Expression And Regulation Of Genes Related To Lipid And Protein Synthesis And Accumulation In Soybean

Soybean is an important economic crop in the world,which is rich in protein and oil.The functional components related to soybean protein and oil have important pharmacological effects in anticancer,tumor inhibition,antioxidation,antivirus,immune regulation,cardiovascular disease and so on.The physiological processes of soybean oil and protein synthesis are very complex,involving many genes and enzymes.At present,the biochemical pathways of lipid synthesis have been studied thoroughly,but the regulation mechanism is not clear;and the key genes and pathways affecting soybean protein synthesis are not clear.Genetically engineered soybean oil and protein anabolism,including increasing seed oil/protein content and improving fatty acid/amino acid composition,can meet the growing human demand for soybean oil and protein,and can make soybean oil and protein have more nutritional value and special industrial application value.In this study,the seeds of “ Guichun103” soybean varieties developed 20,30,40 and50 days after flower were used as the research material,Four mRNA and miRNA libraries were constructed,and RNA-Seq and miRNA sequencing were carried out.The differentially expressed genes in the process of protein and lipid accumulation,as well as the miRNAs involved in the regulation of lipid and protein synthesis were analyzed and mined.It is the theoretical basis for the cultivation of good soybean varieties and the improvement of oil and protein content in soybean production.The main results of this study are as follows:?1?The contents of oil and protein in soybean seeds were determined by acid hydrolysis and Automatic Kjeldahl method.and the contents of oil and protein in 20d?DD₂0?,30d?DD₃0?,40d?DD₄0?and 50d?DD₅0?were 7.03%,15.79%,17.64% and20.36% respectively and the contents of protein in 20d?DD₂0?,30d?DD₃0?,40d?DD₄0?and 50d?DD₅0?were 28.03%,30.45%,32.88% and 35.49% respectively.?2?The total clean reads obtained from 20 d,30d,40 d,and 50 d transcriptome sequencing were 107 548 920,111 670 776,109 339 672,and 108 884 270,and Q20 was98.19%,98%,98.12%,and 97.98% respectively.?3?In three comparison group?DD₂0-vs-DD₃0,DD₃0-vs-DD₄0,DD₄0-vs-DD₅0?,the total number of differential expression genes was 24767,In DD₂0-vs-DD₃0 was 4759 differential expression genes(DEGs,of which 1801 DEGs were up-regulated and 2958 DEGs were down-regulated.In DD₃0-vs-DD₄0 was 6245 DEGs,of which 2941 DEGs were up-regulated and 3304 DEGs were down-regulated.In DD₄0-vs-DD₅0was 13763 DEGs,of which 5695 DEGs were up-regulated and 8 068 DEGs were down-regulated.?4?Eighteen genes related to lipid synthesis were screened,among which the expression of FATB?ID100781381,ID100797796?,LACS?ID100803126,ID100802266?,DGAT?ID100817270?genes was continuously up-regulated;the expression of FATB?ID100170693?,KASI?ID547575?and GPAT?ID100817927?genes showed a general up-regulated trend;the expression of ACC?ID100777962?gene continued to rise in the early stage and decreased at 50d;the expression of ACC?ID547859?,GPAT?ID100791297?,DGAT?ID732606?was stable at the early stage and up-regulated at50d;the expression of LPAAT?ID100814107?showed a steady trend;it is speculated that these 13 genes may be positively regulated in lipid synthesis;the expression of SAD?ID100037478?gene was continuously down-regulated from 30 d.The mega-6 fatty acid desaturase?FAD2:ID100805040?gene showed a sustained down-regulated expression,and the FAD2?ID100805040?gene expression was sustained down-regulated expression;these genes were presumed to promote the increase of stearic acid and oleic acid content in soybean grains through negative regulation.?5?Twenty-three genes related to protein synthesis were screened,among which PEPC?ID100793542,ID100814240,ID547756?and CAT?ID100810218,ID100776978?gene expression levels were similar in the early stage,and were slightly up-regulated at50 d.PEPC?100820574?,HSP20?100803161,547852,100812707?,BGS?100507221?and HSP70?100812707?genes expression were down regulated first and then up regulated;GS?100817922?,HSP20?100500475,100526870,547537,100787482,100305751?,BGS?547485,547908?,HSP70?100790356?and BC?547465,100775426,100806840?genes expression were continuously up-regulated,these genes positively regulate proteinsynthesis and may promote the increase of protein content in soybean grains.?6?The AP2-EREBP family transcription factors and ABI3VP1 family transcription factors play a role in the regulation of oil and protein synthesis.In addition,C2C2-Dof transcription factors are involved in the regulation of oil synthesis,and bZIP family transcription factors are involved in the regulation of protein synthesis.?7?The sequence length of palmitoyl-acyl carrier protein thioesterase?FATB?obtained by soybean is 2061 bp,CDS sequence is 1269 bp,encoding a total of 422 amino acids,belonging to the PLN02370 superfamily,and has a high similarity to the proteins encoded by the FATB gene of Glycine soja and Cajanus cajan,and the similarity is 100% and 83%,respectively.The cloned FATB gene was constructed into a plant expression vector,and Arabidopsis thaliana was transformed by inflorescence staining.PCR results showed that the FATB gene was successfully transferred into Arabidopsis.?8?The total clean reads obtained from 20 d,30d,40 d,and 50 d miRNA sequencing were 27 011 109,27 860 083,27 420 413 and 21 718 921,respectively,and their Q20 content was 99.3%.?9?In three comparison group?DD₂0-vs-DD₃0,DD₃0-vs-DD₄0,DD₄0-vs-DD₅0?,the total number of differential miRNAs was 515,In DD₂0-vs-DD₃0 comparison group was 162,of which 91 differential miRNAs were up-regulated and 71 differential miRNAs were down-regulated.In DD₃0-vs-DD₄0 comparison group was 171,of which 67 differential miRNAs were up-regulated and 104 differential miRNAs were down-regulated.In DD₄0-vs-DD₅0comparison group was 182 differential miRNAs,of which 70 differential miRNAs were up-regulated and 112 differential miRNAs were down-regulated.?10?Some miRNAs that may be involved in the synthesis of soybean grain lipid were screened,among which gma-miR396b-5p,gma-miR396 c and gma-miR396k-5 targeting genes for LACS promoted target gene expression by negative regulation and increased soybean grain lipid synthesis,while gma-miR408 d targeting gene for GPAT promoted the increase of soybean grain oil content by positive regulation of GPAT expression and its enzyme activity;Gma-miR159e-5p,through negative regulation,promoted the continuous up-regulation of KASI expression and promoted the synthesis of oleic acid in soybean grains.?11?Some miRNAs that may be involved in protein-related synthesis genes werescreened,among which gma-miR156 d,gma-miR156 c,gma-miR156 I targeting PheRS were identified.The expression patterns of gma-miR156 j,gma-miR156 l and gmamiR156 m were continuously up-regulated,which was consistent with the continuous increase of protein content in soybean grains;while gma-miR396b-5p,gma-miR396 c and gma-miR396k-5p might play a role in promoting protein synthesis in soybean grains by negatively regulating the expression of target heat shock protein 70 gene?HSP70?.?12?The RT-PCR results showed that the expression trends of four differentially expressed genes and four differentially expressed miRNAs were basically consistent with the sequencing data.