Cloning,Expression Pattern Analysis And Functional Verification Of The MsPL Gene From Alfalfa

Alfalfa(Medicago sativa)is a high-quality forage grass.It is necessary to develop high-quality hybrid alfalfa varieties using heterosis,and cultivating alfalfa sterile lines is a very important point in hybrid breeding.In 2020,the research team used transcriptome sequencing analysis on the anthers of the sterile and maintainer lines of alfalfa at five stages,and found that c93045.graph＿C0 may be a candidate gene for infertility,a gene for the synthesis of pectin lyase(PL).Sterility mechanism of alfalfaPectin lyase can promote the elongation and growth of plant cell walls,as well as the release of pollen walls.Therefore,it is necessary to study the effects of pectin lyase on alfalfa abortion and its expression patterns in alfalfa,in order to provide support for the study of the mechanism of alfalfa male sterility,and to be helpful in alfalfa molecular breeding.In order to study the relationship between pectin lyase and alfalfa sterility,studies on pollen viability and anther dehiscence revealed that the sterile line had fewer pollen grains,weaker pollen viability,and even malformations,while the maintainer line had more pollen grains,stronger pollen vigor,and plumpness.The selection of materials for male sterile and maintainer lines was confirmed to be correct.Semithin sectioning,in vitro germination of pollen tubes,measurement of pectin lyase activity,and determination of deesterified pectin content were performed on the anthers of the two lines to further study the effect of pectin lyase on the anthers.It was found that the degradation of the tapetum of male sterile anthers during the pre and late tetrad stage was not completely delayed until the pollination stage,and the microspores were deformed and dried up.Most of the pollen grains of male sterile lines do not germinate,while a small portion of the germinated pollen tubes have a shorter length.The enzyme activity of pectin lyase in the anthers of male sterile lines at the pre and post tetrad stage was significantly lower than that of the maintainer lines,and the content of deesterified pectin in the anthers of male sterile lines at the post tetrad stage was higher.In order to further study the MsPL gene at the gene level,a full-length cloning was performed,and the gene was named MsPL.Bioinformatics analysis was performed.The total length of MsPL was 1305 bp,encoding 434 amino acids,and containing pectin lyase Amb＿The conserved domain of all is highly homologous to leguminous plants and has the closest molecular distance to Medicago terrestris.Subcellular localization confirms that the protein encoded by the MsPL gene plays a role in cell membranes and cell walls.The results of Real-time PCR showed that the expression of MsPL gene in the anthers of male sterile lines and maintainer lines was significantly lower in the pre and late tetrad stage and significantly different in the early monocyte stage,while there was no significant difference in the expression amount of MsPL gene in other stages and stems,leaves,and roots.It is speculated that the MsPL gene mainly acts in the tetrad stage.Construction of prokaryotic expression vector and verification of prokaryotic expression showed that the protein encoded by MsPL had pectin degrading enzyme activity in vitro.Construction of RNAi vector and overexpression vector were used to introduce them into alfalfa using Agrobacterium tumefaciens,and genetic transformation analysis was conducted to analyze the function and mechanism of MsPL on alfalfa.The results showed that the pollen of overexpression positive plants was fertile,and the pollen tube length was elongated,The activity of pectin lyase increased.However,RNAi positive plants have semi sterile pollen,shortened pollen tube length,and decreased pectin lyase activity.Therefore,it is speculated that inhibiting the expression of MsPL gene in alfalfa anthers will lead to abnormal metabolism of pectin in pollen tubes,tapetum,and other structures in the anthers,thereby affecting pollen tube elongation and tapetum degradation.It is proved that MsPL gene has an impact on the fertility of alfalfa.