Detection Of The Bt Gene Cassettes In Transgenic Cotton Of China

Transgenic insect-resistant cotton, which area was over70%of total cultivated area of cotton, is thelargest genetically modified crops planting in China. There are many transgenic cotton varieties inChina, and more than1600biosafety certificates have been granted by the Ministry of Agriculture(MOA). But only a few detection methods and molecular characteristics were reported till now. In thisstudy, a new Bt gene cassette was identified and construct-specific detection method was established.Also more than200cotton materials from four provinces were analysed using this method. The mainresults were summarized as follows:1. A new Bt gene cassette was identified and the construct-specific detection method was developed.The new Bt gene cassette-cry1Aa gene cassette was obtained from cotton Nannong6F1using theprimers pairs of the regulatory elements and cry1A gene.The full lenth was2381bp, includingCaMV35S promoter, cry1Aa gene and7S UTR terminator, which located1-277bp,278-2125bp and2126-2382bp, respectively. Thus we got a new Bt gene cassette-cry1Aa gene cassette from Nannong6F1, The construct-specific detection method for cry1Aa gene cassette was established. when comparedwith other two Bt gene cassettes reported previously. Eight of eighteen cottons were have the cry1Aagene cassette using the method developed.2. Construct-specific multiplex PCR detection method for three Bt gene cassettes was developed andmore than200cotton lines were analysed from four provinces of China. Based on comparing thecry1Ac and cry1Ab/Ac gene cassettes, a construct-specific multiple PCR detection method for all thosethree cassettes was developed using the joint region of Bt gene and7S UTR as the target. The Bt gene ofcry1Ac gene and cry1Ab/Ac gene cassettes were designed as forward primers, which were located in3765-3782bp,2016-2036bp, repectively. Also, combined to detection of cry1Aa gene cassette primerpairs, the construct-specific multiple PCR detection method for the three Bt gene cassettes wasestablished. The cry1Ac, cry1Ab/Ac, cry1Aa gene cassettes were amplified by this method,were183bp,329bp and462bp, respectively.The limit of detection of this method was22copies, equivalent to0.05ng cotton genome. A total of204cotton lines from four provinces of China were identified using thismethod. The materials have cry1Ac gene cassette, cry1Ac+cry1Ab/Ac gene cassettes, cry1Ac+cry1Aagene cassettes, and all the three Bt gene cassettes were173,16,11and4, respectively. Also,60seedsfrom a selected cotton were detected individually using the multiplex PCR method developed: Thematerials have cry1Ac gene cassette, cry1Ab/Ac gene cassette, cry1Aa gene cassette, cry1Ac+cry1Aagene cassettes, without cry1Ac+cry1Ab/Ac+cry1Aa gene cassettes were28,1,16,3and12,respectively.