论文部分内容阅读
【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】 A cDNA library of Aspergillus niger H1 was constructed and the genes related to phosphate solubilization were screened out. 【Method】 The double-stranded cDNA of A. niger H1 was synthesized by SMART technique and ligated into pBluescript II SK (+) vector. The recombinant plasmid was transformed into E. coli HST08 to obtain the primary cDNA library of Aspergillus niger. The clones with ability to dissolve phosphorus were screened with poorly-soluble phosphorous media, sequenced and the gene sequence was analyzed by Blast. In the phosphate-insoluble liquid medium, the effect of the clones on the pH and soluble phosphorus content of the solution and the organic acid production experiment were carried out. 【Result】 The cDNA library of Aspergillus niger H1 was successfully constructed. The initial library capacity was about 5.65 × 106 cfu / mL and the recombination rate was about 99.15%. Clone 61 Among them, the cDNA sequence of clone H-54 was 839 bp in length, and the sequence of amino acid residues coding for the gene was 179 n · t. The clone E. coli HST08 H-54 was cultured in liquid phosphorus-poor medium to increase the expression of organic acids and increase the types of organic acids. After 12 h cultivation, formic acid and acetic acid started to be produced in the solution. After 24 h, malic acid and α-ketoglutaric acid were produced in the solution. After 36 h incubation, the pH value of the solution decreased from 6.32 to 3.93 and the soluble phosphorus content reached 0.105 mg / mL. 【Conclusion】 One soluble phosphorus-related gene was obtained from Aspergillus niger H1 and named as psgA.