| Although researchers have been studying the breeding of Alfalfa,which is a preferred herbage resource in the development of grass planting and animal husbandry,the research progress of Alfalfa on fertility heredity basis and inheritance pattern is slow due to the difficulty in the selection of high quality male sterile lines and supporting maintainers in the process of hybrid breeding.To maximize the obvious hybrid vigor of Alfalfa and comprehensively reveal the mechanism of its male sterility at an early date,this study took cytoplasm nuclear interaction male sterility line MS-JN1 A anther and maintainer line MS-JN1 B anther of Alfalfa as materials,utilized high throughput sequencing to perform differential expression analysis for 5 anther development stages,screened differentially expressed genes to conduct enrichment analysis and function annotation,and selected 4 differential candidate genes related to the male sterility of Alfalfa in the glycometabolism pathway to carry out gene expression profile analysis in anthers.Then,it concretely analyzed the spatial and temporal expression profile of β-galactosidase(β-GAL)gene in Alfalfa,and synthetically discussed the relationship between the amount ofβ-GAL gene expression and the anther development of Alfalfa by combining the measured results of β-GAL activity and galactose content.Furthermore,it cloned the overall length of c DNA in the β-GAL gene of Alfalfa to analyze complete sequence feature,protein structure and subcellular localization,and established plant overexpression vector and RNA interference expression vector by reverse genetics approach to perform function analysis for the β-GAL gene.This study not only revealed the control process of β-GAL gene in the anther development of Alfalfa,but also provided theoretical foundation for making better use of the male sterility line and hybrid vigor of Alfalfa to breed the high-quality hybrid and expand the alfalfa industry in production practice.The research results were mainly as follows:1.The gene expression profiles of two-line Alfalfa in the 5 anther development stages were obtained via transcriptome sequencing.(1)A total of 50,561 pieces of unigene annotation information were gained,and 11,859 differentially expressed genes were screened,of which1,151 were specifically expressed in the sterile line anther and 1,358 were specifically expressed in the maintainer line anther at the tetrad stage.(2)Enrichment analysis showed that the differentially expressed genes related to male sterility mainly participated in the metabolic pathways of carbohydrate,starch and sucrose,pentose and glucuronic acid conversion,and ribosome.(3)The 4 significantly different genes in the pathway related to glycometabolism were specifically expressed intensively at the first 4 stages,of which c112871.graph＿c0 was annotated as a member of the glucoside hydrolase family 35 after comparison.2.The spatial and temporal expression profile of β-GAL gene in Alfalfa was further analyzed via real-time fluorescent quantitative PCR.(1)It was found that the β-GAL gene was expressed in each tissue,and it was specifically expressed in the maintainer line anther at the tetrad stage,indicating that β-GAL gene had strong space-time specificity and tissue specificity.(2)The determination of β-GAL activity showed that the maximum value of enzyme activity occurred at the pollen mother cell stage in the sterile line,while the peak value of enzyme activity appeared at the tetrad stage in the maintainer line and was remarkably higher than any other stages and tissues.(3)The galactose content showed a significant difference between the two lines at the tetrad stage,and also reached the maximum value along with the rise of enzyme activity at the tetrad stage in the maintainer line,suggesting that β-GAL activity in Alfalfa,to some extent,affected the change of galactose content.3.A β-GAL gene was isolated from Alfalfa via TA clone technology,and it was named as MsGAL.(1)The overall length of c DNA sequence was 2,499 bp,and it had the closest genetic relationship with Medicago truncatula,with the similarity reaching 96.28%,followed by high homology with Trifolium pratense and Cicer arietinum.(2)It included 832 amino acids,the relative molecular weight of its encoding protein was about 93.3k Da,the theoretical isoelectric point was 8.46,and it belonged to a kind of non-transmembrane hydrophilic protein.The N-terminal of amino acid sequence had a signal peptide and contained a typical structural domain of glucoside hydrolase family 35,while the C-terminal had a structural domain of galectin.(3)Subcellular localization analysis indicated that the secreted protein of MsGAL gene coding was targeted to the cytoderm.4.Double digestion technique was used to establish the overexpression vector of p BI121-MsGAL,homologous recombination method was used to establish the interference expression vector of p RNAi-MsGAL,and MsGAL gene was transformed into tobacco plants by Agrobacterium-mediated leaf disc method.(1)The β-GAL activity of tobacco plants after overexpression of MsGAL gene was about 4 times higher than the wild type ones,and combining the down-regulated expression pattern of MsGAL gene in the sterile line,it could be known that MsGAL gene promoted tissue differentiation and cell elongation,and positively regulated the growth and development of plants.(2)When suppressing the expression of MsGAL gene,the enzymatic activity showed a decline in varying degrees,so it was speculated that the suppression expression of MsGAL gene would result in abnormal glycometabolism pathways such as galactose in Alfalfa,and further influence the energy metabolism of cell wall tissues such as anther wall and pollen wall,and even the metabolic process of substance such as lipid and also the process of pollen development,but the in-depth action mechanism of MsGAL gene still needed further research. |
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