Research Of Differential Proteome Of Male Sterile Solanum Melongena.L Anther And Cloning And Expression Analysis Of Related Genes

Eggplant（Solanum melongena L.） is one of the indispensable vegetables in daily life. It has strong heterosis and it would improve the yield of eggplant with enhanced stress resistance and high quality. Male sterile line, as female parent, is a kind of important way to utilize heterosis of eggplant. Anther indehiscent is one of the important forms of functional male sterility. Clearing the mechanism of eggplant male steril has important theoretical significance and practical application value for creation and utilization of heterosis breeding and germplasm resources.The eggplant male sterile line F16-5-6 and fertile 142 were analyzed through two-dimensional electrophoresis and mass spectrometry identification technology during the same period, and differential expressed proteins associated with anther growth and eggplant male sterility were obtained. One of the differential expressed proteins COI1 and two genes JAZ1 and MYC2 of its pathways were cloned, and the bioinformatics and fluorescence quantitative PCR expression were analyzed to explore the possible reasons of the eggplant anther dehiscence on the protein level and gene level. These studies lay the foundation for further exploration in eggplant functional mechanism of male sterility. The main results were as follows:1. Using 2D-PAGE technology, eggplant anther protein were separated, and four significant differential protein points were selected from the 43 protein points to analyze MALDI-TOF/TOF-MS/MS spectrometry identification. Finally the four significant differential protein points were identified successfully. Through database retrieval to these differential protein points, it has been found that the four proteins are GTP enzyme switch made protein, iron superoxide dismutase（sod）, COI1 protein and pyruvate kinase. They were all down-regulated protein.2. Genes cloning, bioinformatics and expression analysis of the differentially expressed genes related to anther development and anther dehiscence in eggplant.（1） Taking the first chain cDNA of stigma total RNA of stem apex as template from eggplant anther, full length of COI1 gene were gotten, whose ORF sequence was 1812 bp and encoded 603 amino acid residues. Througt predicting the protein had an F-box domain in N-terminal and there were five leucine-rich repeats in the C-terminal, it is indicated that the protein belonged to the plant COI1 protein family. Meanwhile, the full length of two key genes JAZ1 and MYC2 in the eggplant anther jasmonate signal transduction pathways were cloned, respectively. ORF of JAZ1 was 759 bp encoding 252 amino acids residues, and the protein contained two conserved functional domains TIFY（ZIM） and CCT₂（Jas）. ORF of MYC2 was 2076 bp encoding 691 amino acids residues and the protein contained a bHLH conserved domains. he analysis of the evolutionary relationships of plants with a variety of homologous genes indicated that the three key genes of JA signal transduction pathway had a high similarity with homologous genes of different plants.（2） Using fluorescence quantitative PCR method to detect the expression level of eggplant JA signal pathway key genes COI1, JAZ1, MYC2 in anther of sterile line and fertile line in different periods, respectively. It was found that the expression level of COI1 in fertile material 142 was gradually decrease over time and the expression of JAZ1 kept a low level. While the expression of MYC2 increased at first and then decreased. It showed that JA signal passed as a receptor before flowering through COI1 gene and it subsequently enhanced. From the declination of COI1 and the coordinately growth of MYC2 it could conclude that: the expression level of COI1 after flowering was lower than that before flowering. From that it was presumed that presume JA signal had been conducted to downstream before flowering. Instead, in sterile material F16-5-6, it showed that the expression level of COI1 remained a low state, and it was significantly lower than the expression of fertile material 142. The expression level of inhibiting factor JAZ1 continued to increase. At the same time, the high expression of MYC2 decreased with it, and caused the block of JA signal. In summary, it was conculed that key genes COI1, JAZ1, MYC2 of JA signal pathway were related with anther dehiscence, and these genes could play a role in the formation of male sterility in eggplant.