Molecular Cloning Of Male Sterile Gene BnMS1 In Brassica Napus And Research On Its Function

Male srerility is a phenomenon that in the process of sexual reproduction in plants, anthers, pollen and male gametes are abnormal but the function of the female bud is normal.Male srerility of rapeseed is wildly used in domestic and international hybridization.because of the genome of brassica napus is complicated,so far there is no report on map-based cloning of male sterility gene on brassica napus.Rapeseed and Arabidopsis belong to the Brassica species of Cruciferae plant.Comparative genomics studies have shown that the rape and Arabidopsis thaliana share highly homologous genome,which means that we can make use of fertility-related genes in Arabidopsis thaliana cloning male sterile gene in Rape and conducting research on male sterility.So far there are many fertility-related genes separated and cloned from Arabidopsis thaliana, such as MS2,MS5,Coll, spl, MS1, eMSl,dde2-2,mmdl and so on. The gene of MS1 expresses during the period of spores release in tapetal tissues,and the expression is low in abundance.The pollen of MSI mutation is abortion and the development of the other floral organs are not affected, which can be have a great use in production.In this research,we carried some works on MS1 gene,as follows:1.The cloning of male sterile gene BnMS1 in Brassica napus and its bioinformatics analysis.According to the coding sequences of Arabidopsis MS1 gene,a series of primers were designed to clone the homologous sequences in Brassica napus. PCR Walking method was used to clone the full sequence of MS1 gene of Brassica napus. The total lengh of the BnMSl gene cloned here is 3424bp,and we deduce there are three exons located at 521bp-+821 bp,1189bp-+1471 bp and1876bp-+3296 bp;while introns located at 822bp-+1188 bp,and 1472bp-+1875 bp.The MS1 from Brassica napus shared a similarity of 89.5% in encoding sequence and 60.3% in full sequence with that from Arabidopsis thaliana. According to the results we deduced that it has an open reading frame(ORF) of 2004bp;encoding a protein of 667 amino acid, with protein molecμlar weight of 26kD and isoelectric point of 8.01.The similarity between the MS1 from Brassica napus and that from Arabidopsis thaliana is 93% in amino acid sequence.2.A plant expressed vector with AntiMS was constructed. Cloning the third exon sequence between 2402-+2919bp of BnMSl gene and liagased with pFGC5941 Vector reversed. Recombinant plasmid was transformed into Agrobacterium tumefaciens.Using Floral-Dip transformation method to invert the expressed vector with Anti-MS into Arabidopsis thaliana. Identificat of the transgenic plants by PCR amplification and use RT-PCR amplification to analysis the expression of MS1 gene.Stain and Observ the pollens under the microscope. The results:14 transgenic plants have been selected,11 of them are positive and there are no significant difference between positive transgenic Arabidopsis thaliana and col-o Arabidopsis thaliana on Phenotype,pollen activity and expression of MS1 gene.3.A plant expressed vector with amiRNA of MS1 was constructed. Clone the amiRNA of MS1 then liagsed with pFGC5941 vector.Recombinant plasmid was transformed into Agrobacterium tumefaciens.Using Floral-Dip method to invert the expressed vector with amiRNA into Arabidopsis thaliana. Identificat of the transgenic plants by PCR amplification and using RT-PCR to analysis the expression of MSI gene.Stain and Observe the pollens under the microscope. The results:we have selected 27 transgenic plants,25 of them are positive. Some strains of transgenic Arabidopsis showed intermediate male sterility,and the Pods are shorter and the number of pollen grains in early flower anthers are fewer than col-0 Arabidopsis.there are no significant difference between the other transgenic Arabidopsis thaliana and col-o Arabidopsis thaliana.4.Construct a expressed vector with the promoter of BnMSl.Using PCR to amplified the promoter sequence of BnMSl then connecting the promoter sequence with the pGUS-eGFP double-labeled plant expression vector, at last it was transferred to agrobacterium tumefaciens for future study. Identification of the transgenic Agrobacterium tumefaciens by PCR amplification.the results showed that the promoter sequence was transferred into Agrobacterium,so the vector was successfully constructed.