Genetic Transformation And Functional Analysis Of Male Sterility Related Genes In The Photo-thermo-sensitive Male Sterility Line 337S

Male sterility is the most important and effective way to utilize heterosis in self-pollination and cross-pollination crops.To a great extent,whether wheat heterosis can be widely utilized depends on our understanding and the different types of male sterility in wheat,including nuclear sterility,cytoplasmic infertility,and photo-temperature-sensitive sterility.The main use of male sterility in wheat production is photo-thermo-sensitive male sterility in China.The study of the inheritance and regulation mechanism of photo-thermo-sensitive male sterility in wheat will broaden our understanding of wheat male sterility and expand the utilization of wheat heterosis.In this study,we cloned several genes whose expression significantly was up-regulated in photo-thermo-sensitive male sterile line 337 S under short-day and low-temperature conditions.Overexpression vectors of three candidate genes Ta MS337S-3,Ta MS337S-4 and Ta MS337S-5 were constructed by using Gateway technology.Different immature embryos of wheat were genetically transformed with gene gun and Agrobacterium tumefaciens method.And we analyzed the differences of transgenic positive seedlings in phenotype.Three candidate genes were transformed and analyzed in Arabidopsis,The main results are as follows: 1.Transformation of immature embryos by gene gun: There are 59 stains of resistant seedlings which use Huamai 2152 as recipient were obtained from the Ta MS337S-3 gene by transformational experiment,PCR test identified 13 positive seedlings;There are 46 stains of resistant seedlings which use Huamai 2566 as recipient were obtained from the Ta MS337S-5 gene by transformational experiment,9 of them were positive confirmed by PCR test;There are 39 stains of resistant seedlings which use Kenong 199 as recipient were obtained from the Ta MS337S-5 gene by transformational experiment,2 of them were positive confirmed by PCR test.2.Agrobacterium-mediated genetic transformation of wheat immature embryos: There are 49 stains of resistant seedlings which use Huamai 2152 as recipient were obtained from the Ta MS337S-3 gene by transformational experiment,2 of them were positive confirmed by PCR test;There are 14 stains of resistant seedlings which use Huamai 2566 as recipient were obtained from the Ta MS337S-4 gene by transformational experiment,2 of them were positive confirmed by PCR test;There are 49 stains of resistant seedlings which use Kenong 199 as recipient were obtained from the Ta MS337S-5 gene by transformational experiment,5 of them were positive confirmed by PCR test.3.The difference of phenotype in transgenic positive seedling: Compared with the corresponding recipient parent,the number of tillers of the positive seedlings transfected with Ta MS337S-3 gene increased significantly.The growth potential was more prosperous,and the vegetative growth period was prolonged significantly,indicateding that Ta MS337S-3 gene was one of the key genes controlling the transformation from vegetative growth to reproductive growth.It was speculated that Ta MS337S-3 gene might be related to the fertility of 337 S.Compared with the recipient parents,the positive plants of Ta MS337S-4 gene were shorter and thinner,the spikelet was smaller significantly.The stamens of some florets were shorter,and the pollen did not disperse,so the seeds could not be formed by normal filling.This indicated that Ta MS337S-4 gene had a negative effect on the growth of spikelet and the floret development of wheat.Therefore,it was speculated that Ta MS337S-4 gene could be related to the sterility of 337 S.There was no significant difference in the phenotype between the positive seedlings transgenic with Ta MS337S-5 gene and the recipient materials.It is not clear whether the Ta MS337S-5 gene is related to fertility.4.The effects of three candidate genes on the growth and development of Arabidopsis thaliana were studied by using the T3 generation of Arabidopsis with Ta MS337S-3 and Ta MS337S-5 genes and the T2 generation of single copy Arabidopsis thaliana with Ta MS337S-4 gene as materials.The results showed that two of the transgenic plants of Ta MS337S-3 Arabidopsis haved a significant increase in the number of branches,which was different from the wild type significantly,and the flowering time was about a week later than the wild typ,which was similar to the phenotype trend of the transgenic wheat.The Ta MS337S-4 gene delayed significantly in the development of Arabidopsis thaliana.Compared with the wild type,the flowering time of Arabidopsis thaliana was nearly 20 days late,and the plant was relatively weak.The phenotypic values of pod length,pod number,plant height and seed quality were smaller than those of the wild type significantly,and similar to the phenotypic trend of transgenic wheat.The traits of five Arabidopsis lines transfected with Ta MS337S-5 gene were similar to those of wild type,and also similar to the phenotypic trend of transgenic wheat.Based on the phenotypic difference analysis results of transgenic wheat and transgenic Arabidopsis positive seedlings,it can be concluded that Ta MS337S-3 and Ta MS337S-4 genes are associated with the fertility of photo-thermo-sensitive male sterility line 337 S,and the Ta MS337S-5 gene has little effect on the fertility of 337S.