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》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基因的抗蟲棉,在美國、加拿大、澳大利亞等國被批準(zhǔn)種植或允許用作食品飼料原料,但在我國尚未獲得批準(zhǔn)種植和應(yīng)用。目前國內(nèi)外尚沒有轉(zhuǎn)基因棉花MON757特異性的純雜合檢測和定量檢測方法報道。為了檢測我國棉花中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檢測方法和實時熒光定量PCR(quantitative real-time PCR,q RT-PCR)檢測方法。所建立的MON757轉(zhuǎn)化體純雜合定性PCR檢測方法的檢測引物由分別位于MON757外源基因插入位點兩側(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檢測方法具有良好的可重復(fù)性和靈敏度,其檢測下限(limit of detection,LOD)為11個拷貝,定量下限(limit of quantitative,LOQ)為44個拷貝。用此方法對2014年湖北省的49份商品棉花種子進行了檢測,結(jié)果有5份種子樣品檢測到MON757轉(zhuǎn)化體。采用MON757轉(zhuǎn)化體純雜合定性PCR檢測方法對這5份種子樣品各60個單粒分別進行了檢測,同時采用q RT-PCR檢測方法對這5份種子樣品進行定量測定。結(jié)果表明,有1份含量在1.5%左右,4份含量超過了20%,兩種方法的測定結(jié)果基本一致。本研究建立的MON757轉(zhuǎn)化體純雜合定性PCR檢測方法和q RT-PCR檢測方法在田間棉花植株和種子中MON757轉(zhuǎn)化體純雜合狀態(tài)的測定以及混合樣品中MON757轉(zhuǎn)化體含量的測定方面都具有重要的應(yīng)用價值。
[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】： 中國農(nóng)業(yè)科學(xué)院植物保護研究所/農(nóng)業(yè)部轉(zhuǎn)基因環(huán)境安全及植物抗性監(jiān)督檢驗測試中心(北京)/植物病蟲害生物學(xué)國家重點實驗室;
【基金】：轉(zhuǎn)基因新品種培育重大科技項目(No.2014ZX08012-01B)
【分類號】：S562

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