The Screening Of Proteins Related To Male Sterility Of Salvia Miltiorrhiza And Their Function Analysis

Salvia miltiorrhiza Bunge,a perennial herb in Labiatae family and Salvia genus, is one of the most famous and traditional Chinese medicine. S. miltiorrhiza distributes in many areas of China and can be used to treat cerebrovascular disease,coronary heart disease,irregular menstruation,angina pectoris and so on. Cross breeding through male sterile plants has unique advantages in various aspects. Since the male sterile S. miltiorrhiza named Sh-B was found in 2002, a new methods could be used for its utilization of heterosis. Employing proteomics technology, we studied the differentially expressed proteins in the flower buds of the male sterile S. miltiorrhiza, in the hope of providing theoretical basis and technical support for the understanding of the mechanism of male sterility in S. miltiorrhiza and for the application of the male sterile S. miltiorrhiza in breeding. The main results are as follows:1. An iTRAQ facilitated quantitative proteomic analysis on the floral buds of male sterile and fertile S. miltiorrhiza in three developmental stages(squaring stage, initial bloom stage and full bloom stage) was performed and 2075 proteins were identified. 70, 184, and 173 differentially expressed proteins between male sterile S.miltiorrhiza and fertile S. miltiorrhiza were identified in squaring stage, initial bloom stage and full bloom stage, respectively. Bioinformatics analyses were performed and the result shows that the differentially expressed proteins were mainly significantly enriched in cellular component categories such as photosystem、photosystem I and chloroplast thylakoid membrane, in molecular function categories such as oxidoreductase activity and chlorophyll binding, in biological process categories such as photosynthesis、oxidation-reduction process and photosynthetic electron transport chain, in pathway such as stilbenoid, diarylheptanoid and gingerol biosynthesis、photosynthesis- antenna proteins and photosynthesis.2. Based on the result of the iTRAQ analysis and bioinformatics analyses, PSAB(Photosystem I)、PSAC(Photosystem I)、PSBE(Photosystem II)、PETA(photosynthetic electron transfer A)、PETB(photosynthetic electron transfer B)、PETD(photosynthetic electron transfer D)and RPL14(ribosomal protein L14)were selected from the differentially expressed proteins, and the Real-Time qPCR were performed in each of the three developmental stages(squaring stage, initial bloom stage and full bloom stage) to analyze the expression of the selected genes in transcriptional level. Of the seven selected proteins, six genes(except RPL14) showed similar mRNA and protein expression patterns at all three developmental stages.3. Full-length sequence PSAB and PETB were cloned using cDNA obtained by RT-PCR and sequencing was performed to ensure the accuracy of the sequence. PSAB and PETB were ligated to the pGBKT7 vector respectively after it was digested by double enzyme digestion to form the pCsGFPBT-PSAB and pCsGFPBT-PETB recombinant plasmid. After that, the pCsGFPBT-PSAB and pCsGFPBT-PETB over-expression vector were confirmed by Sequencing and double enzyme digestion verification.