Differential Proteome Analysis Of Male Sterile Wolfberry Anther And Cloning Of Three Related Genes

Wolfberry (Lycium barbarum L.) is one of important edible economic crops. The utilization of heterosis is the effective way to improve wolfberry production, ease the contradictions of quality and yield and enhance stress resistance (tolerance). Male sterility is an important genetic tool in utilization of heterosis. Therefore, understanding of male sterility mechanism has important theoretical significance and application value.Using2D-DIGE and mass spectrometry technolgies, we studied differential proteome of male sterile wolfberry anther at the stage of early tetrad. We obtained differently expressed proteins which may be related with anther development and male sterility. Two of those differentially expressed proteins, Lb14-3-3and LbCHS genes cDNA were isolated, analyzied by bioinformatics and relatively quantified by qRT-PCR. Also, we cloned the R2R3MYB geae LbMYB103open reading frame (ORF) of wolfberry. The main results are as follows:1. We optimized two-dimensional electrophoresis conditions and anther total protein extraction method of wolfberry. Using TCA-acetone precipitation combined Tris-HCl to extract wolfberry anther protein, with13cm pH4-7srips, about350μg loading quantity, lysis buffer containing2mol/L thiourea and silver nitrate staining can obtain reproducible, high-quality2-DE maps of anther protein of wolfberry. Anther proteins are mainly concentrated in the pH4-7and15.0-85.0kDa range. This2-DE condition can be effectively applied to analysis the wolfberry anther proteome.2. Using2D-DIGE technology,13cm pH4-7IPG strips, over1760protein spots of wolfberry anther were separated, and52protein spots differentially expressed more than1.5folds (p<0.05) were selected to further analysis by MALDI-TOF/TOF-MS/MS, among which,45protein spots were successfully identified as40different proteins, with a success rate of86.0%. By analysis their function annotations of these successfully identified proteins, we classified these proteins. They were involved in protein synthesis and folding (20%), carbohydrate and energy metabolism (18%), signal transduction (9%), stress response (9%), amino acid metabolism (7%), unknown function (7%), anther development (4%), transcription factors (3%), fatty acid metabolism (2%), nucleic acid metabolism (2%) and flavonoid synthesis (2%). The wolfberry anther development is subject to a complex regulatory networks, multiple genes differentially expressed in multiple metabolic pathways affect pollen fertility.3. Among significant differently expressed protein spots, spot No.14and26,27,28were identified as glutamine synthetase (GS) and ascorbate peroxidase (APX) respectively by mass spectrometry. Enzymes activity analysis results showed that in male sterile mutant anthers, GS and APX activities were only about22%and33%of that in the wild type, respectively.4. Cloning and analysis of expressions of differentially expressed proteins related anther development and male sterility in wolfberry.①By RACE technology, Lb14-3-3gene was cloned and its cDNA full-length was1005bp, its ORF was768bp, which encoded256amino acids. At transcription level, Lb14-3-3gene was predominantly expressed in anther, fruit and flower, and with a low expression in the roots, stems and leaves. The transcription level of male sterile mutant anther was lower than that of the wild type at the early tetrad stage, which was consistent with the change at protein level.②The LbCHS gene was cloned with full-length of1448bp and its ORF was1167bp which encoded389ammo acids. LbCHS belongs to the CHS gene family. Gene expression analysis showed that LbCHS was highly expressed in reproductive organs of wolfberry, including flower and anthers, and low in the fruit. It can not be detected in vegetative organs. At early tetrad stage, LbCHS transcription level declined in male sterility relative to the wild type, which was similar to the trend at protein level.5. The ORF sequence of LbMYB103gene was cloned with975bp encoding324amino acids. Blastp analysis showed that, LbMYB103genes encoded protein having R2R3domain of MYB transcription factor family. Quantitative real-time RT-PCR analysis indicated that LbMYB103mRNA accumulated with high level in flowers and anthers, and no transcripts detected in roots, stems, leaves and fruits. The relative expression level in male sterile mutant was declined relative to the wild type in early tetrads. From the preliminary analysis above, we speculate LbMYB103may be involved in the regulation of wolfberry anther development.