Screening And Functional Identification Of 4CL Gene Family In Mulberry

Lignin is widely present in plants,and is a class of aromatic polymers formed by oxidizing polymerization.It has important functions in terms of plant growth and development,resistance.The biosynthesis of lignin is to start from the phenylene metabolic pathway,in which 4-coumarate-coa ligase(4CL)plays an important regulatory role.As more and more 4CL genes were isolated and identified from plants,4CL genes were found to form homologous family genes.This present study was firstly performed based on the screening of 4CL homologs using online Morus DB and then four 4CL genes named Mm4CL2,Mm4 CL 3,Mm4 CL 5 and Mm4 CL 6 were cloned and functionally identified in Morus multicaulis.The main research results are as follows:1.4CL gene family members named Mn4CL1 to Mn4CL6 were screened using HMMER Search against online Morus DB with validated 4CLs from different plants.The putative proteins of Mn4 CLs were aligned and the conservative Box I(SSGTTGLPKGV)and Box II(GEICIRG)domain were detected.Systematic analysis showed that Mn4CL1,Mn4CL2,and Mn4CL4 belong to Class I,mainly involved in lignin biosynthesis.Mn4CL3 belongs to Class II,mainly involved in biosynthesis of flavonoids.Mn4CL5 and Mn4CL6 are far from other 4CLs with divergent amino acids,belonging to 4CL like proteins.2.According to the CDS sequences of Mn4 CL genes obtained from mulberry,specific primers were designed and homologous cloning was carried out in the Morus multicaulis Hu Sang 32.Four mulberry 4CL genes named Mm4CL2、Mm4CL3、Mm4CL5 and Mm4CL6 were cloned with complete open reading frame length between 1644 and 1779 bp.The putative physical and chemical properties of the protein sequences showed that the length of the protein was between 539 and 592 aa,the molecular weight was between 59.73 and64.50 k D,and the equidistant was between 6.03 and 9.25.3.qRT-PCR was used to analyze the expression profile of 4CL genes in different tissues and organs(leaves,tender buds,stems,bark,flowers)in mulberry.Mm4CL2,Mm4CL3,Mm4CL5,Mm4CL6 were expressed in leaves,tender buds,stems,bark and flowers to different degrees.The expression levels of Mm4CL2 and Mm4CL3 were higher in tender bud,stem,bark and flower,but lower in leaf.The Mm4CL5 has a higher expression in the leaves and stems,and the expression of Mm4CL6 is high in bark and flowers.The expression of Mm4CL5 in the leaves is the highest,and Mm4CL6 has the lowest expression in the stem.4.Recominant plasmids p ET28a-Mm4CL2/Mm4CL3/Mm4CL5/Mm4CL6 were successfully constructed.The His-Tagged Mm4CL2/Mm4CL3/Mm4CL5/Mm4CL6 were purified and analyzed in vitro respectively.The Knobloch method was used to analyze the enzymatic activity of the purified recombinant protein in vitro.Enzymatic assay showed that the Mm4CL2 protein can catalyze the p-cinnamic acid,caffeic acid,and ferulic acid to generate corresponding cinnamyl Co A respectively,but could not catalyze sinapic acid.The Mm4CL3 showed substrate specificity for p-cinnamic acid and hardly utilized other hydroxycinnamic acids which further suggested its possible role in flavonoid biosynthesis.In this study,the primary function of Mm4CL2 and Mm4CL3 was successfully identified.Our results provided evidence to understand the roles of 4CL gene family in the process of lignin biosynthesis.