草酸青霉菌I1的cDNA文库构建及其溶磷相关基因的筛选

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

【目的】构建草酸青霉菌I1的cDNA文库,筛选溶磷相关基因。【方法】利用SMART技术构建草酸青霉菌I1的初级cDNA文库,通过难溶磷培养基筛选具有溶磷能力的转化子,测序并进行生物信息学分析。在难溶磷液体培养基中,进行转化子对溶液pH值、可溶磷含量的影响和产有机酸试验。【结果】成功构建了草酸青霉菌I1的初级cDNA文库,其库容量约为5.29×106cfu.mL-1,重组率为99%;利用难溶磷固体培养基筛选,得到具有溶磷圈的转化子48个,其中转化子I-4的cDNA序列全长536 bp,为一个新的序列,基因编码氨基酸残基序列长129 n.t。转化子E.coli HST08 I-4在液体难溶磷培养基中培养,提高了有机酸的表达量,并增加了有机酸的种类,在培养12 h后,开始产生乙酸,24 h后,溶液中产生乳酸、苹果酸和α-酮戊二酸,培养36 h,溶液pH值由6.32降到3.69,可溶磷含量达到0.1076 mg.mL-1。【结论】从草酸青霉I1中筛选到一个溶磷相关基因pstI。

[Objective] The objective of this study is to obtain phosphate-dissolving related genes from eDNA library of Penicillium oxalicum I1. [Method] A primary eDNA 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 eDNA library reached 5.29×10^6 cfu.mLl, 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 eDNA of transformant 1-4 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 a-ketoglutarate were secreted in 24 h. The transformant 1-4 decreased the pH value of medium from 6.32 to 3.69 and released soluble phosphate up to 0.1076 mg.mLl in 36 h. [Conclusion]A phosphate-dissolving related gene, designatedpstl, was screened from the eDNA library of P. oxalicum I1.