Analysis Of The Function Of 2-I05 Gene Related To Dominant Geneic Male Sterility In Brassica Napus L.

Having obtained the cDNA sequence of male sterility related gene(2-I05) of B.napus,I used bioinformatics methods to analyze the sequence,and then constructed an antisense inhibition vector to transform into Arabidopsis thaliana and Brassica napus,so that I can identify the function of gene 2-I05 and verify how it impacts on the growth and fertility of these plants.Main conclusions are listed as follows:1.Using bioinformatics methods,I analysed the male sterility related gene 2-I05 (EU306599) of B.napus.cDNA sequence of 2-I05 encodes 357 amino acids and the protein is predicted that it locates in the nucleus.In Arabidopsis thaliana,there is another protein named RAD23-1ike which is homologous to the protein 2-I05 encodes,and RAD23-1ike protein is involved in DNA nucleotide excision repair procedure.I also analysed the phylogeny of RAD23-1ike protein's amino acid sequences which came from nine different plants,the results showed that the evolution of the amino acid sequences consisted with that of plants.Genetic variation of RAD23-like protein's amino acid sequences is much smaller if these sequences come from coordinal plants than that come from interfamily plants.2.I constructed an antisense inhibition vector and then,the anti-sense sequence of gene 2-I05 was transferred into Arabidopsis thaliana Columbia by flower dipping method, and twenty three transgenic plants were got after Hyg(30mg/L) resistance screening.But due to the poor culture conditions,ultimately,only four Arabidopsis thaliana seedlings of T₁ generation survived.PCR detection indicated that two of them were positive transgenic plants.3.After Hyg(30mg/L) resistance screening,PCR detection of the resistant Arabidopsis thaliana plants of T₂ generation,UV treatment and RT-PCR analysis,I found that:in the role of constitutive promoter 35S,the expression of the endogenetic homologous genes which encoding RAD23-like protein were suppressed obviously in flower buds and leaves of wild-type Arabidopsis thaliana.UV treatment results showed that Arabidopsis tkaliana plants of T₂ generation were more sensitive to UV than wild-type Columbia.So,the function of 2-I05 gene is mainly involved in DNA nucleotide excision repair after UV damage.However,the experimental results did not confirm our predecessor's assumption that 2-I05 gene involved in UV damage repair could cause plant infertility,but 2-I05 gene might be involved in regulating the length of the stamen.4.I used two kinds of transgenic methods to transfer the plant expression vector which containing anti-sense sequence of gene 2-I05 into Brassica napus.But,because the vector has Hyg resistant gene and Hyg is too toxic to B.napus explants,I have not found a suitable Hyg screening concentration;as a result,the follow-up work could not go on. However,these two kinds of transgenic methods were compared and analyzed.