| Cotton(Gossypium.L)is an important fiber and oil crop in the world.The present research on cottonseed oil mainly focused on the analysis of key gene families involved in the oil metabolism and location of QTLs related to oil content.However,the identification of oil-related candidate genes and their functional verification is still not explored.Therefore,this study used the data of the cottonseed oil content in backcross inbred lines of G.hirsutum × G.barbadense to locate the QTL related to the cottonseed oil content,and integrated bioinformatics and functional genomics analysis to screen out candidate genes for preliminary functional analysis.1.The oil content data of shelled cottonseed in five environments were measured by nuclear magnetic resonance instrument,and the phenotypic data of parents and populations were analyzed descriptively.The results showed that the oil content of parent G.barbadense Hai 7124 was significantly higher than that of G.hirsutum CCRI 36.The coefficient of variation of the five populations was more than 10%,indicating that the variation within the population was rich and suitable for QTL analysis.2.Using ICi Mapping for mapping,9 QTLs related to oil content were identified.Among them,the QTL located on the D03 chromosome have higher phenotypic interpretation rate(PVE)and LOD values of 34.73% and 16.88,respectively,and they are all detected in 5 environments.Therefore,the QTL on the D03 chromosome is regarded as a stable QTL.3.Referring to the previous studies,it was found that cottonseed oil began to accumulate rapidly at20 days after ovule development.Based on this,the stable genes were screened.Combined with the transcriptome data of high-oil material Hai 7124 and low-oil material TM-1,9 genes highly expressed at20 days of ovule development were screened in the QTLs interval of D03 chromosome.It was found that Gh＿D03G1072 was not expressed at the early stage of rapid oil accumulation,but was highly expressed during the period of rapid oil accumulation(ovule development after 20 days).The gene was named GhHSD1.4.The HSD1 in wheat,corn,Arabidopsis thaliana,peanut,soybean,cotton and other different species of orthologous genes encoding proteins to construct a phylogenetic tree analysis,the results show that the HSD1 gene in cotton is the same as the genes in most oil crops They are clustered into a large group and have a long relationship with starch crops.This result indicates that the evolution of HSD1 is accompanied by the differentiation between species.Further analysis of the conserved domains of the HSD1 revealed that protein sequences of different species have typical conserved functional domains,which are unique to oil body sterol proteins: there is a conserved enzyme activity site in the middle of the protein sequence.Yxxx K,secondly,is a co-coenzyme binding site at the N-terminus of the protein,the sequence is Gxxx Gx G.5.The total oil content and the contents of dodecanoic acid,myristic acid,palmitic acid,stearic acid and oleic acid in the positive clones of 35S::GhHSD1 and 35S::Gb HSD1 transgenic yeast were analyzed.The results showed that the total oil content and fatty acid content of transgenic yeast lines were significantly higher than those of the control.Compared with wild type,GhHSD1 gene transferred into Arabidopsis thaliana increased the oil content of Arabidopsis thaliana seeds,and increased the contents of oleic acid,linoleic acid,linolenic acid,arachidic acid and eicosapentenoic acid in Arabidopsis thaliana seeds.In summary,the candidate gene GhHSD1(Gh＿D03G1072)was identified through integrated data analysis of stable QTL interval and transcriptome.Additionally,preliminary functional quantification of the candidate gene was carried out,which laid the foundation for identifying the excellent sites related to cottonseed oil content and the key genes causing the difference of seeds oil content between G.hirsutum and G.barbadense.Our results will be critical for future breeding programs that aim to improve cottonseed oil contents. |
