草酸青霉菌I1的cDNA文库构建及其溶磷相关基因的筛选

doi:10.3864/j.issn.0578-1752.2012.18.012

Abstract

Abstract: 【Objective】The objective of this study is to obtain phosphate-dissolving related genes from cDNA library of Penicillium oxalicum I1. 【Method】 A primary cDNA library of P. oxalicum I1 was constructed using SMART (switching mechanism at 5′ end of RNA transcript) technique. Transformants with phosphate-dissolving activities were screened on the insoluble phosphate medium, and the sequence of the bioinformation was analysed. To study the phosphate-dissolving mechanisms of the cloned gene, the changes of the pH value, the soluble phosphate content and the production of organic acids were analyzed in the insoluble phosphate liquid medium inoculated with the transformants harboring the phosphate-dissolving gene. 【Result】A cDNA library of P. oxalicum I1 was successfully constructed. Titer tests showed that the content of constructed P. oxalicum I1 cDNA library reached 5.29×106 cfu•mL-1, in which the percentage of recombinants was 99%. Forty-eight transformants with phosphate-dissolving activities were screened on the solid medium with insoluble phosphate. The corresponding gene in one of these transformants was identified. The full length cDNA of transformant I-4 was 536 bp, encoding a predicted protein with 129 amino acid residues. The expression of phosphate-dissolving gene in E. coli could enhance organic acids secretion and improve the phosphate solubilizing activity. It was found that acetic acid was secreted in 12 h, and lactic acid, malic acid and α-ketoglutarate were secreted in 24 h. The transformant I-4 decreased the pH value of medium from 6.32 to 3.69 and released soluble phosphate up to 0.1076 mg•mL-1 in 36 h. 【Conclusion】A phosphate-dissolving related gene, designated pstI, was screened from the cDNA library of P. oxalicum I1.

Key words: Penicillium oxalicum, cDNA library, phosphate-solubilizing related gene, organic acid

TANG Chao-Xi, GONG Ming-Bo, LI Shun-Peng, ZHU Chang-Xiong. Construction of cDNA Library of Penicillium oxalicum I1 and Screening of Phosphate-Dissolving Related Gene[J].Scientia Agricultura Sinica, 2012, 45(18): 3792-3800.

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Construction of cDNA Library of Penicillium oxalicum I1 and Screening of Phosphate-Dissolving Related Gene

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