【摘要】：【目的】cry1Aa基因是2011年本實(shí)驗(yàn)室在我國棉花商品種子中發(fā)現(xiàn)的1種未經(jīng)批準(zhǔn)的轉(zhuǎn)基因成分,本研究旨在通過整合結(jié)構(gòu)解析,建立特異性檢測方法,對(duì)市售棉種進(jìn)行初測,明確該外源基因在我國棉種中的存在情況。【方法】通過Genome walking、長鏈PCR分離獲得了轉(zhuǎn)cry1Aa基因棉花轉(zhuǎn)化體(定名為NN6轉(zhuǎn)化體,簡稱NN6)的外源基因整合結(jié)構(gòu),同時(shí)基于插入位點(diǎn)基因組及外源DNA連接區(qū),建立了NN6轉(zhuǎn)化體特異性的定性聚合酶鏈?zhǔn)椒磻?yīng)(Polymerase chain reaction,PCR)方法和實(shí)時(shí)熒光定量PCR方法�！窘Y(jié)果】NN6整合結(jié)構(gòu)主要包括cry1Aa、aad和Npt II基因,插入棉花基因組7號(hào)染色體37 169 450位,產(chǎn)生91 bp基因組缺失;所構(gòu)建的定性PCR檢測方法能特異、快速地對(duì)棉花單株和種子單粒中NN6轉(zhuǎn)化體的純/雜合狀態(tài)進(jìn)行鑒定,實(shí)時(shí)熒光定量PCR檢測限為44拷貝,能滿足國內(nèi)外對(duì)轉(zhuǎn)基因標(biāo)識(shí)的需要;對(duì)含有cry1Aa基因的3個(gè)市售棉種進(jìn)行檢測,其純合度分別為1.51%,5.21%和21.09%�！窘Y(jié)論】本研究解析了NN6的整合結(jié)構(gòu),并建立特異性檢測方法,為我國轉(zhuǎn)基因棉花新品種選育及轉(zhuǎn)基因安全管理提供技術(shù)手段。
[Abstract]:[objective] cry1Aa gene is an unapproved transgenic component found in cotton commercial seeds in our country in 2011. The purpose of this study was to establish a specific detection method for commercial cotton seeds by integrated structural analysis. [methods] the transgenic cotton transformants with cry1Aa gene (named NN6 transformant, NN6) were isolated by Genome walking, long chain PCR. At the same time, a qualitative polymerase chain reaction (Polymerase chain reaction,PCR) method and a real-time fluorescence quantitative PCR method were established based on the insertion site genome and the exogenous DNA junction region. [results] the integrated structure of NN6 mainly includes cry1Aa,. Aad and Npt II genes were inserted into cotton genome 7 at 37,169,450, resulting in 91 bp genome deletion. The constructed qualitative PCR detection method can identify the pure / heterozygous status of NN6 transformants in cotton plants and seeds quickly. The detection limit of real-time quantitative PCR is 44 copies, which can meet the needs of transgenic identification at home and abroad. The homozygosity of three commercial cotton varieties containing cry1Aa gene was 1.51% and 21.09%, respectively. [conclusion] the integrated structure of NN6 was analyzed and the specific detection method was established. To provide technical means for breeding and safety management of transgenic cotton varieties in China.
【作者單位】： 中國農(nóng)業(yè)科學(xué)院植物保護(hù)研究所/植物病蟲害生物學(xué)國家重點(diǎn)實(shí)驗(yàn)室/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)測試中心(北京);
【基金】：國家科技重大專項(xiàng)——轉(zhuǎn)基因生物新品種培育(2016ZX08012-002)
【分類號(hào)】：S562

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