黑曲霉H1的cDNA文库构建及其溶磷相关基因的筛选

Construction of cDNA library of Aspergillus niger H1 and screening of phosphate-dissolving related gene

【目的】构建溶磷黑曲霉H1的cDNA文库,并从中筛选溶磷相关基因。【方法】利用SMART技术合成黑曲霉H1的双链cDNA并将其连接于pBluescript II SK(+)载体上,将重组质粒转化E.coli HST08,得到黑曲霉的初级cDNA文库。利用难溶磷培养基筛选具有溶磷能力的克隆子,测序并利用Blast分析基因序列。在难溶磷液体培养基中,进行克隆子对溶液pH值、可溶磷含量的影响和产有机酸实验。【结果】成功构建了黑曲霉H1的cDNA文库,其初级库容量约为5.65×106cfu/mL,重组率约为99.15%;通过难溶磷固体培养基筛选,得到具有溶磷圈的克隆子61个,其中克隆子H-54的cDNA序列长839 bp,基因编码氨基酸残基序列长179n.t。克隆子E.coli HST08 H-54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,溶液中开始产生甲酸和乙酸,在24 h后,溶液中产生苹果酸和α-酮戊二酸,培养36 h,溶液pH值由6.32降到3.93,可溶磷含量达到0.105 mg/mL。【结论】从黑曲霉H1中获得1个溶磷相关基因,将其命名为psgA。

[Objective]To obtain phosphate-dissolving genes from cDNA library of Aspergillus niger H1.[Methods] The double-stranded cDNA was synthesized using switching mechanism at 5end of RNA transcript technique and ligated to the vector pBluescript II SK（＋）.We transformed recombinant plasmid into E.coli HST08,resulting in a primary cDNA library.We screened clones with phosphate-dissolving activities on the insoluble phosphate medium and blasted the sequence in National Center for Biotechnology Information（NCBI）.To study the phosphate dissolving mechanisms of the cloned gene,we analyzed the changes of the pH value,the soluble phosphate content and the production of organic acids in the insoluble phosphate liquid medium inoculated with the clones harboring the phosphate-dissolving gene.[Results] A cDNA library of A.niger H1 was successfully constructed.Titer tests showed that the content of constructed A.niger H1 cDNA library reached 5.65×106 cfu/mL,in which the percentage of recombinant clones was 99.15%.We screened 61 clones with phosphate-dissolving activities on the solid medium with insoluble phosphate.The corresponding gene in one of these clones was identified.The full length cDNA of clone H-54 was 839 bp,encoding a predicted protein with 180 amino acid residues.The expression of phosphate-dissolving gene in E.coli enhanced organic acids secretion and improved the phosphate solubilizing activity.Formic acid and acetic acid were found in 12 h,and malic acid and α-ketoglutarate were secreted in 24 h.The clone H-54 decreased the pH value of medium from 6.32 to 3.93 and released soluble phosphate up to 0.105 mg/mL in 36 h.[Conclusion] We had obtained a phosphate-dissolving gene designated psgA from Aspergillus niger H1.