| Barley is a "Diversified crop" that combines food,feed,nutrition,health care,brewing and processing.It has a wide range of research and application prospects.On the basis of systems biology,with the help of metabolomics research technology,the genetic basis of complex biological traits of barley can be analyzed from a new perspective,the relationship between metabolites and barley phenotypic traits can be understood,explore important genes that control important biological traits,improving genetic improvement efficiency of barley traits.In this experiment,we used the DH population of “Huaai 11×Huadamai 6” as the materials,conducted mQTL analysis of metabolites from mature seeds of 122 strains using high-density genetic linkage map,screened some key genes of important metabolic pathways in DH population,and the obtained candidate genes were cloned and verified in vitro.The main study results were as follows:1.Mature seed mQTL analysis: Mature seed samples of barley DH population were tested by extensive targeted metabolomics analysis,and a total of 1150 metabolites including flavonoids,polyphenols,amino acids and their derivatives,nucleic acids and their derivatives were obtained,these included 555 species of annotated metabolites.Using high-density genetic linkage map to detect 1150 metabolic traits,among which 799(69.48%)metabolic traits were mapped to 1116 mQTLs,and the number of mQTLs mapped to these metabolites ranged from 1-6,the most metabolites were located in one mQTL,accounting for 48.96%.The highest phenotypic variation explain rate of the 1116 mQTLs was the polyphenol metabolite wt00516,which was located on the538.20Mb-546.65 Mb interval of chromosome 7H,which explained the phenotypic variation rate of up to 92.0%.In addition,a total of 945 mQTLs of 1116 mQTLs were located on chromosomes 2H and 7H,and 14 mQTL hotspots were discovered for flavonoids,polyphenols,amino acids and their derivatives,and nucleic acids and their derivatives.2.Screening and cloning of metabolite candidate genes: Combined with the results of genome,gene annotation information,chemical structure of related metabolites and its homologous genes in rice and other model crops,a total of 13 candidate genes related tothe four metabolites of flavonoids,polyphenols,amino acids and their derivatives,nucleosides and their derivatives were screened out.Seven candidate genes with single exons and obvious target bands were selected for cloning and sequence analysis.The three decarboxylase genes(Hv TDC-1,Hv TDC-2,Hv TDC-3)were found to have a full length of 1533 bp in both parents,Huaai 11 and Huadamai 6,encoded a protein with containing 510 amino acid residues.The gene of a glycosyltransferase Hv UGT-1 was1512 bp in both parents,encoded a protein containing 503 amino acid residues.The sequences of the four genes had no sequence difference between the two parents;The other two glycosyltransferase genes(Hv UGT-2,Hv UGT-3)and one acyltransferase gene(Hv AT-1)had different degrees of sequence variation in two parents,Among them,Hv UGT-2 had a full length of 1524 bp in both parents,encoded a protein encompassing507 amino acid residues,and there was a difference of 19 base,which lead to the difference of 6 amino acid residues in the amino acid sequence;Hv AT-1 had a full length of 1377 bp in both parents,encoded a protein of 458 amino acid residues,with 11 base differences,resulted in a difference of 6 amino acid residues;The full-length sequences of Hv UGT-3 in Huadamai 6 and Huaai 11 were 1140 bp and 1131 bp,encoded 379 amino acids and 376 amino acids,respectively.The sequence of the Huaai 11(Hv UGT-3a)was identical to the sequence of the reference genome by sequence alignment,but the sequence of the Huadamai 6(Hv UGT-3b)differented from the reference genome by multiple bases,and a base G was inserted at 1090 bp,which made the subsequence transcoding mutation and leaded to its delayed termination,moreover,there were two transmembrane in the first 50 amino acid residues of the protein encoded by Hv UGT-3a and Hv UGT-3b,this domain ultimately affected the tertiary conformation of its protein.3.Functional verification of metabolic related genes: The prokaryotic expression vector of seven candidate genes were constructed,and determined suitable protein expression conditions.In vitro enzyme activity assays were performed on proteins that induce expression.Six candidate genes(HvUGT-1,Hv UGT-2,Hv TDC-1,Hv TDC-2,Hv AT-1,Hv TDC-3)were found that had corresponding enzyme activities.The glycosyltransferase genes Hv UGT-1 and Hv UGT-2 can effectively transfer glucosides from UDP to the hydroxyl groups of tricin to complete glycosylation modification.However,the activity of Hv UGT-2 gene in parents was different,and its activity in Huaai11 was much larger than that of Huadamai 6.The decarboxylase genes Hv TDC-1,Hv TDC-2,and Hv TDC-3 can decarboxylate tryptophan to form tryptamine,and it had consistently high activity in both parents.The acyltransferase gene Hv AT-1 can acylate apigenin-7-O with propionyl Co A as a coenzyme in both parents.Since metabolites in mature seeds accumulated during the filling stage,we used q RT-PCR to analyze the expression levels of six candidate genes with enzyme activity in seven different periods of grain filling of Huaai 11 and Huadamai 6.The expression levels of Hv UGT-2,Hv TDC-1,Hv TDC-2 and Hv TDC-3 were found to be significantly different between the parents.The Hv TDC-1 and Hv TDC-2 genes were only overexpressed in the fifth stage of Huaai 11 filling.However,the expression levels in the seven stages of Huadamai 6 were extremely low,which was consistent with the difference in the content of related metabolites in mature seeds,indicated that the difference in the relevant metabolic traits in mature seeds was caused by the difference in gene expression.In this study,the key candidate genes involved in the expression regulation of barley metabolic pathway were cloned and functionally verified by metabolomics research,which provided a reference for further systematically analysing the genetic basis of metabolites and phenotypic traits in barley. |
PDF Full Text Request