：蘇云金芽胞桿菌(Bacillus thuringiensis, Bt)是一種已被廣泛用于農業害蟲生物防治的昆蟲病原細菌。本研究從永春天湖山礦區采集的159份樣品中分離出1株Bt，光學顯微鏡檢測發現菱形伴孢晶體。以cry1/7/9、cry2、cry3、cry4/10、cry5、cry6、cry8、cry11和cry1I的通用引物對該Bt分離菌進行Cry毒素基因分析，利用SDS-PAGE和雙向電泳-質譜法(2-dimensional electrophoresis/mass spectrometry,2-DE/MS)對Bt分離菌進行蛋白鑒定。結果表明，分離菌含有cry1基因型，經克隆、測序后可知，菌株中的cry1亞基因型與cry1Ac有極高的同源性。分離株表達的晶體蛋白約為130 kD，通過蛋白質譜分析表明，殺蟲蛋白為Cry1Ac，與PCR-RFLP所得出的結果一致；通過質譜分析還鑒定出分子伴侶GroEL、F0F1型ATP-β亞基合成酶、延伸因子Tu和丙酮酸脫氫酶。生物信息學分析結果表明，這些蛋白主要參與幫助新生肽的正確折疊、能源生產和轉換，以及提高細胞的應激能力以抵御外界不良刺激。本研究從煤礦中分離鑒定出Bt菌，為發現新資源、新型殺蟲蛋白基因以及利用微生物治理重金屬污染提供基礎資料。%Bacillus thuringiensis (Bt) is an insect pathogenic bacteria that has been widely used in biological control of agricultural insect pests. In this study, one Bt strain was isolated from 159 samples collected from coalfields in Tianhu Mountain, Yongchun County, Fujian Province. Bipyramidal parasporal crystal of isolated Bt strain was observed under microscope. PCR results showed that isolated Bt strain carried cry1 type gene using general primers of cry1/7/9, cry2, cry3, cry4/10, cry5, cry6, cry8, cry11 and cry1I genes, then cry1 gene was cloned and sequenced. Homology analysis of nucleotide sequence of cry1 showed that it was of high similarity with the reported cry1Ac genes. Results of SDS-PAGE and 2-dimensional electrophoresis/mass spectrometry (2-DE/MS) showed that isolated Bt strain encoded approximately 130 kD crystal protein, which was then identified as insecticidal protein Cry1Ac by mass spectrometry (MALDI-TOF/TOF-MS). Molecular chaperone GroEL, ATP F0F1 synthase subunit beta, elongation factor Tu and pyruvate dehydrogenase were identified by MALDI-TOF/TOF-MS, respectively. Further understanding of protein functions were conducted through bioinformatics analysis, leading to correct folding of nascent peptides, energy production and conversion, as well as improving the ability of cells to withstand adverse external stimuli. This work provides basic data for discovering new Bt resources, new insecticidal protein and using microbial to control heavy metal pollution.
12.利用RNAi技術研究水稻瘤矮病毒P8蛋白在病毒裝配中的作用
- 劉華敏;鄭立敏;賈東升;李俊欽;陳紅燕;魏太云;
- 《農業生物技術學報》 | 2014年第011期
摘要：Rice gall dwarf virus (RGDV) is a member of the genus Phytoreovirus in the family Reoviridae, which is transmitted by leafhopper Recilia dorsalis and Nephotettix cincticeps in a persistent-propagative manner. The viral genome of RGDV encodes 6 structural proteins and 6 nonstructural proteins. The structural protein P8 is a component of viral outer capsid protein, but the function of this protein in viral infection in insect vector is still unknown. Here, RNA interference (RNAi) induced by double-stranded RNA(dsRNA) synthesized in vitro was used to investigate the role of RGDV P8 protein in viral infection in the cultured cells derived from R. dorsalis. Immunofluorescence assay of RGDV infection in cultured cells after the treatment of dsRNAs from P8 gene (dsP8) howed that dsP8 did not only knock down the expression of P8, but also reduced the accumulation of RGDV in the insect vector cells. Then viral dsRNA genome and proteins were detected by SDS-PAGE and Western blot, the results indicated that the treatment of dsP8 significantly decreased the synthesis of viral dsRNA genome and the expression of P8 protein in insect vector cells. Thus, the results suggested that structural protein P8 of RGDV might play a role in viral assembly and multiplication in insect vector cells. This provides important theoretical basis for controling the spreading of rice gall dwarf disease by using RNAi technology.%水稻瘤矮病毒(Rice gall dwarf virus, RGDV)是呼腸孤病毒科(Reoviridae)植物呼腸孤病毒屬(Phytoreovirus)的一個成員，由介體電光葉蟬(Recilia dorsalis)和黑尾葉蟬(Nephotettix cincticeps)以持久增殖型方式傳播。其基因組編碼6個結構蛋白和6個非結構蛋白。已知結構蛋白P8是病毒外層衣殼的主要成分，但其在病毒侵染介體細胞過程中的功能尚未弄清。本研究利用體外合成的dsRNA誘導的RNA干擾(RNA interference, RNAi)技術，研究RGDV P8蛋白在病毒侵染電光葉蟬培養細胞中的功能。通過免疫熒光標記技術對dsP8處理后的電光葉蟬培養細胞進行RGDV侵染觀察，發現dsP8處理不僅抑制了P8在細胞內的正常表達，同時也抑制了病毒在細胞內的積累。隨后通過SDS-PAGE凝膠電泳和Western blot分別檢測病毒的dsRNA基因組和病毒蛋白水平的變化，發現dsP8處理顯著降低了病毒雙鏈RNA (double-stranded RNA, dsRNA)基因組的合成水平和病毒P8蛋白的表達量。由此推測RGDV P8蛋白參與了病毒粒體的組裝過程和病毒在介體昆蟲細胞內的有效增殖和侵染過程。本研究結果為利用RNAi技術控制水稻瘤矮病的傳播提供了重要的理論依據。
13.殺蟲抗生素的研究進展
- 陳園;張曉琳;黃穎;李曉敏;
- 《農業生物技術學報》 | 2014年第011期
摘要：Natural biological control agents such as insecticides that exhibit high efficacy, low toxicity, superior selectivity and environmental friendliness, have maintained an important role in integrated pest management of agriculture. This paper provided an overview of recent promising insecticides from natural sources. They were avermectins, spinosyns, meibemucins, and ivermectin. The researches on their efficacies, biochemical properties, mechanism of actions, as well as recent advances in the molecular genetics of the producing microorganisms at home and abroad would be discussed here.%殺蟲抗生素作為生物源殺蟲劑，具有高效、低毒、選擇性強、環保等優點，在農業害蟲綜合治理中占有重要地位，是無公害作物首選農藥之一。本文主要從生理生化、作用機制、分子遺傳研究等方面，介紹幾種應用前景廣闊的殺蟲抗生素近年來國內外的研究進展。
14.玉米根際土壤芽胞桿菌的多樣性
- 劉國紅;朱育菁;劉波;車建美;唐建陽;潘志針;陳澤輝;
- 《農業生物技術學報》 | 2014年第011期
摘要：為了解玉米(Zea mays)不同品種根際芽胞桿菌種群多樣性信息，本研究采用稀釋平板法，對10個玉米品種根際的芽胞桿菌進行了分離，并對其進行16S rRNA序列分析。結果表明，從10個玉米品種根際共分離到69個形態差異的芽胞桿菌菌株；16S rRNA序列分析表明，69個菌株鑒定為23個種，歸屬于3個屬，芽胞桿菌屬(Bacillus)、賴氨酸芽胞桿菌屬(Lysinibacillus)和嗜冷芽胞桿菌屬(Psychrobacillus)，其中芽胞桿菌屬的種類和數量最多。玉米不同品種根際的芽胞桿菌的種類數量不同，QB662× QB2219和QB1013×QB446根際分離到的芽胞桿菌種類最多，均為8種；J106×QB572的芽胞桿菌菌落含量最高。玉米根際的優勢種群主要為嗜氣芽胞桿菌(B. aerophilus)、阿氏芽胞桿菌(B. aryabhattai)、簡單芽胞桿菌(B. simplex)和蘇云金芽胞桿菌(B. thuringiensis)，其他芽胞桿菌種類僅在一種或少數的玉米品種出現。QB662×QB2219根際的芽胞桿菌種類香農-威納(Shannon-Wiener)指數(H)最大；QB948×QB48根際的芽胞桿菌種群香農-威納多樣性指數次之，但均勻度指數最高；J106×QB572的多樣性指數和均勻度指數皆最低。菌株FJAT-17411、FJAT-17472、FJAT-17430和FJAT-17442與GenBank中已報道16S rRNA基因序列的相似性為97%~98%，可能為芽胞桿菌的新種。本研究結果表明，玉米根際具有較為豐富的芽胞桿菌種群多樣性，并存在一些潛在的新的微生物菌種資源。本研究對芽胞桿菌資源的開發和利用提供良好的實驗材料和理論基礎。%The aim of this study was to determine the diversity of the cultivable Bacillus species population in the rhizosphere of maize (Zea mays). Bacillus species were isolated from 10 soil samples by using the dilution method on NA medium and 69 isolates were obtained through morphological difference, and identified by 16S rRNA. Phylogenetic analysis based on 16S rRNA gene sequencing showed that the 69 isolates were grouped into 3 genera (Bacillus, Lysinibacillus and Psychrobacillus) with 23 distinct species. Most of these species were owned to the genus Bacillus. The number of species isolated from every maize variety was different. The most number of Bacillus species (8 species) occurred in the varieties QB662 × QB2219 and QB1013 × QB446, and the most colonies occurred in the variety J106 × QB572. The dominant populations in the maize rhizosphere were B. aerophilus, B. aryabhattai, B. simplex and B. thuringiensis, other species only occurred in one maize variety or few maize rhizosphere. The highest Shannon-Wiener index was QB662×QB2219, the second was QB948×QB48 but owning the highest evenness index. The lowest Shannon-Wiener index and evenness index was J106×QB572. Strains FJAT-17411, FJAT-17472, FJAT-17430 and FJAT-17442 may be the new species, which had lower similarities with the known Bacillus spcies in GenBank database. In conclusion, the result of this study showed that the population diversity of the cultivable bacteria was abundant and there were some potential novel species in the rhizosphere of maize. The findings provide an important scientific base for the development and utilization of Bacillus resources.
15.濃香型白酒老窖泥中好氧芽胞桿菌的分離及系統發育樹的初步構建
- 鄭琦;章秋萍;何新新;胡杰;黃小紅;
- 《農業生物技術學報》 | 2014年第011期
摘要：Production of Luzhou-flavor liquor is based on pits where ample microbial communities are living, and interactions among all sorts of microbes especially Bacilli in it make fermented sapor of Luzhou-flavor liquor very unique. In this study, 23 aerobic Bacilli were isolated from 300-year pit mud of cellar affiliate to Luzhou laojiao, Luzhou, Sichuan Province. Microscopic and colonial morphology analysis showed that the difference between these aerobic Bacilli were not significant. Through PCR amplification of 16S rDNA gene and NCBI blast, a phylogenetic tree was preliminarily constructed. The results showed that the strains of aerobic Bacilli were mainly Bacillus thuringiensis, B. cereus and B. anthracis. The genetic relationship of these strains was inconformity. As a whole, the phylogenetic relationship was close and the genetic polymorphism was low between these Bacilli isolated from 300300-year pit mud. Aged pit mud was always in the surroundings of anaerobic or facultative anaerobic, studying on the survival condition and genetic relationship of the aerobic Bacilli in aged pit mud was helpful to get a insight in the change of surrounding environment during the liquor fermentation, and improve the quality of liquor. The result provides basic data for research on the change of surrounding environment during the liquor fermentation, and improving the quality of liquor.%濃香型白酒的生產是以窖池為基礎，窖池窖泥中棲息著龐大的微生物群系，微生物間相互作用形成濃香型白酒的獨特風味，尤其以芽胞桿菌為主。本研究從瀘州老窖酒廠300年窖池窖泥中分離出23株好氧芽胞桿菌屬(Bacilli)，經光學顯微鏡和菌落形態觀察，23株好氧芽胞桿菌形態差異不顯著。通過16S rDNA基因擴增，NCBI基因比對，構建系統發育樹，結果表明，23株好氧芽胞桿菌主要為蘇云金芽胞桿菌(Bacillus thuringiensis, Bt)、蠟樣芽胞桿菌(B. cereus, Bc)和炭疽芽胞桿菌(B. anthrax)，菌種間親緣關系存在差異；窖泥中分離出的23株好氧芽胞桿菌親緣關系近，遺傳多樣性較低。本研結果為了解白酒發酵過程中老窖泥周圍環境的變化、提高白酒品質提供基礎資料。
16.插入序列ISRso21在青枯雷爾氏菌中的分布以及插入位點多態性分析
- 車建美;林海云;劉波;林營志;
- 《農業生物技術學報》 | 2014年第011期
摘要：插入序列(insertion sequence, IS)元件可以插入到基因或DNA序列，其可能在生物體進化中起著重要的作用。ISRso21是在青枯雷爾氏菌(Ralstonia solanacearum)FJAT-1458菌株中發現的一個新的插入序列。根據序列分析可知，該插入序列是屬于ISL3家族中的一個成員。本研究發現，福建閩北地區菌株ISRso21的陽性率高于其他地區的趨勢，不同地區的青枯雷爾氏菌ISRso21的分布有顯著性差異， ISRso21的分布可能與菌株分離個體的地理來源有關。不僅如此，由于ISRso21在青枯雷爾氏菌插入位點的不同，從而導致青枯雷爾氏菌致病性的差異。研究表明，由于ISRso21的插入導致表現型轉換系統轉錄調控因子A(phcA)的重排可能在青枯雷爾氏菌的致病性起著極其重要的作用。在所有菌株中， ISRso21在phcA基因上游中插入的檢出率達到4.71%，其中無致病力菌株和強致病力菌株中的檢出率分別為28.57%和0.00%。而ISRso21在二氨基庚二酸脫羧酶基因中的插入可能是為了更好地適應環境。ISRso21在不同致病性青枯雷爾氏菌中的插入位點的研究，可能為揭示青枯雷爾氏菌致病性機制提供新的研究途徑。%Insertion sequence (IS) elements of organisms have the ability to move into genes or DNA sequences as a single unit, implying that these elements play an important role in the evolution of the organisms. A new IS (ISRso21) was isolated and characterized from Ralstonia solanacearum strain FJAT-1458. Sequence analysis indicated that the ISRso21 was closely related to the members of ISL3 family. The results of insertion sites polymorphism of ISRso21 showed that the frequency of positive strains isolated from Northern of Fujian Province was higher than that isolated from other areas. There was significant difference in the distribution of ISRso21 among R. solanacearum isolated from different geography. Therefore, the present of ISRso21 in R. solanacearum strains might be associated with geographic origin. Moreover, the different insertion sites of ISRso21 in R. solanacearum genome were associated with the different pathogenicity. The results indicated that genomic rearrangements of phenotype conversion transcriptional regulator A (phcA) which was inserted by ISRso21, might play a major role in the control of pathogenicity. In upstream of phcA gene of R. solanacearum, the inserted detection rate of ISRso21 in all strains was 4.71%. The detection rates in avirulent strains and virulent strains were 28.57% and 0.00%, respectively. The insertion of ISRso21 in diaminopimelate decarboxylase gene might make the R. solanacearum strains adapt betterly to the environment. The different insertion sites of ISRso21 in the R. solanacearum strains need to be furtherly investigated, which can provide an alternative way to reveal the pathogenicity of R. solanacearum.
17.編者按
- 《農業生物技術學報》 | 2014年第011期
摘要：植物病蟲害一直是困擾農業生產的一大難題，嚴重影響著農產品的產量和品質。19世紀70年代以來，化學農藥的發明在防治植物病蟲害方面發揮了重要的作用，但由于其過度和不合理的使用，導致了人體健康、污染環境和害蟲產生抗性等一系列問題。生物農藥是指利用真菌、細菌、昆蟲病毒、轉基因生物和天敵等生物活體或其代謝產物。
18.藤黃輪絲鏈霉菌T0907-107抑菌活性研究
- 肖順;張婷婷;盧云彬;劉國坤;張紹升;
- 《農業生物技術學報》 | 2014年第011期
摘要：從海產蝦殼上分離篩選拮抗放線菌、研究其抑菌活性具有重要的實踐意義。藤黃輪絲鏈霉菌(Streptomyces luteoverticillatus)T0907-107是分離自海產蝦殼上并經初篩和鑒定的一株鏈霉菌，為了進一步開發和利用該菌，本研究采用極性不同的5種溶劑(丙酮、乙酸乙酯、正丁醇、乙醇和甲醇)，對該菌株的發酵液菌體活性物質進行了提取，對粗提物的理化性質和抑菌活性進行了初步研究。結果表明，其發酵液活性物質大部分存在于菌體中，采用甲醇和丙酮浸提的效果好，其抑菌圈直徑分別為26.20和22.50 mm；對粗提物進行耐熱性實驗，結果表明，粗提物是一種能夠耐60℃高溫和一定酸堿的物質；粗提物對蛋白酶K穩定；菌體及粗提物對多種植物病原菌物如煙草炭疽病菌(Colletotrichum nicotianae Sacc.)、煙草赤星病菌(Alternaria alternata (Fries) Keissler)、煙草立枯絲核病菌(Rhizoctonia solani Kühn)、枇杷炭疽菌(C. gloeosporioides (Penz.) Sacc.)和白菜炭疽病菌(C. higginsianum Sacc.)有較強抑制作用。研究結果表明，甲醇和丙酮是較合適的萃取溶劑，該菌發酵液菌體粗提物極性強、穩定性好、有較廣的抑菌譜，顯示出好的開發前景。本研究為進一步鑒定該活性物質及開發和利用該菌提供了基礎資料。%It has important practical significance to isolate and screen antagonistic actinomycetes from marine shrimp shell and study their antimicrobial activity. Streptomyces luteoverticillatus strain T0907-107 was isolated and identified from shrimp shell. In order to furtherly exploit and utilize the bacteria, antimicrobial activity substance was extracted from the thalli of strain T0907-107 which was collected from fermentation broth by 5 different solvents, acetone, ethyl acetate, n- butanol, ethanol and methanol. Antimicrobial activity and stability of the antimicrobial metabolites produced from strain T0907-107 were carried out. The results showed that acetone and methanol were the best solvents for the crude extract, and the inhibition zones were 26.20 and 22.50 mm, respectively. After protease K treatment, the crude extract still had inhibitory effects to the fungus. The crude extracted substance was material of thermostability (60℃) and had a good performance of acid and alkali resistance. The thalli of the strain and the crude extracted substance presented a strong antifungal activity against half of tested plant pathogenic fungi such as Colletotrichum nicotianae Sacc., Alternaria alternata(Fries)Keissler, Rhizoctonia solani Kühn, C. gloeosporioides (Penz.) Sacc. and C. higginsianum Sacc..The results showed that the acetone and methanol were good extraction solvents, the crude extracted substance had strong polarity and a strange of thermostability, acid and alkali resistance, which indicated that the bacteria had good development prospect. This study provides basic data for further identification of the active material and the exploitation and utilization of the strain T0907-107.
19.埃及伊蚊免疫應答相關基因mapk-like的dsRNA原核表達及其飼喂后表達水平的變化
- 吳松青;劉昭霞;蘇偉超;王官棟;陳慧成;吳志卯;關雄;張靈玲;
- 《農業生物技術學報》 | 2014年第011期
摘要：mapk-like是一種信號轉導相關促有絲分裂激活蛋白激酶家族基因，與線蟲(Caenorhabditis elegans)中參與抵御Cry毒素的p38 MAPK含有類似的模序，其是否參與埃及伊蚊(Aedes aegypti)抵御蘇云金芽胞桿菌(Bacillus thuringiensis, Bt)過程有待驗證。為簡便并大量獲得RNAi驗證所需的dsRNA，本研究利用RNaseⅢ缺陷型大腸桿菌(Escherichia coli) HT115高效和廉價的優點，根據已測序的促有絲分裂激活蛋白激酶家族基因mapk-like基因序列，設計特異性引物，PCR擴增大小為361 bp的目的片段mapk-like (GenBank登錄號：AAEL003728-RA)，將其連接到克隆載體pMD18-T上，利用限制性內切酶XbaⅠ和XhoⅠ將其插入到原核表達載體pLitmus28i的2個T7啟動子之間，成功構建可誘導形成目的雙鏈RNA (dsRNA)的原核表達重組載體pLitmus28i-mapk-like，并轉化大腸桿菌HT115，經IPTG誘導，1 mL菌液的dsRNA產量可達1.18μg，電泳檢測條帶清晰，質量良好。此外，利用殼聚糖包裹dsRNA形成納米微粒，上清經電泳檢測表明，殼聚糖的聚沉效率良好，納米微球可有效防止dsRNA從飼料瓊脂塊中游離出來，提高dsRNA的穩定性。埃及伊蚊幼蟲攝取mapk-like基因的dsRNA后，相對轉錄表達水平為65.55%，表達量明顯下調，飼喂效果良好。研究結果將有利于進一步篩選獲得相關抵御基因，為建立一個基于RNA沉默防治蚊媒病的研究體系提供了基礎資料。%Mapk-like, the signaling transduction related mitogen-activated protein kinase gene, shares a conserve motif with p38 MAPK that involves in cellular defense of Caenorhabditis elegans against Bacillus thuringiensis(Bt) Cry toxins. Little validation work, however, had been done in its important role for defense response of Aedes aegypti to Cry toxins. Therefore, RNaseⅢ-absent Escherichia coli HT115, with the high-efficiency and low-cost, was applied to establish a convenient and economical method for mass production of dsRNA for RNA silencing. Using specific primers, mapk-like gene (GenBank No. AAEL003728-RA) was amplified. Then 361 bp PCR products were cloned into the cloning vector pMD18-T and subcloned into the expression vector pLitmus28i, which contained 2 T7 promoters located in each side of multiple cloning sites with the digestion of restriction endonuclease XbaⅠ/XhoⅠ. The recombinant plasmid pLitmus28i-mapk-like was transformed into the HT115. dsRNAs of 1.18 μg/mL of bacteria liquid with high quality were obtained after 0.5 mmol/L IPTG induction. In addition, nanoparticles were prepared by mixing resulted dsRNA and chitosan and the supernatant was examined by agarose gel electrophoresis. These results indicated a good coagulation effect for chitosan, by which nanoparticles could protect dsRNA from dissociation from the mixture of feed andagarose and the stability was improved. Finally, the relative expression levels of mapk-like gene after the effective dsRNA feeding decreased to 65%. These results provide potential for use in RNAi, screening for more defense related gene, and basic research for RNA silencing to control mosquito transmitted diseases.
20.基因工程菌誘導表達蘇云金芽胞桿菌Vip3Aa蛋白的條件優化
- 宋飛飛;李夢楠;陳凡冰;關雄;黃志鵬;
- 《農業生物技術學報》 | 2014年第011期
摘要：Vegetative insecticidal protein (Vip) is secreted by Bacillus thuringiensis (Bt) during the period from vegetative growth to logarithmic metaphase. To obtain efficient expression condition of Vip3Aa protein, 3 main factors (concentration of isopropyl- β- d- thiogalactoside(IPTG), inducing time and inducing temperature) were screened for the optimization inducing condition of recombinant Escherichia coli BL21-pCzn1-Vip in producing the Vip3Aa by orthogonal experiment. The highest expression level of target protein could be attained with the condition of 200μg/mL IPTG inducing for 8 h at 21℃. The data were analyzed by direct calculation and one-way ANOVA method. R value (resulting from direct calculation) of inducing temperature was the highest among the 3 factors, followed by the inducing time and the concentration of IPTG, wihch indicated that the inducing temperature was the most important factor to affect expression level of target protein. P value from one-way ANOVA of inducing temperature and inducing time was lower than 0.01 and 0.05, respectively, and the P value of the other factor was higher than 0.05, which were similar as the results of direct calculation. The amount of the expressed target protein at the optimal inducing condition was up to 27.6% of total protein, which was 2.3 times of the production before optimization. It can provide the basis for the Vip3Aa manufacture, in-depth theoretical research and practical application.%營養期殺蟲蛋白(vegetative insecticidal protein, Vip)是在蘇云金芽胞桿菌(Bacillus thuringiensis, Bt)營養生長至對數中期到穩定期期間產生的一類蛋白。為獲得蘇云金芽胞桿菌Vip3Aa蛋白的高效表達條件，本研究利用正交試驗綜合考察了大腸桿菌(Escherichia coli)重組基因工程菌BL21-pCzn1-Vip誘導過程中誘導劑濃度、誘導時間和誘導溫度對Vip3Aa蛋白表達量的影響。結果表明，該工程菌目的蛋白的最優誘導表達條件：誘導溫度21℃、誘導時間8 h、誘導劑(異丙基-β-d-硫代半乳糖苷(isopropyl-β-d-thiogalactoside, IPTG))濃度為200μg/mL；直觀分析表明，影響目的蛋白表達量的最主要因素為誘導溫度，其次為誘導時間，誘導劑濃度影響最小；方差分析結果表明，誘導溫度的P值＜0.01、誘導時間的P值＜0.05、誘導劑濃度的P值＞0.05，與直觀分析結果相一致；利用優化后誘導表達條件進行培養，測得目的蛋白最高表達量可占總蛋白的27.6%，相當于常規誘導方法的2.3倍。本研究為大量制備高純度Vip3Aa蛋白及其深入的理論研究和實踐應用提供了基礎資料。
21.鴻雁線粒體DNA全基因組序列測定及分析
- 穆春宇;陳國宏;黃正洋;陳陽;王彬;蘇燕輝;李洋;孫志明;徐琪;趙文明;
- 《農業生物技術學報》 | 2014年第012期
摘要：線粒體基因組(mitochondrial genome, mtDNA)具有進化速率快、多態性豐富、無重組、母系遺傳等特點，是開展群體遺傳學、系統發育學、分子生態學、分類學等研究的理想分子標記。本研究根據鴻雁(Anser cygnoides)同屬近緣物種豆雁(Anser fabalis)線粒體全基因組序列(EU009397.1)設計引物，采用直接測序技術對鴻雁線粒體基因組全序列進行了綜合分析，結果顯示，鴻雁線粒體基因組序列(GenBank登錄號：KJ124555)全長16739 bp，包含22個tRNA、2個rRNA基因、13種蛋白質編碼基因和一個D-loop區。堿基組成T占22.49%，C占32.24%，A占30.21%，G占15.06%，無明顯的AT偏好性。22種tRNA都為典型的三葉草結構，參照原雞(Gallus gallus)、黑尾地鴉(Podoces hendersoni)的12SrRNA，對鴻雁12SrRNA的二級結構進行了預測，含有4個結構域、37個莖環和13個突出部。對D-loop控制區序列分析發現，含有LSP/HSP、ETAS1-2、Goose hairpin、E-box、F-box、D-box、C-box、Bird similarity-box、CSB1-box、CSB-like和OH。以原雞作為外群，采用鄰接法(N-J)和最大擬然法(ML)算法以及貝葉斯法，基于線粒體基因組全序列分別構建系統進化樹，結果顯示，鴻雁與灰雁(Anser anser)、豆雁(Anser fabalis)、白額雁(Anser albifrons)和加拿大黑雁(Branta canadensis)處于一個分支，親緣關系較近。該研究結果豐富了鴨科線粒體基因組全序列，為研究鴻雁的系統發生和分子進化研究以及種質資源保護和合理利用提供新的基礎資料。%Mitochondrial genome (mtDNA) has advantages in rapid evolution, rich polymorphism and maternally inheritance without gene recombinations, which has become an ideal molecular markers of population genetics, phylogenetics, molecular ecology and taxonomy. In this study, the primers were designed based on the mitochondrial genome sequence of Bean goose (Anser fabalis) which was a closely related species of Swan goose (Anser cygnoides). Swan goose mitochondrial genome sequence was analysed by direct sequencing techniques. The results showed that whole mitochondrial genome sequence was 16 739 bp (GenBank accession No. KJ124555) in Swan goose, including 22 tRNA genes, 2 rRNA genes, 13 protein-coding genes and a D-loop region. Base composition of T, C, A and G were 22.49%, 32.24%, 30.21%and 15.06%, respectively. Besides, the base preference of AT was not determined. 22 kinds of tRNA were all typically cloverleaf structures. Compared to 12SrRNA of Red Junglefowl (Gallus gallus) and Mongolian Ground Jay (Podoces hendersoni), we found the secondary structure of 12SrRNA included 4 domains, 37 stem-loops and 13 salients in the Swan goose, and LSP/HSP, ETAS1-2, goose hairpin, E-box, F-box, D-box, C-box, Bird similarity-box, CSB1-box, CSB-like and OH in the D-loop control region. Finally, taken Red Junglefowl as an outgroup, the phylogenetic tree was constructed based on mitochondrial genome sequences using Neighbor-joining (N-J) algorithm, Maxium-likelihood (ML) algorithm and Bayesian model. The results showed that Swan goose, Greylag goose, Bean goose, white-fronted goose and Canada goose had close genetic relationship. The findings enrich the ducks mitochondrial genome sequences and provide a theoretical basis for the study of geese phylogeny.
22.利用簡單重復序列（SSR）標記分析太湖稻區現代粳稻品種的遺傳多樣性
- 羅兵;徐港明;孫海燕;楊志剛;沈宗根;高陽;王小虎;端木銀熙;
- 《農業生物技術學報》 | 2014年第012期
摘要：The japonica rice (Oryza satiua ssp. japonica) resources are rich in Taihu area. Along with large-scale popularization of rice varieties, the genetic basis of breeding material is became narrower and the genetic similarity is increased, thus the japonica rice breeding has became more difficult. Genetic diversity of 42 japonica rice varieties, collected from Taihu area, was analyzed by 24 pairs of simple sequence repeat (SSR) primers. The SSR primers were located at 12 rice chromosomes, and 2 pairs of SSR primers were selected at each chromosome. The results showed that 23 pairs of SSR primers performed polymorphism in the 42 materials. A total of 105 alleles loci were detected among 42 varieties using the 23 pairs of SSR primers. The average number of detected alleles by each primer pair was 4.57 with a range from 2 to 8. The effective alleles were 56.43 with average 2.45. The value of allelic polymorphism information content (PIC) varied widely from 0.083 0 to 0.807 9 with an average value of 0.496 6. The number of polymorphic loci each variety ranged from 6 to 19, and the number of allele loci ranged from 27 to 55. The genetic similarity coefficient of SSR markers among 42 japonica rice varieties ranged from 0.391 to 0.990, with average 0.610, and the genetic similarity coefficient of 74.45% tested materials ranged from 0.50 to 0.80. Furthermore, the UPGMA(unweight pair group method with arithmetic mean) clustering analysis at the genetic similarity value of 0.50 grouped the 42 japonica rice varieties into two groups. The 19 conventional japonica rice varieties could be grouped, and they were divided into 2 subgroups at the level of genetic similarity coefficient 0.58. Another group was composed of the 23 japonica hybrid rice varieties, and it also was divided into two subgroups at the level of genetic similarity coefficient 0.70. Based on the all analysis, it was concluded that the genetic background comparability of the japonica rice varieties in Taihu area was high and the diversity was low. Therefore, introducing and making use of the new genetic resources and creating breeding materials should be strengthened. The genetic diversity of japonica rice based on SSR markers can provide technical support and theoretical basis for breeding new rice varieties in Taihu area.%太湖地區有豐富的粳稻(Oryza satiua ssp. japonica)種植資源，隨著品種大面積推廣應用，育種材料的遺傳基礎趨窄，相似性增高，使粳稻育種突破困難。本研究利用分布于水稻(Oryza satiua L.)12條染色體上的24對簡單重復序列(simple sequence repeat, SSR)引物對太湖地區的42份粳稻品種進行遺傳多樣性分析。結果表明，有23對SSR引物在42份粳稻材料間表現出多態性；23對引物共檢測到105個等位基因，每對SSR引物檢測到等位基因為2~8個，平均為4.57個，有效等位基因共有56.43個，平均每個位點為2.45個。每個多態位點的多態信息含量(polymorphism information content, PIC)變幅為0.0830~0.8079，平均為0.4966；每個粳稻材料多態性位點數的變幅為6~19，等位基因總數的變幅為27~55；42份粳稻品種間的遺傳相似系數變幅為0.391~0.990，平均為0.610，遺傳相似系數在0.50~0.80之間的材料占全部的74.45%，供試材料相似度高；非加權配對算術平均法(unweighed pair group method with arithmetic mean；UPGMA)聚類結果顯示，遺傳相似系數為0.50，42份粳稻材料可以分為兩個類群，一個類群包含19份常規粳稻，另一個類群包括其他23份雜交粳稻。結果顯示，太湖地區的粳稻品種總體上遺傳背景相似度高，遺傳多樣性不夠豐富，育種工作有待進一步加強新的基因資源引進和利用，創新水稻育種材料。本研究結果為新品種選育提供技術支持和理論根據。
23.黃芩苷對熱應激條件下豬近端腎小管（LLC-PK1）細胞凋亡率及B細胞淋巴瘤-2基因（Bcl-2）和Bcl-2相關X蛋白基因（Bax）表達的影響
- 孫春玲;國曉瞳;趙園;陳健偉;叢霞;王新;姜忠玲;高善頌;田文儒;
- 《農業生物技術學報》 | 2014年第012期
摘要：The objective of this study is to explore baicalin influence B-cellymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) expression as well as apoptosis rates of heat-stressed pig kidney proximal tubular cells (LLC-PK1). Readily cultured LLC-PK1 cells were randomly divided into seven groups. The cells in groupⅠwere used as blank control cultured at 37℃and cells in groupⅡwere heart-stressed at 42℃for 1 h. Cells in groupsⅢ,Ⅳ,Ⅴ,ⅥandⅦwere cultured additionally with different concentrations (0.01, 0.1, 1, 10 and 100μg/mL) of baicalin at 42℃for 1 h, respectively. Expression of Bcl-2 mRNA and Bax protein were detected with Real-time PCR and Western blot, respectively. Apoptosis rates of LLC-PK1 cells were determined with Annexin V-FITC/PI. Results showed that as compared to groupⅠ, Bcl-2 mRNA in groupⅡincreased significantly (P<0.05), and Bax mRNA and its protein increased extremely significantly (P<0.01), and both ratios between Bcl-2 and Bax mRNA and their proteins decreased significantly (P<0.01), and the apoptosis rates increased extremely significantly(P<0.01). However, there was no evident influence on Bcl-2 protein expression (P>0.05). Compared with groupⅡ, Bcl-2 mRNA expression significantly increased in groupⅣand Ⅵ (P<0.05) and extremely significantly in groupⅤ(P<0.01), but not in groupⅢor Ⅶ (P>0.05), while the Bal-2 protein expression had no difference (P>0.05). Similarly, compared with groupⅡ, both Bax mRNA and its protein expression decreased significantly in groupsⅢ, Ⅳand Ⅶ (P<0.05) and decreased extremely significantly in groupⅤandⅥ(P<0.01). Ratios between Bcl-2 and Bax mRNA and their proteins in all groups except groupⅢhad a significant increase (P<0.05), while the ratio between Bcl-2 and Bax protein in groupⅤincreased extremely significantly (P<0.01). The apoptosis rates decreased extremely significantly in groupⅤ and Ⅵ (P<0.01), and decreased significantly in group Ⅳand Ⅶ(P<0.05). The results indicated that certain concentration (0.1~100 μg/mL) of baicalin protectsed heat-stressed LLC-PK cells by down-regulating Bax expression, increasing the ratio between Bcl-2 and Bax and decreasing apoptosis rates. This experiment reveals some heat-resistant mechanism of baicalin on LLC-PK1 cells exposed to heat stress which will provide valuable reference for its clinical application.%為探討不同濃度黃芩苷對熱應激條件下豬近端腎小管細胞(pig kidney proximal tubular cells, LLC-PK1)細胞凋亡率及B細胞淋巴瘤/白血病-2基因(B-cellymphoma-2, Bcl-2)和Bcl-2相關X蛋白基因(Bcl-2 associated X protein, Bax)表達的影響，將培養的LLC-PK1細胞隨機分為7個組，Ⅰ組為37℃空白對照組，Ⅱ組為42℃單純熱應激1 h組，Ⅲ、Ⅳ、Ⅴ、Ⅵ和Ⅶ組分別為用不同濃度黃芩苷(0.01、0.1、1、10和100μg/mL)處理組后42℃熱應激1 h組，運用實時熒光定量PCR和Western blot分別檢測Bcl-2和Bax基因及蛋白的表達，流式細胞儀(Annexin V-FITC/PI雙染法)檢測細胞凋亡率。結果表明，與Ⅰ組相比，Ⅱ組能顯著誘導LLC-PK1細胞Bcl-2 mRNA的表達(P＜0.05)，極顯著誘導細胞Bax mRNA和蛋白的表達(P＜0.01)，能極顯著降低細胞Bcl-2和Bax mRNA和蛋白的比值(P＜0.01)，極顯著增加細胞凋亡率(P＜0.01)，但對細胞Bcl-2蛋白的表達無顯著影響(P＞0.05)。黃芩苷處理組與Ⅱ組相比，Ⅳ、Ⅴ和Ⅵ組LLC-PK1細胞Bcl-2 mRNA的表達量均升高，其中Ⅴ組差異極顯著(P＜0.01)，Ⅳ及Ⅵ組差異顯著(P＜0.05)，Ⅲ及Ⅶ組差異不顯著(P＞0.05)，而細胞Bcl-2蛋白的表達量與Ⅱ組相比均差異不顯著(P＞0.05)；同樣與Ⅱ組相比，黃芩苷處理的各組細胞Bax mRNA及蛋白的表達量均降低，其中Ⅴ及Ⅵ組差異極顯著(P＜0.01)，其余各組差異顯著(P＜0.05)；除Ⅲ組外，其他各組細胞Bcl-2和Bax mRNA及蛋白的比值與Ⅱ組相比均顯著升高(P＜0.05)，其中Ⅴ組細胞Bcl-2和Bax蛋白的比值差異極顯著(P＜0.01)；細胞凋亡率僅有Ⅲ組與Ⅱ組相比差異不顯著(P＞0.05)，而Ⅴ及Ⅵ組細胞凋亡率極顯著低于Ⅱ組(P＜0.01)，其余的Ⅳ和Ⅶ組顯著低于Ⅱ組(P＜0.05)。一定濃度范圍內的黃芩苷(0.1~100μg/mL)可能通過下調熱應激條件下LLC-PK1細胞Bax的表達，從而提高Bcl-2和Bax的比值，降低細胞的凋亡率，對細胞起到保護作用。本研究從分子水平研究黃芩苷緩解熱應激對豬LLC-PK1細胞的損害作用，可為明確其解熱機制提供理論基礎，并為其在臨床上的應用提供有價值的參考資料。
24.非生物脅迫下多年生黑麥草qRT-PCR分析中內參基因的選擇
- 嚴海東;蔣曉梅;張新全;黃琳凱;
- 《農業生物技術學報》 | 2014年第012期
摘要：提高實時熒光定量PCR(Real-time quantitative PCR, qRT-PCR)準確性的先決條件是選擇表達穩定性較高的內參基因。本研究以多年生黑麥草(Lolium perenne L.)為材料，利用qRT-PCR技術檢測翻譯起始因子4A(eukaryotic initiation factor 4 alpha, eIF4A)、TNF結合蛋白Ⅰ(Tat binding protein-1, TBP-1)、泛素連接酶(ubiquitin-conjugating enzyme, E2)、多聚泛素酶基因(ubiquitin, UBQ)、生物鐘受體蛋白(zeitlupe, ZTL)和甘油醛三磷酸脫氫酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)6個候選內參基因在不同非生物脅迫下的表達情況。運用geNorm(ver.3.5)、Normfinder(ver.0.953)、BestKeeper(ver.1.0)、Delta Ct和RefFinder軟件綜合分析表明，在干旱、鹽、熱脅迫及ABA處理下，eIF4A基因表達穩定性最高；在澇脅迫下，UBQ基因表達穩定性最高。因此，eIF4A和UBQ候選基因可作為不同脅迫下的內參基因，為進一步開展多年生黑麥草基因表達的定量研究了提供了一定的理論依據。%The selection of high stablely expressed reference genes is an important prerequisite for improving the accuracy of Real-time quantitative PCR (qRT-PCR). We investigated the expression of 6 candidate reference genes including eukaryotic initiation factor 4 alpha (eIF4A), Ti-binding peptide-1 (TBP-1), ubiquitin-conjugating enzyme (E2), ubiquitin (UBQ), zeitlupe (ZTL) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) under various abiotic stresses in perennial ryegrass (Lolium perenne L.). The analysis with geNorm (ver. 3.5), Normfinder (ver. 0.953), BestKeeper (ver. 1.0), Delta Ct and RefFinder revealed that under drought, salt, heat and ABA stresses, eIF4A gene was the most stable reference gene;under waterlogging stress, UBQ gene was the most stable reference gene. As a result, eIF4A and UBQ genes were selected as reference genes for perennial ryegrass under different abiotic stresses, which provides a foundation for gene expression investigation of perennial ryegrass.
25.中間錦雞兒CiMYBJ2基因克隆及表達分析
- 邢丹丹;韓曉敏;馮宗琪;楊杞;齊力旺;劉福軍;王瑞剛;李國婧;
- 《農業生物技術學報》 | 2014年第012期
摘要：MYB transcription factor is one of the largest family of transcription factors in plants which play impotant roles in regulation of plant growth and development, and response to many environmental stimuli. In this study, a gene named CiMYBJ2 (GenBank accession No.KJ937783) was cloned by RACE technique from Caragana intermedia and its expression level along with the promoter was analysed. The full-length cDNA of CiMYBJ2 was 1 368 bp with the 3'UTR of 196 bp and the 5'UTR of 203 bp. The ORF of CiMYBJ2 was 969 bp, encoding a protein of 322 amino acids which was hydrophilia. The full-length gDNA was 1 285 bp, including two introns and three exons. The expression pattern of CiMYBJ2 analysed by qRT-PCR showed that it was altered by heat, dehydration, NaCl and drought treatments. After heat treatment, the expression of CiMYBJ2 decreased at 1 and 3 h, and then maintained a lower level expression. the change of gene expression at different time points was extremely significant compared with the 0 h (P<0.01). After dehydration treatment for 1 h, the expression of CiMYBJ2 increased shortly, and then gradually decreased at 3 h, and the expression difference was not significant compared with the 0 h (P>0.05). After 8 h , the expression of the gene showed extremely significant comapred with that of the 0 h (P<0.01), and the expression level of the gene at 24 h reached only half of that at 0 h (P<0.05). Under NaCl treatment, the transcript level of CiMYBJ2 began to increase after 1 h, and reached the maximum at 3 h. After that the overall trend kept downward, the expression dropped to the lowest level at 8 h, and expression of the gene was extremely significant at different time points except for 12 h (P<0.01). After drought treatment of 6 d, the expression of CiMYBJ2 increased slightly, and dropped significanly after compared with 0 d (P<0.01), reached the minimum expression at 15 d. The promoter sequence of CiMYBJ2 with 873 bp in length was obtained by genome walking technique and many abiotic stress-related cis-elements were found. These results indicated that CiMYBJ2 might be involved in response of C. intermedia to abiotic stresses. The research results provide a basis for the further study of gene function and expression regulation.%MYB轉錄因子是植物體內最大的轉錄因子家族之一，參與植物生長發育的調控、對逆境的響應等生理過程。本研究通過RACE技術從中間錦雞兒(Caragana intermedia)中克隆了一個MYB轉錄因子CiMYBJ2(GenBank登錄號：KJ937783)，并對其在植物中的表達及其啟動子進行了初步研究。本研究克隆得到該基因cDNA全長1368 bp，3'UTR長196 bp，5'UTR長203 bp。其中開放閱讀框長969 bp，編碼一個含322個氨基酸的親水蛋白。gDNA序列長1285 bp，有2個內含子和3個外顯子。采用實時熒光定量PCR技術分析了CiMYBJ2的表達模式，結果發現，在高溫、脫水、NaCl、干旱等處理條件下，CiMYBJ2基因對不同脅迫均有響應。在高溫處理后，CiMYBJ2基因的表達量在1和3 h明顯降低，之后維持一個較低值，在不同時間點基因表達量與0 h相比差異均達到極顯著水平(P＜0.01)；在脫水處理1 h后CiMYBJ2基因的表達量上升，且與0 h相比差異極顯著(P＜0.01)，然后逐漸降低，在3 h CiMYBJ2基因的表達量與0 h相比差異不顯著(P＞0.05)，在8和12 h該基因的表達量與0 h相比差異均達到極顯著(P＜0.01)，在24 h的表達量是0 h的一半，且差異顯著(P＜0.05)；在NaCl處理1 h后CiMYBJ2基因的轉錄水平開始增加，3 h達到最高，之后總體呈下降趨勢，在8h表達量最低，在1、3、8和24h該基因的表達量與0h相比差異極顯著(P＜0.01)，在12 h CiMYBJ2基因的表達量與0 h相比差異不顯著(P＞0.05)；干旱處理6 d后，CiMYBJ2基因的表達量上升，之后一直下降，且與0 d相比差異達到極顯著水平(P＜0.01)，15 d表達量最低。研究結果暗示CiMYBJ2基因可能在植物對脅迫的響應過程中起作用。利用染色體步移技術克隆了中間錦雞兒CiMYBJ2基因啟動子序列，長度為873 bp，分析顯示，該啟動子具有響應多種非生物脅迫的順式作用元件。研究結果為進一步研究CiMYBJ2基因功能和表達調控提供了依據。
26.轉基因生物對腸道微生物影響的研究進展
- 元延芳;鄒世穎;郎天琪;徐國建;賀曉云;黃昆侖;
- 《農業生物技術學報》 | 2014年第012期
摘要：腸道微生物平衡對于維持機體的生理平衡發揮重要作用，與宿主的營養代謝、免疫調節有著重要的關系。腸道健康直接反映機體的健康狀態。外源物質可通過腸道菌群、腸壁通透性、粘膜免疫、腸道代謝物等影響機體健康。對轉基因生物進行腸道健康的安全評價可以監測轉基因生物通過腸道產生的非期望效應，為轉基因生物的安全性提供有力保證。本研究匯總了近年來國內外對轉基因生物在腸道微生物平衡方面的研究，完善了非期望效應的評價內容，在一定程度上也進一步完善了轉基因生物安全評價體系。%The gastrointestinal microflora play an important role in maintaining the physiological balance, and has an important relationship with nutrition metabolism, immune regulation of host. The gastrointestinal health reflects the body's health directly. Exogenous substances may influence the gastrointestinal health by altering intestinal microflora, intestinal permeability, mucosal immunity, intestinal metabolites. Safety assessment of gastrointestinal health about genetically modified organisms (GMOs) is used to monitor the unintended effect, which provides guarantee to the safety of GMOs. This paper summarized the researches on the unintended effect of gastrointestinal health caused by GMOs. The safety assessment of gastrointestinal health will continuously improve and perfect the system of safety assessment of GMOs.
27.橡膠樹甾醇14α-去甲基化酶基因（HbCYP51）的克隆及機械損傷和外源激素對其表達的影響
- 吳春太;黎瑜;代龍軍;聶智毅;康桂娟;曾日中;
- 《農業生物技術學報》 | 2014年第012期
摘要：Sterol 14α-demethylase (CYP51), an important member of the cytochrome P450 family, is the key enzyme in sterol biosynthesis pathway. The objective of the study was to clone CYP51 gene and illustrate its function in latex production and drainage, and in defense of rubber tree against both tapping and ethephon stimulation. The target gene, encoding sterol 14α- demethylase, was separated from the rubber latex transcriptome library of elite Hevea brasiliensis material CATAS 7-33-97 and the mRNA expression difference, which stimulated by tapping and ethephon or jasmonic acid, was identified by Real-time quantitative PCR (qRT- PCR). BLAST analysis indicated that the cloned gene from Hevea brasiliensis was related to cytochrome P450 and named as HbCYP51 (GenBank No. KM203677), which had high homologies with the sterol 14α-demethylase reported in Populus trichocarpa (90%) and Malus domestica (88%). DNA sequence analysis showed that the size of the HbCYP51 cDNA was 2 305 bp, which included an open reading frame (ORF) of 1 461 bp, and encoded a polypeptide of 486 amino acids residues. The genome sequence length of HbCYP51 gene was 5 271 bp, consisting of 3 exons and 2 introns, and the first intron presented in 5'-UTR of the gene. The phylogenetic analysis suggested that HbCYP51 and CYP51 of Solanaceae (Nicotiana tabacum, Solanum chacoense, Solanum lycopersicum and Petunia × hybrida) were in the same branch group, indicating that the HbCYP51 had closer genetic relationship with other CYP51 of Solanaceae than that of Geum rivale, Fragaria vesca and Arabidopsis thaliana. qRT-PCR analysis showed that HbCYP51 was induced in the latex. HbCYP51 was regulated by tapping, ethephon and jasmonic acid, and significantly up- regulated by mechanical damage at the 7th tapping and ethephon at 48 h, and extremely significantly up-regulated by jasmonic acid at 24 h. The result confirmed that tapping, ethephon and jasmonic acid activated the expression of HbCYP51. Correlation analysis revealed that times of tapping had significantly positive correlation with expression of HbCYP51 gene (P<0.05), and with latex yield (P<0.01), but there was no significant correlation between latex yield and expression of HbCYP51 gene. In conclusion, the HbCYP51 gene might play an important role in the defense of rubber tree, and is directly or indirectly involved in the defense response against tapping and ethephon.%甾醇14α-去甲基化酶(sterol 14α-demethylase, CYP51)是細胞色素P450家族的重要成員之一。基于從巴西橡膠(Hevea brasiliensis)膠乳轉錄組文庫分離出的目的基因，利用實時熒光定量PCR方法研究橡膠樹在不同機械損傷(割次)中和外施乙烯利和茉莉酸后，其膠乳中HbCYP51 mRNA表達差異，探討不同刺激方式和水平對HbCYP51基因表達的影響。BLAST結果表明，本研究克隆了1個與細胞色素P450相關的基因，該基因與毛果楊(Populus trichocarpa)(90%)和蘋果(Malus domestica)(88%)中已報道的CYP51同源性最高，命名為HbCYP51(GenBank登錄號：KM203677)，含有3個外顯子和2個內含子，其cDNA全長2305 bp，包括1461 bp的開放閱讀框(ORF)，推測編碼1個含486個氨基酸的蛋白。定量RT-PCR分析表明，膠乳HbCYP51基因是誘導型表達，并被割膠、乙烯利和茉莉酸調控表達，其中受24 h茉莉酸誘導上調表達最顯著(P＜0.01)，首次證明了割膠、乙烯利和茉莉酸可激活HbCYP51的表達。相關性分析表明，割膠刀次與HbCYP51基因的表達量和膠乳產量均呈極顯著正相關(P＜0.01)，而膠乳產量與HbCYP51基因的表達量無顯著相關。研究結果初步說明，HbCYP51可能是巴西橡膠的重要防御因子，直接或間接參與對割膠和乙烯利的防御反應。
28.快速鑒定主要禽類性別的一對新的通用引物
- 劉宏祥;胡艷;姬改革;李慧芳;
- 《農業生物技術學報》 | 2014年第012期
摘要：家禽的性別一般可以通過翻肛法或其他常規方法進行。翻肛方法對個體的應激較大，而且鵝、鴿等家禽進行翻肛鑒定錯誤率較高。本研究針對禽類性染色體上的染色質解螺旋蛋白DNA結合蛋白1(chromodomain helicase DNA binding protein 1, CHD1)基因，設計了一對引物gCHD，對雞(Gallus gallus)、鴨(Anas platyrhynchos)、鵝(Anser anser)和鴿(Columba livia)的性染色體CHD1基因進行PCR擴增。擴增結果顯示，雄性禽類只有一條帶，雌性禽類有兩條帶，其中較長的帶與雄性的條帶位置相同。對擴增產物進行克隆測序，序列分析結果表明，較長的條帶序列位于Z染色體，命名為CHD1-Z條帶；較短的條帶序列位于W染色體，命名為CHD1-W帶。在鑒定的4個禽類物種中，CHD1-Z條帶與CHD1-W條帶大小相差均為150 bp左右，其缺失的序列位于內含子中。結果表明，設計的gCHD引物可以作為禽類的通用引物，通過擴增產物凝膠電泳后的條帶數目可快速、準確地鑒定禽類的性別。該方法可以提前鑒定出禽類的性別，提高養殖效率；而且為特定性別的胚胎研究提供了可靠的性別鑒定手段。%Poultry sex can be identified by anal swelling or other conventional methods. However, there's a problem that individuals can be injured effortlessly by anal swelling and with higher error rate for geese and pigeons especially. A new pair of primers named gCHD was designed in this study aiming at chromodomain helicase DNA binding protein 1 (CHD1) gene which links to sex of poultries and CHD1 fragments of chickens (Gallus gallus), ducks (Anas platyrhynchos), geese (Anser anser) and pigeons (Columba livia) were amplified. Via agarose gel electrophoresis, the production showed one band for male individuals and two bands for female individuals with the longer band location similar to band of males. Cloning sequence of production and sequence alignments between productions of chickens showed that the longer bands was located in Z chromosome and the shorter bands in W chromosome, named CHD1-Z and CHD1-W, respectively. In these 4 identified species, the length of CHD1-W was about 150 bp shorter than that of CHD1-Z, and the deficiency was in the 14th intron for CHD1-Z mRNA and in the 7th intron as to CHD1-W mRNA. Compared to molecular method, the correct rate of anal swelling measure for chickens, ducks, geese and pigeons were 100%, 93.75%, 87.50% and 87.50%, respectively. These results indicated that the new pair of primers gCHD could be the powerful universal primers to identify sex of poultry by counting the number of bands different between 2 sexes of poultry. This molecular sexing method can not only identify the sexes of poultry in advance for enhancing cultivation efficiency, but also provides a reliable sexing tool for specific study on specific gender of embryos.
29.DNA技術在農業上的應用
- 房經貴;
- 《農業生物技術學報》 | 2014年第012期
摘要：With the development of molecular biology, some DNA-based technologies have showed great potentiality in promoting the efficiency of crop breeding program, protecting germplasm resources, improving the quality and outputs of agricultural products, and protecting the eco-environment etc., making their roles in modern agriculture more and more important. To better understand the application of DNA technologies in agriculture, and achieve the goals to promote their utilities in modern agriculture, this paper describes, in some different way, the applications of molecular markers, transgenic engineering and gene's information in agriculture. Some corresponding anticipations for their development prospects are also made.%隨著分子生物學的發展，基于DNA分子的多種技術在提高作物育種效率、保護種質資源、改善農產品品質、提高農產品產量和保護生態環境等方面都顯示出很大潛力，在農業生產中的作用日趨重要。為更好地了解DNA技術在農業上的應用情況，并達到促進DNA技術為現代化農業服務的目的，本研究對DNA分子標記技術、轉基因技術以及基因信息等在農業上的應用進行了較有特點的介紹，并對其發展前景作了進一步展望。
30.絨山羊成纖維細胞生長因子5基因（FGF5）毛囊特異性表達載體的構建及轉染胎兒成纖維細胞
- 康健;胡廣東;權富生;張涌;
- 《農業生物技術學報》 | 2014年第012期
摘要：The quality and yield of cashmere goat wool are closely related to the growth and development of skin hair follicles. This study aimed to construct an eukaryotic expression vector and prove whether it could specifically express fibroblast growth factor 5 gene (FGF5) in the hair follicles of cashmere goat (Capra hircus). The keratin associated protein 6-1 (KAP6-1) promoter and FGF5 gene coding sequence were obtained by PCR. The vector pEGFP-N1-KF, a hair follicles specific expression vector of FGF5, was constructed by inserting the KAP6-1 promoter into the CMV promoter-less pEGFP-N1, and then connecting FGF5 to the downstream of KAP6-1 promoter, using Porcine teschovirus 2A peptide (P2A) to link FGF5 and enhanced green fluorescent protein (EGFP) gene. The constructed vector was transfected into cashmere goat fetal fibroblast cells and two methods were used to verify its functionality including qRT-PCR and Western blot. The enzyme digestion results showed the vector was correctly constructed. The qRT-PCR results indicated that the FGF5 gene could express in fetal fibroblast cells. The Western blotting results confirmed that the fusion protein could be availably spelited into FGF5 and GFP protein by the functionality of P2A. The result showed that the vector pEGFP-N1-KF which could specifically express FGF5 gene was successfully constructed and could normally expressed in cashmere goat fetal fibroblast cells.%絨山羊的絨毛品質、產量與皮膚毛囊的生長發育密切相關。本研究旨在構建絨山羊(Capra hircus)成纖維細胞生長因子5基因(fibroblast growth factor 5, FGF5)的毛囊特異性表達載體，并證明其表達的有效性。分別以絨山羊基因組和cDNA為模板，利用PCR方法克隆角蛋白關聯蛋白6-1(keratin associated protein 6-1, KAP6-1)基因的啟動子和FGF5基因的編碼區序列(coding sequence, CDS)，并將這兩個元件連接到去除CMV啟動子的真核表達載體pEGFP-N1上，FGF5與增強綠色熒光蛋白(enhanced green fluorescent protein, EGFP)基因表達框之間以豬捷申病毒(Porcine teschovirus, PTV)2A(P2A)相連，構建毛囊特異性表達載體pEGFP-N1-KF。表達載體轉染絨山羊胎兒成纖維細胞后，對細胞表達產物進行qRT-PCR和Western blot檢測。酶切鑒定結果表明，載體pEGFP-N1-KF構建成功；qRT-PCR結果表明， FGF5正常表達；Western blot結果顯示，P2A能夠有效剪切FGF5和EGFP融合蛋白。表明成功得到了絨山羊FGF5基因的毛囊特異性表達載體并在絨山羊胎兒成纖維細胞中能正常表達。本實驗為進一步研究絨山羊FGF5基因在毛囊發育和周期調控中的作用提供了技術支持。

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