Analysis Of T-DNA Flanking Sequences And Structure Twin T-DNA Vector System In Cotton(Gossypium Hirsutum L.)

Cotton(Gossypium L.) is one of the most important fiber crops, and is also one of the main oil crops in the world. The cotton fiber is the basic raw material for textile industry. The cottonseed being as a secondary product, had many uses. Study of mutants play a substantial role in functional genomics. Agrobacterium mediated T-DNA insertion is one of the important strategies to create mutants. The T-DNA tags not only disturbed gene expression, but also simply and rapidly cloned genes in order to study their functions. And, since 1997, the widespread use of insect-resistant cotton in agriculture results in increasing of resistance in insects. Single Bt cotton is replaced by polyvalent transgenic cotton.In this reseach, we generated cotton mutants with promoter trap system by using Agrobacterium-mediated transformation. We use these mutants to isolate T-DNA flanking sequences and analyze the copy number of T-DNAs. Then we study gene’s functions with T-DNA flanking sequences and obtained their biological information from cotton genome. The twin T-DNA system is a vital and highly efficient method to delete marker gene after infiltration into plants. We constructed an expressionvector pCAMBIA2300 SDT, carried nptII marker gene and cry1C* or mGFP5* target genes. Transgenic(cry1C* and mGFP5*) cotton were obtained by Agrobacterium-mediated method, transformed tissues were selected in kanamycin(kan). We obtained the following results.a. Up to now, 1000 independent transformants carrying the promoter trap system were obtained from two cotton cultivars. Approximately 67.47% lines were found positive in PCR analysis. 50.56% lines had a single copy and each transformants contained 1.83 copies of T-DNA, conformed by southern bolting.b. we isolated 274 and 366 flanking sequences from left border and right border respectively, using FPNI-PCR. 152(55.47%) and 268(57.44%) of these sequences map to the cotton genome.c. We analyzed integrated characterizations of flanking sequences in cotton genome, the lack sequences were worse left border than right border. The right border were retained 3 bp has high frequency, about 23.51%.d. We analyzed distributions of flanking sequences in cotton genome, T-DNA integration have preference. T-DNA insertions were biased towards to integrate into genes and transposon elements, and undifferentiated towards A or D subgenome.e. The plant expression vector pCAMBIA2300 SDT was constructed basing on binary vector pCAMBIA2300 S. In this vector, the nptII resistance gene was marker gene, and the Cauliflower mosaic virus 35 S promoter to upregulate the target gene’s expression. The DNA fragment of ―T-DNA-LB-RB‖ was integrated into multiple cloning sites(MCS) from pCAMBIA2300 S. The modified vector was named pCAMBIA2300 SDT.f. The target genes mGFP5* or cry1C* were ligated into pCAMBIA2300 SDT. The reconstructive vector was named pCAMBIA2300SDT-cry1C*/mGFP5* and used to transform to obtain transgenic cotton.