甘蔗内生固氮菌Klebsiella variicola DX120E溶磷基因GDH和pqqE的克隆及溶磷特性分析

Cloning of Phosphorus-soluble Gene GDH and pqq E from Sugarcane Endogenous Nitrogen-fixing Bacteria Klebsiella variicola DX120E

葡萄糖脱氢酶(GDH)和吡咯喹啉醌(PQQ)对溶磷微生物溶解无机磷具有重要作用。本研究为了探讨甘蔗内生固氮菌变栖克雷伯菌(Klebsiella variicola)DX120E的溶磷机制,从该菌中克隆溶磷基因GDH和pqqE的ORF,并进行了生物信息学分析,同时还研究了该菌对不同磷源的利用能力。结果表明:克隆得到甘蔗内生固氮菌Klebsiella variicola DX120E GDH和pqqE基因的ORF分别为2373 bp和1143 bp,编码氨基酸分别为790个和380个。生物信息学分析结果表明,GDH蛋白为稳定蛋白,pqqE蛋白为不稳定蛋白。GDH蛋白是一个与细胞信号传导有关的膜受体蛋白,具有PQQ_membr_DH、PQQ_mGDH功能域和PQQ_DH_like超家族蛋白结构;pqqE基因编码的蛋白是胞内蛋白,含有1个PQQ_syn_pqqE功能域和Radical SAM超家族蛋白的结构。内生固氮菌Klebsiella variicola DX120E对不同磷源的利用和GDH和pqqE基因在不同磷源培养时的qRT-PCR分析表明,该菌对不同磷源的溶磷能力表现为FePO_(4)>AlPO_(4)>Ca_(3)(PO_(4))_(2),且溶解FePO_(4)的能力与溶解Ca_(3)(PO_(4))_(2)、AlPO_(4)等难溶磷源的差异均显著(P<0.05)。在几种磷源条件下,GDH和pqqE基因相对表达量均增加,且2个基因的表达量变化趋势一致。GDH和pqqE基因在以FePO_(4)为磷源条件下的表达量与以Ca_(3)(PO_(4))_(2)为磷源的表达量差异显著(P<0.05)。本研究可为进一步研究内生固氮菌与甘蔗的互作和溶磷机制提供参考。

Glucose dehydrogenase(GDH)and pyrroloquinoline quinone(PQQE)play an important role in the dissolution of inorganic phosphorus by phosphorus-soluble microorganisms.The phosphorus-soluble mechanism of the nitrogen-fixing bacteria Klebsiella variicola DX120 E in sugarcane was investigated.The ORF of the phosphorous-soluble gene GDH and pqqE were cloned from the bacterium and analysised by bioinformatics.At the same time,the utilization ability of the bacteria to different phosphorus sources was studied.The ORF of GDH and pqqE was 2373 bp and 1143 bp,the coding amino acids were 790 and 380,respectively.Bioinformatics analysis showed that GDH was a stable protein,but pqqE was an unstable one.GDH was a membrane receptor protein related to cell signaling,with PQQmembrDH,PQQmGDH functional domain and PQQDHlike superfamily protein structure.pqqE encoded proteins called intracellular proteins,structure containing PQQsynpqqE functional domain and Radical SAM superfamily proteins.The utilization of different phosphorus sources and the qRT-PCR analysis of GDH and pqqE in different phosphorus sources showed that under different conditions,the ability of the bacteria to dissolve phosphorus was FePO_(4)>AlPO_(4)>Ca_(3)(PO_(4))_(2) and the ability to dissolve FePO_(4)was significantly different from that of Ca_(3)(PO_(4))_(2) and AlPO_(4)(P<0.05).Under the condition of several phosphorus sources,the relative expression of GDH and pqqE increased,and the expression change trend of the genes was the same.The expression of GDH and pqqE in phosphorus source was significantly different from that in Ca_(3)(PO_(4))2(P<0.05).This study would lay a foundation for further study on the interaction and phosphorus dissolution mechanism between endophytic nitrogen-fixing bacteria and sugarcane.