Transcriptomics And Metabolomics Studies On Cytoplasmic Male Sterility In Alfalfa

Alfalfa（Medicago sativa L.）is an excellent pasture in the legume family Alfalfa.Since the discovery of the cytoplasmic male sterile line MSJN1A and its homozygous maintainers MSJN1B,research team has been studying the mechanism of male sterility in alfalfa.In this experiment,early-stage anthers of two lines were studied by making semi-thin sections of anthers,using liquid chromatography-mass spectrometry（LC-MS）to explore the metabolites,using pathway annotation and co-expression analysis of differentially expressed genes（DEGs）and metabolites（DMs）using transcriptome sequencing results.The results indicated more differences in the pathways of glucose metabolism,phenylpropane metabolism,fatty acid metabolism,energy metabolism,and so on.Subsequently,the role of MsGDSL esterase/lipase in anther development was specifically analyzed.The spatiotemporal expression characteristics of this gene were combined with the determination of recombinant lipase activity at different periods of anther development with prokaryotic expression to investigate the role of this gene in anther development in a comprehensive manner.Transgenic alfalfa plants with semi-sterile pollen were constructed by RNAi method.The theoretical basis for the creation of novel alfalfa sterile lines was provided.The main results are as follows.1.From the semi-thin sections,the tapetal cell of the anther of sterile lines,degraded abnormally after meiosis;at mid-uninucleate,the central large vesicles of mononucleated microspores disappeared,and pollen abortion appeared obviously.It suggested that the pollen abortion in sterile lines was related to the delayed degradation of tapetal cell and the abnormal structure of the microspore vesicles.2.Using non-targeted metabolome sequencing technology,we obtained metabolites in the early stage of two alfalfa lines.401 and 405 metabolites were detected in the late tetrad and mid-uninucleate and 39 metabolites were consistently up-regulated,and 88 metabolites were down-regulated.Differential ploidy analysis indicated the presence of 45 versus 37 differentially significant metabolites in the two periods,respectively.The differential metabolites were mainly enriched in sucrose and starch metabolism,phenylpropane metabolism,fatty acid metabolism,and energy metabolism.The combined analysis showed that DEGs were coenriched with DMsin the above metabolic pathways.As shown in the results of q RT-PCR assay,there was down-regulated expression of MsGDSL,MYB 35,MsXTH,MsPME,C₄H,MsPrx,MsPHD,CYP703a2,and MsUGPD in the sterile lines during early stage of anther.It is hypothesized that downstream metabolite content is regulated by upstream genes,which ultimately affects pollen fertility.3.The sequence of MsGELPs gene was cloned and analyzed for its full length.It belongs to the GDSL esterase/lipase family,with a sequence length of 1095 bp,encoding 364 amino acids,and is highly expressed in early stage of anther.Phylogenetic tree analysis showed that the gene is conserved,subcellularly localized to the cell wall,and can hydrolyze palmitic acid.It is hypothesized that this gene affects anther development by regulating lipid metabolism.4.The recombinant vectors of p ET-32a-MsGELPs and RNAi-MsGELPs were designed and constructed.After gene silencing in wild-type alfalfa plants,lipase activity decreased and pollen showed semi-sterility,which was consistent with the expected results.