Haplotype Analysis Of TaGW2 And Agrobacterium Mediated Genetic Transformation System Optimization

Wheat is one of the most important food crops in China.Yield,quality,safety and other factors of wheat directly affects China's food security and social stability.Grain weight as yield factor,has been one of the key concerns in breeding.With the development of the breeding work for many years,the traditional breeding technology has is not easy to achieve great-leap-forward development.Since 1992,Vasil won the first transgenic plants,the genetic transformation of food crops,economic crops has gradually become a hot point.But the development of wheat transgenic technology is still slow.Especially in recent years,the safety of genetically modified food is concerned.Our country also put forward new requirements for the study of marker-free transgenic crops,which brings great challenge to wheat transgenic technology.In this study,the grain weight related gene TaGW2 as target gene,aiming at the difficulties of wheat tissue culture and screening marker is difficult to remove the problem of transgenic vector construction and genetic transformation of TaGW2 gene haplotype analysis,and obtains the following results:1.We screened 54 High grain weight materials in 361 candidit materials,and the TaGW2 gene was anylized by haplotype.A total of 6 haplotypes were detected,and TaGW2-6AS was found in 895 and 316 of the wheat XuKe occurred into the T base between 976 and 977,leading to the gene in 987 bit code termination.The sequencing of the seventh intron,6BS was detected in 3 haplotypes,with polymorphism in the intron 459,478,594,679,821bp;6DS detected 2 haplotypes with polymorphism in 709 and 713bp in the intron2.Using the method of homologous cloning,in large grain material "pindong 34" cloned the TaGW2 gene of early termination,and this gene was successfully constructed in the three plant expression vector:PCXSN-TaGW2,DRB-bar-TaGW2 and PTCK303mm-TaGW2,in which PCXSN-TaGW2 vector containing hygromycin selection marker,DRB-bar-TaGW2 containing double right border with herbicide resistance,PTCK303mm-TaGW2 marker-free vector..3.Xiaoyan 22,Z50 and Zhengmai 366 as receptor material,the genetic transformation of immature embryo tissue culture and Agrobacterium mediated,and the browning and hydration phenomenon of culture system optimization.The Xiaoyan 22 and Z50 in three varieties with higher rate of callus.Antibacterial stage Xiaoyan 22 in immature embryo genetic transformation,the lowest degree of browning.Added to the culture medium of lipoic acid in the inhibitory phase,can effectively reduce the browning rate of callus,but the addition of lipoic acid significantly improve callus hydration degree,resulting in callus regeneration difficulties.The addition of betaine can reduce the hydration degree of callus,and can reduce the Z50 Callus Browning Degree,but can enhance the Xiaoyan 22 Callus Browning degree.4.The Xiaoyan 22 as receptor material,carries on the genetic transformation of mature embryo tissue culture and Agrobacterium mediated,and studied the effects of different inoculation methods and different culture medium on callus morphological results are as follows:in different inoculation methods,cutting embryo inoculation method compared with embryos from scratch,the more faster,but The two day of the 15 rate difference is not significant;in the induction of lipoic acid added more than 0.1mM medium will reduce the mature embryo browning rate;adding 0.1mM lipoic acid in the inhibitory stage can reduce the cutting embryo callus,and callus decreased the degree of hydration in the induction medium;the addition of 5mg·L-1 can improve the silver nitrate Scraping the embryo rate of the callus.In general,by adding 5mg·L-1 AgN03 in scraping embryo,callus induction stage,color yellow,water phenomenon is light,is conducive to the continued growth of callus.