本文選題：轉(zhuǎn)基因棉花 + MON��； 參考：《農(nóng)業(yè)生物技術(shù)學(xué)報(bào)》2016年06期

【摘要】：轉(zhuǎn)基因棉花(Gossypium hirsutum)MON757轉(zhuǎn)化體是孟山都公司研發(fā)的轉(zhuǎn)cry1Ac基因的抗蟲(chóng)棉,在美國(guó)、加拿大、澳大利亞等國(guó)被批準(zhǔn)種植或允許用作食品飼料原料,但在我國(guó)尚未獲得批準(zhǔn)種植和應(yīng)用。目前國(guó)內(nèi)外尚沒(méi)有轉(zhuǎn)基因棉花MON757特異性的純雜合檢測(cè)和定量檢測(cè)方法報(bào)道。為了檢測(cè)我國(guó)棉花中MON757轉(zhuǎn)化體的存在情況,本研究以轉(zhuǎn)基因棉花MON757轉(zhuǎn)化體的插入位點(diǎn)基因組序列和外源插入片段/基因組連接區(qū)序列為靶標(biāo),建立了特異性的MON757轉(zhuǎn)化體純雜合定性PCR檢測(cè)方法和實(shí)時(shí)熒光定量PCR(quantitative real-time PCR,q RT-PCR)檢測(cè)方法。所建立的MON757轉(zhuǎn)化體純雜合定性PCR檢測(cè)方法的檢測(cè)引物由分別位于MON757外源基因插入位點(diǎn)兩側(cè)基因組和外源插入片段上的3條引物組成,能準(zhǔn)確地鑒別棉花植株和種子中MON757轉(zhuǎn)化體的純合、雜合狀態(tài)。建立的MON757轉(zhuǎn)化體特異性的q RT-PCR檢測(cè)方法具有良好的可重復(fù)性和靈敏度,其檢測(cè)下限(limit of detection,LOD)為11個(gè)拷貝,定量下限(limit of quantitative,LOQ)為44個(gè)拷貝。用此方法對(duì)2014年湖北省的49份商品棉花種子進(jìn)行了檢測(cè),結(jié)果有5份種子樣品檢測(cè)到MON757轉(zhuǎn)化體。采用MON757轉(zhuǎn)化體純雜合定性PCR檢測(cè)方法對(duì)這5份種子樣品各60個(gè)單粒分別進(jìn)行了檢測(cè),同時(shí)采用q RT-PCR檢測(cè)方法對(duì)這5份種子樣品進(jìn)行定量測(cè)定。結(jié)果表明,有1份含量在1.5%左右,4份含量超過(guò)了20%,兩種方法的測(cè)定結(jié)果基本一致。本研究建立的MON757轉(zhuǎn)化體純雜合定性PCR檢測(cè)方法和q RT-PCR檢測(cè)方法在田間棉花植株和種子中MON757轉(zhuǎn)化體純雜合狀態(tài)的測(cè)定以及混合樣品中MON757轉(zhuǎn)化體含量的測(cè)定方面都具有重要的應(yīng)用價(jià)值。
[Abstract]:Transgenic cotton Gossypium hirsutum)MON757 transformant was developed by Monsanto Company and developed by Monsanto. It has been approved to grow or be used as feedstuff in USA, Canada, Australia and other countries, but it has not been approved and applied in our country. At present, there are no pure heterozygosity detection and quantitative detection methods for MON757 specificity of transgenic cotton at home and abroad. In order to detect the existence of MON757 transformants in Chinese cotton, the genomic sequence of insertion site and exogenous insert fragment / genomic linkage region of transgenic cotton MON757 transformants were used as targets in this study. A specific PCR detection method for pure heterozygosity of MON757 transformants and a real-time fluorescent quantitative PCR(quantitative real-time PCRQ RT-PCR method were established. The detection primers for pure heterozygous PCR detection of MON757 transformants were composed of three primers located on the genomes of the two sides of the insertion site of the exogenous gene of MON757 and on the inserted fragments of the exogenous gene, respectively. The homozygosity and heterozygosity of MON757 transformants in cotton plants and seeds can be identified accurately. The established Q RT-PCR assay has good reproducibility and sensitivity. The detection limit of detection limit is 11 copies, and the limit of quantitative limit is 44 copies. This method was used to detect 49 commercial cotton seeds in Hubei province in 2014. The results showed that MON757 transformants were detected in 5 seed samples. The pure heterozygosity qualitative PCR assay of MON757 transformants was used to detect 60 single seeds of the 5 samples, and the Q RT-PCR method was used to detect the 5 samples quantitatively. The results showed that the content of 1 phr was 1.5% or so and the content of 4 phr exceeded 20 phr. The results of the two methods were basically consistent. The qualitative PCR and Q RT-PCR detection methods for pure heterozygosity of MON757 transformants in cotton plants and seeds were developed in this study. They were used to determine the homozygous status of MON757 transformants in cotton plants and seeds and the content of MON757 transformants in mixed samples. It has important application value.
【作者單位】： 中國(guó)農(nóng)業(yè)科學(xué)院植物保護(hù)研究所/農(nóng)業(yè)部轉(zhuǎn)基因環(huán)境安全及植物抗性監(jiān)督檢驗(yàn)測(cè)試中心(北京)/植物病蟲(chóng)害生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室;
【基金】：轉(zhuǎn)基因新品種培育重大科技項(xiàng)目(No.2014ZX08012-01B)
【分類號(hào)】：S562

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本文編號(hào)：1968231