Cloning And Expression Pattern Of IlCHS And IlCHI Genes Of Iris Laevigata And Construction Of Their Overexpression Vectors

Flower color is one of the important ornamental traits of plants.The coloring of blue flowers is more complicated,and the coloring mechanism of different species is different.Iris laevigata with strong cold tolerance is a valuable blue flower in nature,and the studies of genes related to flower color regulation of I.laevigata can enrich the theoretical basis of flower color regulation mechanism research.CHS and CHI are crucial structural genes in the forwaid of anthocyanin metabolic pathway.By comparing the transcriptome of I.laevigata and I.laevigata var.alba tepals,we screened out CHS and CHI genes with significant differences in anthocyanin metabolic pathway and performed cloning and bioinformatics analysis.The test also examined the expression characteristics of CHS and CHI genes during the flowering stage of I.laevigata and I.laevigata var.alba and the expression pattern of I.laevigata seedlings under abiotic stress,and constructed plant overexpression vectors of CHS and CHI genes,which laid the foundation for further analysis of CHS and CHI gene functions of I.laevigata and the development and utilization of gene resources.The main research results are as follows.1、The sequences of the ORF region of 11CHS and IlCHI genes were cloned by RT-PCR using I.laevigata cDNA as template.The lengths of the gene sequences were 1185bp and 666bp,and encoding 394 and 221 amino acids,respectively.Bioinformatics analysis revealed that the molecular weight of IlCHS amino acid sequence is 42.92 kDa,which is a stable hydrophilic protein containing five conserved structural domains,no signal peptide,no transmembrane structure,dominated by threonine phosphorylation modification,mainly composed of α-helix and radom coil,and the protein tertiary shape is typical rice type Ⅲ structure.IlCHI amino acid sequence with a molecular weight of 23.93 kDa.It is an unstable hydrophilic protein containing two conserved structural domains,contains signal peptide and transmembrane structure,dominated by serine phosphorylation modification,mainly composed of α-helix and radom coil,and the protein tertiary structure is an inverted bouquet structure of the common CHI family.The registration numbers of IlCHS and IlCHI genes in GenBank are MW346643 and MT876539,respectively.Sequence comparison analysis between CHS,CHI proteins of homologous species and IlCHS protein,IlCHI protein using BLASTP function in NCBI revealed that IlCHS protein had the highest sequence similarity with IhCHS protein of I.hollandica at 94.09%,and IlCHI protein had the highest sequence similarity with the FhCHI protein of Freesia hybrida at 76.17%.Through the functional analysis of homologous genes,it could be speculated that IlCHS and IlCHI genes can involve in anthocyanin and flavonoid biosynthesis,respond to external stresses and improve plant resistance.2、The expression patterns of IICHS and IlCHI genes in various tissues and organs of I.laevigata and I.laevigata var.alba during florescence were investigated by qRT-PCR.It was found that the expression of IlCHS in I.laevigata was highest in the perianth segments at full-bloom stage,and the expression of IICHS in I.laevigata var.alba was highest at the bud stage.The IlCHI gene was the most highly expression in the leaves of I.laevigata,and I.laevigata var.alba IlCHI was the most highly expression in the anthers,followed by the leaves.The expression of IlCHS and IlCHI genes were specific,and the expression of IlCHS gene was greater than that of I.laevigata var.alba,and the expression of IlCHI gene was less than that of I.laevigata var:alba in the tepals of I.laevigata at full-bloom stage,which was consistent with the transcriptome detection results of the tepals at full-bloom stage.The results of the expression patterns of IlCHS and IlCHI genes in I.laevigata seedlings under stress(4℃,NaCl,PEG)were as follows.The IlCHS and IlCHI genes of I.laevigata responded under all three stresses.The expression of IlCHS gene was up-regulated in leaves and down-regulated in roots under salt stress in seedlings.The expression of IlCHS gene was up-regulated expression in both leaves and roots under PEG stress.The expression of IlCHS gene was up-regulated to down-regulated expression in leaves and down-regulated expression in roots under low temperature stress.The expression of IlCHI gene was down-regulated to up-regulated in leaves and roots of I.laevi gata seedlings under low temperature stress,and down-regulated under salt and PEG stresses.3、pBI121-IlCHS-GFP,pEH12-IlCHI-GFP and pGWB18-IlCHI,three kinds of plant overexpression vectors,were successfully constructed through traditional double digests technology and the new Gateway technology.