华癸中慢生根瘤菌7653R3个脂质转运蛋白基因的克隆、突变及共生表型

Cloning, mutagenesis and symbiotic phenotype of three lipid transfer protein encoding genes from Mesorhizobium huakuii 7653R

摘要:

[目的] 脂类转移家族蛋白基因编码一类参与脂类转运及代谢的蛋白。本研究旨在构建华癸中慢生根瘤菌3个脂质转运家族蛋白基因的突变株，检测及分析突变体与紫云英共生条件下的表型及功能。[方法] 利用生物信息学分析与预测转脂蛋白的结构特征及功能，采用荧光定量技术检测目标基因在自生和共生条件下的表达特性，通过插入突变技术构建目标基因突变株，并进行植物盆栽实验考察其共生表型。[结果] MCHK-5577、MCHK-2172和MCHK-2779基因编码蛋白属于START/RHO alpha_C/PITP/Bet_v1/CoxG/CalC （SRPBCC）超家族，包含脂类转移结构域，参与脂类转运或代谢，与百脉根等中慢生根瘤菌相应基因的序列相似性达95%以上。这3个基因在共生条件下的表达水平都增高。分别构建了MCHK-5577、MCHK-2172和MCHK-2779基因突变菌株，与野生型菌株7653R相比，接种突变株MCHK-2172mut、MCHK-2779mut和MCHK-5577mut后的植株地上部分生物量和根瘤固氮酶活性显著降低。[结论] 华癸中慢生根瘤菌脂质转移家族蛋白基因在共生互作过程中发挥重要作用，突变后明显影响共生固氮表型。本文的实验结果为深入研究脂类转移蛋白在共生固氮作用中的功能机制奠定了基础。

关键词:转脂蛋白基因;华癸中慢生根瘤菌;插入突变;共生表型

Abstract:

[Objective] Lipid transfer protein superfamily is involved in lipid transport and metabolism. This study aimed to construct mutants of three lipid transfer protein encoding genes in Mesorhizobium huakuii 7653R, and to study the phenotypes and function of mutations during symbiosis with Astragalus sinicus.[Methods] We used bioinformatics to predict structure characteristics and biological functions of lipid transfer proteins, and conducted semi-quantitative and fluorescent quantitative real-time PCR to analyze the expression levels of target genes in free-living and symbiotic conditions. Using pK19mob insertion mutagenesis to construct mutants, we carried out pot plant experiments to observe symbiotic phenotypes.[Results] MCHK-5577, MCHK-2172 and MCHK-2779 genes encoding proteins belonged to START/RHO alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) superfamily, involved in lipid transport or metabolism, and were identical to M. loti at 95% level. Gene relative transcription level of the three genes all increased compared to free-living condition. We obtained three mutants. Compared with wild-type 7653R, above-ground biomass of plants and nodulenitrogenase activity induced by the three mutants significantly decreased.[Conclusion] Results indicated that lipid transfer protein encoding genes of Mesorhizobium huakuii 7653R may play important roles in symbiotic nitrogen fixation, and the mutations significantly affected the symbiotic phenotypes. The present work provided a basis to study further symbiotic function mechanism associated with lipid transfer proteins from rhizobia.

Key words:lipid transfer proteins;Mesorhizobium huakuii;insertion mutagenesis;symbiotic phenotypes