Cloning And Preliminary Functional Analysis Of Pectin Methylesterases Gene GhPME6in Cotton

Cotton is an important economy crop in the world, and is the major source of natural fiber for textile industry. The development process of cotton fiber can be divided into four periods:Fiber cell initiation, elongation, secondary wall thickening and maturity. The secondary wall thickening stage among four developmental periods of cotton fiber has an important relationship with fiber strength formation. However, Most of current studies were focused on cotton fiber initiation and elongation stage, studies on cotton fiber secondary wall thickening stage are still in-depth. Therefore, further study on the genetic mechanism of secondary wall thickening of cotton fiber and cloning of the key genes that controlling cotton fiber strength would have an important contribution not only in elucidating the molecular mechanisms but also for future genetic improvement of fiber strength.Pectin methylesterases (PMEs) are functionally associated with cell wall structural changes and are common in plants, which could catalyze pectin in the cell wall to produce pectin hydrolysate acid and methanol with a mass of free carboxyl. The free carboxyl combined with the Ca2+in cell walls will form a gel, which can increase the stiffness and tenacity of the cell wall. In this study, the upland cotton genetic standard line TM-1was selected as materials. We preliminary revealed the role of GhPME6during the process of cotton fiber development by combination of gene cloning, phylogenetic analysis, Real-time fluorescence quantitative expression analysis, prokaryotic expression and transgenic Arabidopsis. The results of this study are as follows:1、An EST sequence that preferentially expressed during fiber secondary wall thickening was obtained using cotton genome sequece data and subtractive hybridization cDNA library. Bioinformatic analysis showed that the EST sequence encodes partial pectin methylesterase protein sequence.2、The full-length cDNA sequence was successfully cloned from upland cotton TM-1, named GhPME6.3、Comparative genomics analysis using different Gossypium genomes showed that PME6gene was highly conserved during Gossypium evolution process.4、qRT-PCR analysis revealed that GhPME6was highly expressed during the stage of secondary cell wall thickening of fiber development.5、Constructing prokaryotic expression vector pET30a-GhPME6, and inducing GhPME6expression in BL21strain. SDS-PAGE electrophoresis indicated that the molecular weight of GhPME6is about57KDa, which consistented with expected size of target protein. Western blotting detection using anti-His antibody further verified the prokaryotic expression protein His-GhPME6.6、Constructed GhPME6gene plant expression vector of pBI121-GhPME6, and successfully transformated into Arabidopsis thaliana (Ecotype columbia). T1generation of transgenic Arabidopsis positive seedlings were achieved by PCR amplification and Kanamycin resistance screening.