Transgene Structure And Integration Pattern In Genome Of Transgenic Cotton (Gossypium Hisutum L.) Via Agrobacterium-mediated Transformation

Cotton (Gossypium spp.) is the world's leading fiber crops and thus an important cash crop for its natural fiber. Among the four cultivated Gossypium species in the world, the American allotetraploid species (G.hirsutum L.) dominated worldwide cotton production and many excellent cotton cultivars have been developed through genetic engineering, which play an important role in maintaining and further improving lint yield and fiber quality.Successful genetic transformation systems have been established in many plants up to now, from which commercial transgenic crops including maize, soybean, cotton and so on have been developed. Among several plant genetic transformation systems, agrobacterium-mediated transformation has been adopted into a powerful tool for production of transgenic plants due to its characteristics of convenience in manipulation, favorable high transformation efficiency and high genetic stability of transformants. It is already confirmed that the DNA to be transferred (T-DNA) by Agrobacterium is delineated by two similar, identical 25bp direct border repeats, the left border (LB) and the right border (RB). The transfer of DNA from Agrobacterium into plant cell, which is covalently linked to the virulence proteins, drill though the cell membrane and cell wall of the host plants and integrate into the genome. However, the vector backbone sequences are also found to be integrated into the plant genome. The mechanism of T-DNA integration is proved illegitimate recombination other than site-specific one. Partly because of the difficulty in obtaining large numbers of transgenic plants, there is still little knowledge on transgene integration in cotton genome however genetic modified upland cotton varieties are ranked among the most successful genetic modified organisms (GMO) in the world.In this study, independently T0 transgenic cotton plants obtained by Agrobacterium tumefaciens carrying a binary plasmid pPZP-GFP and a segregation T1 population derived from a T0 plant, GFP47, which integrated with multiple copies of transgene and vector backbone sequences in different insertion loci were analyzed by PCR, Southern blot, inverse PCR and sequencing analysis.The genomic DNA isolated from T0 plants and seeds harvasted from GFP47 T0 were introduced from Plant Biotechnology lab of Temasek Life Sciences Laboratory, National University of Singapore. T1 plants were planted in the greenhouse for DNA isolation and thereafter molecular analysis.Southern blot analysis indicated that: (1) quite many T0 tansgenic cotton plants were integrated with vector backbone sequences outside the left border of T-DNA; (2) some plants were integrated more than one copy of T-DNA within a single insertion sites and formed possible tandem repeat transgene structures; and (3) some plants were integrated with abnormal T-DNA units in repeat structures.Inverse PCR were carried out for identifying and sequencing the cotton sequences flanking T-DNA integrated into the genome. For those events with both RB junction and LB junctions characterized, primers were designed to amplify and clone pre-insertion site sequences from the non-transgenic control plants. These pre-insertion sequences were compared with genomic sequences after T-DNA integration. Eight T-DNA integration sites are obtained. Microhomology between T-DNA borders (RB or LB) and genome DNA was present for most of the events characterized. Deletion to genome sequence was a general feature. Filler sequences were also detected for three events. They had no strong homology with vector sequence and hence most likely came from plant genome. Careful examination of the fillers found out that all of them contained small repeats in direct or inverted orientations and one structure resembles palindrome recognition sites of restriction enzymes.PCR results showed that the T0 plant of GFP47 was integrated not only normal T-DNA including target gene (GFP) and selection marker gene (NPTII) but also vector backbone fragment outside the left border of the T-DNA. Besides confirmed the PCR results, Southern blot analysis on T0 plant indicated that the T0 plant was integrated with multi-copy of T-DNA. PCR characterization on 33 T1 individuals showed that some individuals with only normal T-DNA structures were segregated from the population. Further analysis with Southern blot on these individuals with different genotype identified by PCR showed that there were, at least, 4 loci with different integration patterns in the cotton genome and the desirable transgenic event with only single copy of normal T-DNA integration could be segregated from the offsprings of this multiple-sites integration transgenic cotton.