Neolamarckia Cadambalignocellulose Related Gene Co-expression Network Analysis And Function Identification

As an important fast-growing tree species in tropical and sub-tropicalregions of China,Neolamarckia cadamba is of importance to Agricultural industries.In this study,RNA-Seq was performed to investigate differentially expressed genes(DEGs)involved in the xylem development of N.cadamba.DEGs during secondary growth were analyzed and predicted with bioinformatic tools,and the functions of some lignin biosynthetic genes were verified through molecular complementation tests of A.thaliana mutants.A protocol for predicting novel genes involved in abiological process or a pathway was developed by constructing gene co-expressionnetworks.The main results of this study are as follows:1.Total RNA from the apical,middle and basal parts of a one year old N.cadamba stem and a mixed sample of the stem,leaves,roots,cambium and seedlings were used for cDNA library construction for high-throughput RNA sequencing usingIllumina Hi-seq2000.Totally,65,147 unigenes were obtained after denovo assembly,and 10,736 of them were differentially expressed(DEGs)amongdifferent tissues.The GO enrichment analysis shows that some of these DEGs were significantly enriched in the processes of phenylpropanoid metabolism,lignin metabolism,phenylpropanoid biosynthesis,polysaccharide biosynthesis,carbohydrate biosynthesis,cellulose metabolism,and beta-glucan metabolism that are all involved insecondary growth in plants.2.The newly identified DEGs were used for blastx analysis against A.thaliana protein data base with an e-value of 1e-10.DEG sequences will be annotatedthrough this blastx analysis.A gene expression matrix was constructed using the Arabidopsis thaliana GEO database,co-expression network construction and clustering of these DEGs were completed with triple link and WGCNA.Results shows that 31 out of the 55 genes in Triple Link de-composed cluster 1 are well studied fortheir functions in secondary growth.The brown module obtained through WGCNA analysis contains 338 genes,and GO analysis indicates that majority of these genes belong to secondary metabolism related pathways.Thirteen of the 20 hub genes in this module were known to be involved in secondary growth.Therefore,we speculate that rest 24 genes in cluster 1 and the 7 hub-genes in Brown module might be novel genes which also participate in secondary growth.3.According to the analysis of transcriptome data,CSE and HCT were selected to be transformed to A.thaliana mutant cse and wild-type A.thaliana for functional analysis.Results showed that Arabidopsiscse mutant was successfully complemented by expressing NcCSE with fully restored phenotype similar to WT.But the phenotype of cse is only partially restored by NcHCT,suggesting that NcCSE has a similar function to that of AtCSE.The partial complementation of cse with NcHCT indicated that NcHCT may share partial function with NcCSE.It is likely thatboth genes invovled in different pathways that partially shared some key gene components.4.Based on the expression data of A.thaliana collected from public databases,genes that may participate in lignocellulose biosynthesis were predicted according to some well known lignocellulose biosynthesis genes.The result indicated about 50%of the predicted genes have been documented to participate in lignocellulose biosynthesis or regulation,the rest 50% are novel genes that may potentially participateinthis process.