Preliminary Function Analysis Profiles Of The GH3 Family Genes And Their Expression In Chinese Hickory (Carya Cathayensis Sarg.) During The Grafting Process

Chinese hickory(Carya cathayensis Sarg.)is a commercially important oil tree species mainly distributed in Zhejiang and Anhui Provinces of China.Grafting could effectively shorten the juvenile phase,dwarf trees and facilitate the cultivation of Chinese hickory.Previous studies have revealed that auxin played an important role in the grafting process of Chinese hickory.It was known that the Gretchen Hagen 3(GH3)genes played a vital part in the mechanism of auxin homeostasis.In this study,grafted seedlings of Chinese hickory were used as the main experimental materials.The basic bioinformatics characteristics and expression patterns of GH3 family genes were analyzed in Chinese hickory.The Cis-acting elements of CcGH3 family genes promoters were analyzed,and the activity of CcGH3-1 gene promoter was verified.Additionally,the basic function of CcGH3 genes were verified by heterologous transformation in Arabidopsis.The expression patterns of CcGH3 family genes during grafting process were also analyzed in this research.The main results are summarized as following:1.The CDS sequences of twelve CcGH3 genes were cloned.CcGH3 family proteins were highly conserved,especially in the ATP/AMP-dependent acylation active sites,which indicated that the family has strong enzymatic activities related with adenosylation and amination.CcGH3 family proteins had closer phylogenetic relationships with woody plants than herbal plants.CcGH3 proteins were localized in cytoplasm,excepting for CcGH3-x.CcGH3-x showed distant phylogenetic relationship and subcellular localization characteristics compared with other CcGH3 members.2.CcGH3 family genes were dominantly expressed in roots and leaves,but showed low expression in stems.The expression of CcGH3 genes in subfamily II were significantly up-regulated under the induction of auxin,salicylic acid,kinetin and abscisic acid.The expression of eight CcGH3 genes was significantly decreased under the induction of ethephon,gibberellin and methyl jasmonate.It was worth noting that the expression patterns of all four members in subfamily I and IV were similar,which was down-regulated under the induction of the above phytohormones.3.Phytohormone-related cis-acting elements,including auxin,ethylene,abscisic acid,salicylic acid,gibberellin and methyl jasmonate-related cis-acting elements,were widely distributed in the promoters of CcGH3 family genes.Under the induction of indole acetic acid and naphthalene acetic acid,the inducible activity of CcGH3-1 gene promoter was enhanced by more than doubled,indicating that auxin involved in the regulation of CcGH3-1 gene expression.The inducible activities of CcGH3-1 gene promoter were related with its differences in length.Cis-elements inhibiting the expression of CcGH3-1 gene might exist in the promoter region of-2000 to-1501 bp,while the basic inducible sequences were found in the region of-500 to-200 bp.4.Compared with wild type,Overexpression of CcGH3-1 in Arabidopsis thaliana led to the phenotypes of smaller leaves,smaller plant height and shorter pods,while the phenotype was less significantly changed in CcGH3-6a transgenic lines.The expression of several genes in AtGH3,AtILR/ILL and AtPIN family were significantly inhibited,but the expression of AtYUC family genes were increased in CcGH3-6a or CcGH3-1 overexpressed lines,suggesting that CcGH3 genes played important roles in the regulation of auxin homeostasis.5.During the grafting process of Chinese hickory,most CcGH3 genes showed the expression trends of decreasing first and increasing afterwards in both rootstocks and scions.The expressions of CcGH3 genes were maintained at low levels,that was only 10%-40% or even lower than 10% of that at 0 d after grafting,at 3 d and 7 d after grafting;while at 14 d and 30 d after grafting,the expressions of CcGH3 genes were revovered,especially the expressions of CcGH3-3,CcGH3-4 and CcGH3-10 were over 10 times of that at 0d after grafting.The contents of endogenous auxin in rootstocks and scions decreased significantly at 3 d after grafting,which were just 8.8% and 5.5% of those at 0 d after grafting;increased continuously at 7 d and 14 d after grafting;and recovered at 30 d after grafting,which were 82.5% and 152.3% of those at 0 d after grafting.The expression changes of CcGH3 genes might explain partly the dynamics of auxin concentration during the grafting process of Chinese hickory.