Cloning and Functional Analysis of Pectin methylesterases Gene GhPME6 in Upland Cotton

Functional Genomics

poster

Shang, Haihong; Wang, Lin; Li, Junwen; Wang, Shaogan; Yuan, Youlu

Cloning and Functional Analysis of Pectin methylesterases Gene GhPME6 in Upland Cotton Wang Lin，Shang Hai-Hong，Li Jun-Wen， Wang Shao-Gan，Liu Ai-Ying，Shi Yu-Zhen，Gong Ju-Wu，Gong Wan-Kui，Yuan You-Lu* (State Key Laboratory of Cotton Biology, Institute of Cotton Research of CAAS, Anyang, 455000) Pectin Methylesterase (PME) is common in plants which has some connection with the change of the cell wall’s structures. Pectin methylesterases can catalyze the demethylesterification of pectin in cell wall releasing acidic pectins and methanol. The free carboxyl can interact with bivalent ions (Ca2+) , so ridigifying the cell wall. Based on the bioinformatic analysis of the ESTs from the cotton fiber SSH-cDNA library about the secondary wall thickening, we obtained a cDNA sequence, which has the homolog sequence of Pectin methylesterases, and it was designated as GhPME6. The full length of cDNA was amplified by RACE from upland cotton TM-1. And the characteristics of this sequence was analyzed by bioinformatic softwares. The analysis of gene structure indicate that GhPME6 has a ORF which length is 150bp and encodes a polypeptide of 519 amino acid. And GhPME6 were composed of two exons and one intron in comparison of their sequences of genomic DNA and cDNA by DNAMAN. The deduced amino acids showed high identity with the Pectin methylesterases by multiple alignments analysis. The deduced amino acid sequence contains typical structures of PMEI(pectin methylesterase inhibitor) and PME(pectin methylesterase) by using Hmmsearch. PME6 is highly conserved during evolution of the Gossypium’s genome through the analysis of comparing the PME6 in G. raimondii, G. arboreum, G. hirsutum and G. barbadense. The tissue expression analysis by using quantitative RT-PCR revealed that GhPME6 expressed predominantly during the stage of secondary cell wall thickening of fiber development. The result suggested GhPME6 might may play an important role in the cotton fiber strength forming. By using E. coli expression system and SDS-PAGE assay, the result show the molecular weight of the prokaryotic expressed protein is 57 KDa. It is same as expected and can lay the foundation for the deeper study of the PME6’s action during the stage of the cotton fiber strength forming.