甜菜BvPPase基因克隆及表达模式分析

Isolation and expression pattern analysis of BvPPase in Beta vulgaris

本研究采用同源克隆技术分离了高糖型甜菜(Beta vulgaris)‘BS02’液泡膜H+焦磷酸化酶(H+-PPase)基因,命名为BvPPase。该基因包含2 289 bp的开放阅读框,编码762个氨基酸,蛋白分子质量为80.11 kDa,理论等电点为5.25。结构预测表明该蛋白二级结构主要以α螺旋和无规则卷曲为主,包含14个跨膜结构域,同时含有VPPase和HPPase超家族保守结构域。系统进化分析表明,该蛋白属于Ⅰ型H+-PPase家族成员,且与盐角草(Salicornia europaea)、藜麦(Chenopodium quinoa)、菠菜(Spinacia oleracea)等多种藜科植物H+-PPase聚为一类。组织表达模式分析结果显示:经400和600 mmol·L^-1氯化钠(NaCl)、200 mmol·L^-1氯化钾(KCl)灌根处理以及20mmol·L^-1脱落酸(ABA)叶面喷施处理后,该基因的表达量在叶和根中分别达到峰值;此外,在NaCl和ABA处理下,该基因在根中的表达量明显高于其在叶中表达量,而在KCl处理下,正好与之相反,表明该基因能够迅速响应NaCl、KCl和ABA处理,且不同胁迫下组织表达模式的差异暗示该基因可能在响应不同胁迫时发挥着不同的作用。这些结果将为探索甜菜耐盐分子机理及高糖型甜菜耐盐性遗传改良提供科学依据。

In the present study, the vacuole-type H+-pyrophosphatase(H+-PPase) gene was isolated by homologous cloning technology from high-sucrose Beta vulgaris ’BS02’, referred as BvPPase, which contains an open reading frame(ORF) of 2 289 bp, encoding 762 amino acids. The predicted protein molecular weight is 80.11 kDa, and the theoretical isoelectric point is 5.25. Protein structure prediction shows that the secondary structure is mainly α-helix and random coil, with 14 transmembrane domains, and had VPPase and HPPase superfamily conserved domain. Phylogenetic analysis shows that BvPPase belongs to type Ⅰ H+-PPase family, and grouped with various H+-PPases of Chenopodiaceae plants, such as Salicornia europaea, Chenopodium quinoa, and Spinacia oleracea. After root irrigation with 400 or 600 mmol·L-1 NaCl and 200 mmmol·L-1 KCl, and foliar spray with 20 mmmol·L-1 ABA, BvPPase gene expression reached the peaks in leaves and roots, respectively. Additionally, the expression of BvPPase in roots significantly higher than that in leaves after NaCl and ABA treatment, which was the opposite after KCl treatment, indicating that BvPPase gene expression can be induced by NaCl, KCl and ABA, but may play different roles in leaves than in roots in response to abiotic stresses. These results would provide a scientific basis for the study of salt tolerance molecular mechanism and genetic improvement of salt tolerance in high-sucrose B. vulgaris.