Cloning And Functional Identification Of Soybean GmKASⅡfamily Genes

Soybean is an important economic crop and oil crop.Mining and identifying func tional genes that affect oil content and components is of great significance for improvi ng soybean quality.Studies have shown that KASⅡis involved in the accumulation of fatty acids in Arabidopsis,peanut,tobacco and cotton,and affects the changes of fatty acid components.Therefore,in this study,using the Arabidopsisβ-ketoacyl-ACP synt hase gene as a probe,Blast comparison was performed in Soybase,and it was found th at there were 11β-ketoacyl-ACP synthase genes（GmKASII）in soybean,and 11 Bioinf ormatics analysis of these genes,combined with transcriptome sequencing and quantit ative analysis,selected the gene with the highest expression in seeds,Glyma.17G047000,for functional verification in Arabidopsis and soybean.Glyma.17G047000 was fou nd to alter the fatty acid composition,increasing the stearic and oleic acid content.The main findings are as follows:1.Search for the gene sequence of Arabidopsis thalianaβ-ketoacyl ACP synthase（atkasⅡ）in the GEN-BANK database of NCBI,and use this gene as a probe to perfo rm Blast alignment in Soybase,and retrieve a total of 11 soybeans.GmKASⅡgenes ar e divided into 3 subfamilies.All 11 GmKASⅡgenes contain the activation site ofβ-ket oacyl ACP synthase（GPNYSISTACATSNFCI）.2.Analysis of GmKASⅡfamily gene promoters showed that there are hormone-r elated and light-responsive cis-acting elements in the GmKASⅡs promoter region;exc ept for Glyma.Stress-related response elements.3.The expression of 11 GmKAS II genes was searched in the transcriptome datab ase at different stages of grain development（R5,R6,R7）,and it was found that Glym a.The expression of Glyma.17G047000 was the highest in R5,R6 and R7 stages.The specificity analysis of GmKASⅡs gene expression in time and space,q RT-PCR results showed that GmKAS-A（Glyma.17G047000）was highly expressed in all tissues.The expression level of GmKASⅡs gene in soybean embryo matured stage showed a trend of first increase and then decrease,and the expression level was the highest in 20 d e mbryo.4.Combined with the results of RNA-seq and real-time quantitative PCR,four ge nes（Glyma.05G129600,Glyma.08G084300,Glyma.13G112700,Glyma.17G047000）were selected to construct a GFP fusion expression vector.The localization experimen ts in protoplasts showed that these four genes were all localized in chloroplasts.5.Combined with the results of RNA-seq and q RT-PCR experiments,a plant expressi on vector was constructed to overexpress GmKASⅡ-A（Glyma.17G047000）gene in A rabidopsis thaliana;3 T₃ generation transgenic Arabidopsis thaliana with higher expres sion levels were obtained.Lines;fatty acid content detected by gas chromatography:c ompared with the wild type,the palmitic acid of Arabidopsis lines overexpressing GmKASII-A gene was significantly reduced;the stearic acid content was significantly incr eased,and one of the overexpressing lines（OE-3）It was significantly increased by 3.36%;the content of oleic acid was increased,OE-1 and OE-3 were significantly increa sed by 4.65%and 5.29%respectively;the content of linoleic acid and linolenic acid w ere decreased.6.The endogenous gene GmKASⅡ-A（Glyma.17G047000）was overexpressed in soyb ean by using the method of soybean cotyledon nodes mediated by Agrobacterium,and a T₁ generation positive line was obtained.The Soxhlet extraction method was used to detect that the total fatty acid content of GmKASⅡ-A gene transgenic soybean was si gnificantly increased by 2.52%;the content of fatty acid components in the grains of T1 positive lines was analyzed by gas chromatography,and it was found that the conten t of palmitic acid C16:0 was significantly reduced.2.94%;the content of stearic acid C18:0 increased significantly by 2.53%;the content of oleic acid C18:1 increased by 5.41%;the contents of linoleic acid and linolenic acid were decreased to varying degrees,and the decrease of linolenic acid content was smaller than that of linoleic acid.