Cloning And Expression Analysis Of Male Sterility-related Genes In Chinese Cabbage

The male sterile mutants are valuable materials for studying the mechanism of male gametes development in plants.In the previous study,we carried out a transcriptome analysis of the flower buds from the sterile and fertile of the Chinese cabbage male sterile dual-purpose line 'AB01',and a series of differentially expressed genes were identified.Bra036581 and Bra040503 expressed in normal flower buds not in the flower buds of the sterile plants,which suggested that the above two genes might play a certain role in the stamen development of Chinese cabbage.We further cloned the full-length sequences of Bra036581 and Bra040503,and investigated the expression patterns,providing a foundation for investigating the gene functions.The main findings are as follows:1.The full-length DNA and cDNA sequences of Bra036581 and Bra040503 were cloned and results showed that Bra036581 contain no intron and the CDs was 237 bp in length,which translated to an unknown protein with 78 amino acids.The relative mass of the protein was 8.27 KD,the isoelectric point was 7.45,and the signal peptidase site was located at the 24 th amino acid;similar to the cDNA sequence of Bra036581,Bra040503 also didn't contain any introns.The cDNA sequence length is 128 bp in length and the encoded protein contained 128 amino acids.It is also an unkown protein,with 14.31 KD relative mass.The protein PI was 9.70,and the signal peptide cleavage site was at the 23 th amino acid.2.The different tissues and flower organs of sterile and fertile lines were collected and extracted for qRT-PCR analysis.The two genes were only expressed in fertile lines,especially in the anthers,and with lower or no expression in the roots,stems,leaves and other floral organs.3.The promoter regions of Bra036581 and Bra040503 were cloned and the expression vectors were transformed into Arabidopsis thaliana by using the dipping methods.48 and 56 positive plants were obtained,by hygromycin selection and PCR identification.GUS staining showed that the GUS genes driven by the mentioned two promoters expressed exclusively in anthers.4.The fusion vectors contain GFP,the red flurescent protein(REF-AHL22)and Bra036581 or Bra040503 were transferred into tobacco cells.By confocal laser scanning microscope observation,we found that the protein Bra036581 was located on the plasma membrane and nucleus,while the protein Bra040503 was appeared in the nucleus.