Cloning And Functional Verification Of GmSACPD Family Genes Of Soybean

Soybean is one of the most important food and oil crops in the world,and soybean oil occupies the important position in the vegetable oil market.Soybean oil is mainly composed of oleic acid,linoleic acid,linoleic acid,palmitic acid and stearic acid.Oleic acid,linoleic acid and linolenic acid are the main unsaturated fatty acids in soybeans,which is beneficial in regulating blood lipids,preventing blood clots,enhancing immunity,improving vision and promoting brain development.With the advancement of sociaty and the improvement of people’s living standards,people’s demand for high-quality vegetable oils,especially soybean oils,is increasing;on the other hand,soybeans can be used as energy materials in industry and in animal feed ingredients.Therefore,It is imminent to cultivate high-quality soybean germplasm resources with high oil and high unsaturated fatty acids,which is of great significance to meet the daily needs of people’s life and agricultural production.The stearoyl carrier protein desaturase(SAD,also known as SACPD)catalyzes the conversion of stearic acid to oleic acid,which is a key point for regulating the ratio of saturated fatty acids and unsaturated fatty acids.In Arabidopsis,cotton,rapeseed and other plants,it has been proved that regulating the expression of SACPD gene can change the fatty acid content and composition ratio of plants,and at the same time affect the growth,development and resistance of plants.The deletion of soybean GmSACPD-C gene has previously been reported to increase the content of soybean stearic acid and at the same time have a certain impact on the formation of soybean nodules.In order to further verify the function of soybean SACPD genes,three GmSACPD(GmSACPD-A,GmSACPD-B,GmSACPD-C)genes were cloned from soybeans in this experiment,then through the exogenous genetic transformation of the model plant Arabidopsis thaliana,the function of the soybean SACPD gene was further verified.The work of quality resources provides a certain reference value.The main research results of this experiment are as following:1.Use bioinformatics analysis to determine the Arabidopsis homologous genes of three SACPD genes in soybeans,the results showed that the amino acid sequence similarity of GmSACPD-A and GmSACPD-B genes is the highest,which reaches79.56% and 81.8%.Respectively,the similarity of amino acid sequence of GmSACPD-C and ATFTM1 reaches 68.29%.2.The results of real-time quantitative PCR in different soybean tissues showed that the expression of the three soybean SACPD genes was low in roots,stems,leaves and flowers,and the expression levels were higher in the mid to late embryonic stages.The expression levels of GmSACPD-B and GmSACPD-C genes from 10 d embryos to40 d embryos showed an increasing trend.The expression of GmSACPD-B gene in20 d embryos was 3.36 times as much as that in roots,while in 30 d embryos it increased to 10.78 times;The expression level of GmSACPD-A gene first increased and then decreased from 10 d embryo to 40 d embryo.The expression of GmSACPD-A gene in 20 d embryos was 2.53 times as much as that in root,while 30 d,it increased to15.57 times,and at 40 d,the embryo decreased to 6.83 times.The results of subcellular localization analysis showed that the proteins encoded by the three SACPD genes were all located in the chloroplast.3.The soybean SACPD gene was overexpressed in Arabidopsis,and the obtained Arabidopsis mutant was identified for homozygous.Compared with wild type,phenotypic observation of the overexpression lines and mutant lines showed that the Arabidopsis lines overexpressing the GmSACPD gene grew vigorously,with broad leaves and increased branches.The mutant Arabidopsis showed severe growth defects and short growth,and the leaves are narrowed.4.Real-time quantitative PCR test results showed that the expression of KASⅡ,FAT,SACPD,DGAT,FAD,and SSI2 genes in the GmSACPD gene over-expression strains all decreased significantly,and expression of WSD1 gene was increased.The expression levels of KASⅡ,FAT family genes,SAD family genes,DGAT family genes,FAD2 gene and FAD3 genes in mutant Arabidopsis were significantly decreased,and expression level of FAD3 gene were increased;The expression levels of SAUR genes,which is related to leaf shape in the GmSACPD gene-transformed Arabidopsis strains were significantly up-regulated,and the expression levels of SAUR genes in the mutants were significantly decreased.5.The fatty acid content of transgenic Arabidopsis and mutant Arabidopsis seeds was determined by gas chromatography.The results showed that compared with wild type,the palmitic acid and stearic acid content in GmSACPD gene transgenic Arabidopsis were significantly reduced,and the oleic acid content and linolenic acid content were significantly increased.The stearic acid content in the mutant Arabidopsis thaliana was increased,while the oleic acid content decreased.6.The thousand-grain weight of the Arabidopsis lines overexpressing GmSACPD gene and the mutant Arabidopsis lines were measured.The results showed that the thousand-seed weight of GmSACPD gene-transformed Arabidopsis lines was significantly higher than that of the wild type.The thousand-grain weight of Arabidopsis is significantly lower than that of the wild type.