Creation Of GhLMM-suppressing Transgenic Cotton And Identification Of Heterologous Expressing GmTIP2;3 Transgenic Cotton

Cotton is an important fiber and oil crop,which has important economic value.During the process of growth and development,the cotton plants are constantly exposed to diseases and insect pests which affect plant growth and productivity.Therefore,one of the important ways to resist plant damage is the introduction of resistant genes to develop new resistant varieties through transgenic technology.Ghlmm（HEMB）,a cotton lesionmimicmutant discovered earlier by our laboratory,was caused by the termination of coding region of Gh＿D05G2237 gene,which could enhance Verticillium wilt resistance,however,the plants were relatively short and the leaf exhibited necrotic disease spot.It was found that the balance between disease resistance and growth and development is regulated by the dose effect of the gene.Therefore,in order to obtain high resistance to Verticillium wilt with normal growth and development,GhLMM gene was down-regulated moderately in cotton by transgenic technology.In addition,the former members of the laboratory expressed soybean Gm TIP2;3 heterologous in cotton and obtained T₀generation transgenic materials.The main results of this study are as follows:1.Creation of a new transgenic cotton material suppressing GhLMM.The candidate gene Gh＿D05G2237 of the lesionmimicmutant identified by laboratory map-based cloning was used to extract the gene and its partial homologous gene Gh＿A05G3720 gene sequence from the cotton genome.The homology comparison between the two shows that the similarity of CDS and amino acid sequence is 98.53%and 99.07%,respectively.A primer was designed to reverse-link the GhLMM gene into the p Bin GFP4 vector.Candidate genes were integrated into cotton genome by Agrobacterium-mediated hypocotyl transformation to create new transgenic cotton materials.The T₀generation transgenic plants were obtained,and the specific primers were designed to detect the target gene.83 individual plants from 13 clones were harvested,11 of which were positive clones.2.Expression analysis of GhLMM-suppressing transgenic cotton.26 individual plants from 11 positive clones were analyzed by q RT-PCR,and W0 was used as negative control.We found that,in 26 transgenic cotton plants,the expression of GhLMM in 10 individual plants decreased significantly and the expression of GhLMM in 10 individual plants increased significantly,however,the expression of GhLMM in the remaining 6 individual plants did not change.Furthermore,the expression of GhLMM in A and D subgroups was analyzed by using specific primers of A and D subgroups.We found that,the expression of9 individual plants in A and D subgroups decreased significantly and the expression of 2individual plants in A and D subgroups did not show significant change.Further,the expression of 5 individual plants in the D subgroup did not change significantly,however,the expression of 4 individual plants in the A subgroup decreased significantly and the expression of 1 individual plant was remarkably increased.The expression of 7 individual plants in the A subgroup did not show any significant change,however,the expression of 2individual plants in the A subgroup decreased significantly,and the expression of 5individual plants increased significantly.Furthermore,the expression of 3 individual plants decrease significantly in the A subgroup,and increase significantly in in the D subgroup.26individual plants exhibited different expression levels,of which,10 individual plants decreased significantly to 37%～82%,10 individual plants increased significantly to116%～353%,and 8 individual plants decreased to 50%～100%compared with W0,the 8individual plants could be used as the main screening targets.At present,16 positive single plants with 5 positive clones were harvested from T₁generation seeds,corresponding to 2clones with up-regulated expression,2 clones with down-regulated expression,and 1 clone with insignificant changes.3.Identification of transgenic cotton material with heterologous expression of soybean Gm TIP2;3.The T₁generation positive plants of different T₀generation positive clones were selected,and the copy number of the T₁generation plants of Gm TIP2;3 transgenic cotton material was determined by using dd PCR technology,and it was found that:Clone 68 got 2and 4 copies,clone 86 got 4 and 5 copies,clone 126 got 12 copies,and clone 135 got 2,4,and 10 copies.The results show that the T₀generation of the above clones are all multi-copy insertions.The 2 copies of T₁DNA templates 349-1 and 354-2 were further subjected to TAIL-PCR amplification.One of the insertion sites of 349-1 was located at A09:56407164;the insertion site of 354-2 was located at D13:40111508 and D13:40111546,for reverse tandem insertion.To detect T₂positive plants,we designed specific primers at both ends of the two insertion sites,one of which is dominantly homozygous and8 dominantly heterozygous.The primer design can be used for homozygous identification of the genotype of the offspring.This primer can be used to identify homozygotes,and then gene function research will be carried out.