Comparation Of The Metabolome Between Cytoplasmic-nuclear Male Sterile Line And Its Maintainer Line And Cloning And Functional Study Of The Related Genes In Soybean (Glycine Max(L.)Merr.)

It has been found that the utilization of heterosis is an effective way to increase production,and cytoplasmic-nuclear male sterility(CMS)has an important value on this utilization.Although many scholars have conducted extensive research on the CMS of soybean,the molecular mechanism of CMS still requires further explanation.In this study,comparation of the metabolome between cytoplasmic-nuclear male sterile line and its maintainer line and cloning and functional study of the related genes in soybean were carried out.The main results were as follows:1.A total of 612 metabolites were detected based on widely-targeted metabolomics technology in the flower buds of the CMS line NJCMS5A and its maintainer line NJCMS5B of soybean.According to the standard of |log2Fold Change|≥1 and VIP≥1,74 differential metabolites were screened out,of which 30 were found to be up-regulated,and 44 were observed to be down-regulated in the young flower buds of the CMS line NJCMS5A,when compared with those of its maintainer line NJCMS5B.The main compounds included sugars,amino acid derivatives,nucleotides and their derivatives,organic acid,flavonoids,isoflavones,quinic acid and its derivatives,alkaloids and sugar alcohols.The KEGG Pathway enrichment analysis found that the main biological processes were isoflavone biosynthesis,flavonoid biosynthesis,arginine biosynthesis,proline biosynthesis and glycerophospholipid biosynthesis.It was speculated that changes in the aforementioned metabolic pathways had led to abnormal accumulations of the related metabolites,which may be the main reason for the formation of the male sterile line for the NJCMS5A.2.This study selected young flower buds of the CMS line NJCMS5A and its maintainer line NJCMS5B of the soybean as the test material.Then,the flavonoid content and activities of the total ATPase;sucrose phosphate synthase(SPS);phosphoenolpyruvate carboxykinase(PEPCK);ascorbate oxidase(AAO);catalase(CAT);total superoxide dismutase(T-SOD);and peroxidase(POD)were measured using a kit method,along with visible spectrophotometer,UV-spectrophotometer,and microplate reader.The results showed that the total ATPase,PEPCK,POD,and CAT activities,as well as the flavonoid content levels,had significantly decreased.Also,the T-SOD activities had displayed a significant increases.However,the SPS and AAO activities were not found to be significantly different in the young flower buds of the CMS line NJCMS5A when compared with those of its maintainer line NJCMS5B.Therefore,in accordance with the experimental analysis results,it was speculated that the loss of metabolite or enzyme activity related to energy metabolism or stress response may be the main reason for pollen abortion of the CMS line NJCMS5A of the soybean.3.Based on the study of small RNA sequencing and degradome sequencing in the soybean young flower buds of CMS line NJCMS1A and its maintainer NJCMS1B,miR159e and its target genes GmMYB33 and GmMYB33-like were cloned for functional study.The 2000 bp upstream of the soybean miR159e gene was isolated from the soybean,and the cis-acting element of the promoter sequence was analyzed by plant CARE online software.It was found that the promoter contained the TATA box and the CAAT box structures.By applying a qRT-PCR analysis,it was found that miR159e was down-regulated,while the target genes GmMYB33 and GmMYB33-like were up-regulated in the young flower buds of CMS line NJCMS1A,when compared with its maintainer NJCMS1B young flower buds.The overexpression vector of the miR159e and its targeted genes and promoters were further constructed in this study.Then,the Arabidopsis were transformed and the PCR test results showed that positive transgenic plants had been obtained.The histochemical staining revealed that GUS protein had been expressed in the roots,hypocotyl,and leaves of the miR159e Pro::GUS transgenic Arabidopsis seedlings.It was observed that when the miR159e Pro::GUS transgenic Arabidopsis were in the reproductive stage,the GUS protein had been expressed in the rosette leaves,stem leaves,root hairs,and anthers.Therefore,when compared with the WT plants,the miR159e transgenic plants had displayed a delayed flowering of approximately one week,and the plant heights were significantly decreased.However,the plant pollen had developed normally.Furthermore,similar cases were also observed in the targeted gene GmMYB33 and GmMYB33-like transgenic plants.