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Acta Agron Sin ›› 2016, Vol. 42 ›› Issue (02): 199-200.doi: 10.3724/SP.J.1006.2016.00190

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Functional Analysis of Polygalacturonase Genes from Ciboria shiraiana

LI Meng-Jiao,LÜ Rui-Hua,YU Jian,CAI Yu-Xiang,WANG Chuan-Hong,ZHAO Ai-Chun,LU Cheng,YU Mao-De*   

  1. College of Biotechnology, Southwest University, Chongqing 400715, China
  • Received:2015-06-11 Revised:2015-09-06 Online:2016-02-12 Published:2015-10-08
  • Contact: 余茂德, E-mail: yumd@163.com, Tel: 023-68250191
  • Supported by:

    This study was supported by China Special Fund for Agro-scientific Research in the Public Interest (201403064), Fundamental Research Funds for the Central Universities (XDJK2014D016), the Modern Agro-industry Technology Research System (CARS-22), and the Natural Science Foundation of China (31360190).

Abstract:

Polygalactuionase (PG) is a kind of cell wall structural proteins that can catalyes the decompocition of alpha- (1,4)-polymer of galacturonia acid , which makes the cell wall structure disintegration and fruit softening. In the paper, we analyzed the expression patterns of PG genes in the leaf infection by the hypha of C. shiraiana at different growth stages by using qRT-PCR methods. The results showed that the cDNA of CsPG1 gene was amplified from the sclerotia of C. shiraiana by RT-PCR, namely CsPG1 (GenBank accession number: KR296662) with 1143 bp of full length, encoding 380 amino acid residues. On the basis of the highest level of gene expression in the process of infecting rape leaf, the main gene of the CsPG gene family was confirmed. CsPG1 was cloned into pET-28a(+) vector and expressed in E. coli BL21 (DE3). The recombinant CsPG1 protein was expressed in the form of inclusion bodies without activity towards polygalaturonic acid. The optimal urea concentration for dissolving CsPG1 inclusion bodies was 6 mol L–1, CsPG1was renaturated by dilution gradiently at low temperature. We sorted out single band after purified by High-Affinity Ni-NTA Resin, and obtained soluble protein. The specific activity of renatured CsPG1 was 5.02 U mg–1. The result of biological test showed that the recombinant protein accelerated the fruit maturity and softening of Jialing 40 (Morus atropurpurea Roxb.). So we speculated that the CsPG1 protein is related to the infection of C. shiraiana to mulberry fruits. The result reveals the sensitivity difference among mulberry varieties to sclerotial disease, providing the molecular evidence for preventing and controlling the sclerotial disease in mulberry cultivation.

Key words: Mulberry, Ciboria shiraiana, CsPG1, Protein expression activity

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