Study On The Expression Characteristics And Function Of Phenylalanine Ammonia-lyase Genes In Larix Gmelinii

Dahurian larch,Larix gmelinii(Rupr.),belongs to the Larix genus of the Pinaceae,and mainly growns in the Greater Khingan Mountains of China.L.gmelinii has a wide range of uses and economic value in the building,wood processing and paper industry because of its strong mechanical properties.Lignin is the main factor that affects wood properties,which is synthesized by a series of enzyme-catalyzed polymerization.Phenylalanine ammonia-lyase(PAL)is the key enzyme in the lignin biosynthesis pathway,and its expression abundance directly affects the biosynthesis of lignin,which affects growth and development of plants,as well as wood quality.Therefore,it is of great significance to study the expression regulation mechanism and function of LgPAL genes in L.gmelinii.The previous research results confirmed that the LgPAL is encoded by two paralogous genes in L.gmelinii.In this study,for further analyzing the expression characteristics of LgPAL1/2,the 1294 bp of LgPAL1 and 1254 bp of LgPAL2 gene promoter sequences were cloned by chromosome walking.The Plant CARE analysis found that pLgPAL1/2 contains plant hormone-responsive elements such as indole-3-acetic(IAA),gibberellin(GA),ethrel(ETH),and methyl jasmonate(Me JA),as well as stress-related cis-acting elements such as low temperature and drought.Heterologous expression of GUS driven by LgPAL1/2 promoters in Arabidopsis thaliana showed that pLgPAL1::GUS was highly expressed in various tissues and organs of Arabidopsis seedlings;pLgPAL2::GUS was mainly expressed in leaf veins;pLgPAL1/2::GUS were expressed in both flower organs and fruit pods,and its expression is induced by GA,ABA,low temperature and mannitol.For studying the subcellular localization of LgPAL,LgPAL1/2-GFP fusion proteins were transiently expressed in the lower epidermal cells of tobacco.The results showed that LgPAL1/2were localized in the cytoplasmic membrane.The phenotypic analysis of wild-type,atpal1 mutant,LgPAL1-overexpressing lines of Arabidopsis,atpal1 functional complementary lines were analyzed,the results showed that LgPAL1 could rescue the growth inhibition caused by At PAL1 mutation,and the overexpression of LgPAL1 increased various plant growth parameters such as leaf area,leaf length,leaf width and plant height of transgenic Arabidopsis,improved the contents of lignin,sucrose and soluble sugar,and enhanced the vegetative growth of transgenic Arabidopsis.The transcriptome analysis of wild-type,atpal1 mutant and LgPAL1-overexpressing lines of Arabidopsis showed that the overexpression of LgPAL1 resulted in significant changes in the expression levels of genes related to phenylpropanoid biosynthesis,starch and sucrose metabolism,plant hormone signal transduction,plant MAPK signal pathway and plant pathogen interaction pathway,which synergistically regulated plant growth development and adaptation to adversity.This study provides theoretical and experimental basis for analyzing the functional mechanism and expression regulation of LgPAL in L.gmelinii,as well as the development of wood formation genetic resources of L.gmelinii and application in the genetic improvement of crops and forest trees.