Identification And Expression Analysis Of Candidate Genes GhA05KASI For Oil Content In Cotton

Cotton is not only an important fiber crop,but also an important oil crop.The oil content of upland cotton seed ranges from 20%to 40%.The edible oil in China is short,and depends on imports.Increasing the production of cottonseed oil can alleviate this problem.Fatty acids are important components of biofilm,and very important to organism.According to the synthesis pathway of fatty acids,it is found that KASI is a very important enzyme in the synthesis of fatty acids.KASI mainly catalyzes the formation of C₄-ACP from C₁₆-ACP.In this study,candidate gene association analysis identified the GhKAS1 gene which was related to cottonseed oil content in upland cotton.It provided a theoretical basis for improving cottonseed oil content in upland cotton.The main results of this study are as follows:1.According to the phenotypic data in 503 upland cotton germplasms in 8 environments,one marker Gr KASI-3705 was correlated with target traits based on a general linear model using TASSEL 2.1 software.There phenotypic variation explained ranged from 7.46 to 18.38 with the average of 14.23.The marker corresponds to the KAS1 gene predicted in G.raimondii.2.Mapping of KASI gene from G.raimondii to upland cotton genome,6 copies of the gene were found and located on chromosome A05,D05,A06,D06,A09 and D09.Specific primers were designed for these 6 candidate homologous genes.Combined with phenotypic data from 8 environments of 503 natural germplasm materials of upland cotton,a marker Gh A05KASI-3705 was detected in TASSEL 2.1 software based on general linear model.which was consistent with the associated result of the marker Gr KAS1-3705.3.The target gene was analyzed by RT-PCR and q RT-PCR.The gene expression showed significant difference between the high oil and the low oil materials in the 30 DPA period.The expression pattern of the target gene in the low oil materials was the same as that of the expression profile.Two conserved domains related to ketoacyl were found by predicting the protein sequence of GhA05KASI.4.Comparing the sequence of GhA05KASI by resequencing,5 low oil materials and 5 high oil materials.One,one and ten mutations site were found located in the intron region,the 3'UTR region,and the promoter region of the gene,respectively.In addition,it was noteworthy that there was an 11 bp deletion at the 784 bp position in upstream of the codon.Primers were designed for these mutation sites.Associated analysis of oil content phenotypes in upland cotton natural population showed that 3 mutation loci were significantly correlated with oil content phenotypes.Two mutations were located in the intron region,and the another one was the deletion of promoter 11 bp located in promoter region.5.Construction of transgenic vector.In this experiment,the overexpression vector,promoter GUS vector and CRISPR vector were constructed to verify GhA05KASI.