Molecular Characterization Of Genes Involved In Xylan Biosynthesis In Cotton Fiber

Cotton is a very important crop not only for natural fibers but also for oil production.The fiber is an ideal research model for plant cell wall biosynthesis and cell expansion. Mature fiber contains over90%of cellulose, small amounts of hemicellulose, pectin and protein etc. Role of cell wall components especially the hemicellulose constituent that play in the fiber development is obscure.How alteration of hemicellulose affect fiber development and the cellulose-hemisellulose network remains largely unknown.In this thesis, based on reported proteins involved in hemicellulose (xylan) biosynthesis in Arabidopsis and Poplar, a bioinformatics approach was applied to identify20candidates genes putatively implicated in hemicellulose synthesis from cotton EST libraries. A thorough in silico analysis of coded proteins was performed using a large number of programs. Furthermore, two candidate genes encoding proteins involved in xylan backbone synthsis and reducing end synthesis respectively were selected for functional characterization, the main results are as follows:1. Identification of candidate genes involved in xylan biosynthesis by a bioinformatics approach and functional prediction via programsBased on previous researches in Arabidopsis and Poplar and the cotton D-genome sequences, a bioinformatics method was performed to identify20candidate genes encoding proteins involved in xylan biosynthesis from cotton EST libraries.Full-length cDNA sequences were obtained by PCR amplification.Sequence analyses including multiple sequence alignment, transmembrane domain prediction, signal peptide analysis, protein localization prediction,3D structure analysis, conserved domain analysis and phylogenetic analysis etc. showed that the deduced proteins were members of different glycosyltransferase families.Among them, there were two members of GT47family, eight members of GT8family, five ESK family members, four members of GXM family and one gene belonged to RWA family. Most of them were localized in Golgi apparatus and belonged to type II membrane proteins.They all contained conserved domains related to xylan biosynthesis.The above analyses demonstrate that the identified candidate genes may participate in xylan biosynthesis.2. Expression of the candidate genes in cotton tissuesTo investigate if these genes are expressed in fibers,RNAs were isolated from various cotton organs and fibers from different developmental stages and reverse transcribed into cDNAs.Semi quantitative RT-PCR and realtime RT-PCR techniques were employed to study the expression of these genes. The results revealed that these candidate genes were all transcribed in developing fibers, some genes preferentially expressed in secondary wall thickening stage.For example, GhRWA was specially expressed in20DPA fiber. High level mRNA transcripts of GhGT47A and GhGT47B were detected in15DPA and20DPA fibers.It can be speculated that these genes represent good candidates involved in fiber secondary wall xylan synthesis.3. Functional characterization of GhGT47BTo verify the function of these genes in xylan biosynthesis, two genes whose mRNAs were abundant in secondary cell wall stage of fiber were chosen for functional analysis, The first gene, designated GhGT47B, may be involved in xylan backbone synthesis;the second gene, named GhGT47A, may be involved in reducing end synthesis. Sequence alignment have showed that GhGT47B shares high amino acid sequence identity with Arabidopsis IRX10which has been proved to be involved in synthesis of xylan backbone.Stems of irx10mutant displayed the typical irregular xylem phenotype and contained less xylose. A GhGT47B complementation vector was constructed and then introduced into irx10mutant. Stem cross sections of GhGT478-complemented plants, showed that GhGT47B could partially rescue the irx10mutant phenotype. The results above imply that GhGT47B is a functional ortholog of Arabidopsis IRX10with divergent function.4. Generation of transgenic cottonTo further investigate the roles of GhGT47B and GhGT47A in fiber development. Overexpression and RNA-interference vectors of GhGT47A and GhGT47B were constructed and transformed into cotton.Up to date, For GhGT47A overexpression, five transgenic lines were obtained, for GhGT47A gene silencing, two transgenic lines were produced. Phenotypic analyses await further studies.