《植物生理学报》 2017, 53(2): 198-208

基于家族分析的苜蓿逆境应答Ca²⁺ ATPase家族基因筛选与鉴定

张美萍, 杨珺凯, 孙明哲, 贾博为, 孙晓丽^*

黑龙江八一农垦大学, 作物逆境分子生物学实验室, 黑龙江大庆163319

通信作者：孙晓丽；E-mail: csmbl2016@126.com

摘　要：

Ca²⁺ ATPase属于P型ATPase超家族, 在植物对外界逆境的应答中发挥重要作用, 但迄今尚未有苜蓿Ca²⁺ ATPase基因研究的相关报道。为筛选逆境胁迫应答Ca²⁺ ATPases, 本研究鉴定获得了16个苜蓿Ca²⁺ ATPase家族成员, 分布在7条染色体上, 且存在一对旁系同源基因。系统进化分析表明苜蓿Ca²⁺ ATPases分为ACA和ECA亚家族, ACA亚家族分为4组, 且组内成员具有相似的内含子-外显子组成模式。此外, 苜蓿Ca²⁺ ATPase家族具有高度保守的蛋白序列组成, 包括4个功能结构域、10个跨膜结构域和11个保守motif。转录组数据分析发现该家族具有不同的逆境诱导表达模式, 且多数苜蓿Ca²⁺ ATPases家族基因受低温和冷冻胁迫诱导表达, 其中MtACA9和MtACA8上调表达最明显。qRT-PCR结果证实MtACA9和MtACA8表达在低温和冷冻胁迫下显著上调。本研究结果为揭示苜蓿Ca²⁺ ATPase基因在低温冷害应答中的功能提供了重要参考。

关键词：苜蓿; Ca²⁺ ATPase; 家族分析; 逆境应答; 表达分析

收稿：2016-10-08   修定：2016-12-28

资助：国家自然科学基金(31500204)和黑龙江省自然科学基金(C2015035)。

Screening and identification of environmental stress responsive Medicago sativa Ca²⁺ ATPases based on gene family analyses

ZHANG Mei-Ping, YANG Jun-Kai, SUN Ming-Zhe, JIA Bo-Wei, SUN Xiao-Li^*

Crop Stress Molecular Biology Laboratory, Heilongjiang Bayi Agricultural Universtiy, Daqing, Heilongjiang 163319, China

Corresponding author: SUN Xiao-Li; E-mail: csmbl2016@126.com

Abstract:

It is widely accepted that the Ca²⁺ ATPase gene family, belonging to the P-type ATPase superfamily, plays an important role in environmental stress responses. However, up to now, no researches reported the function of alfalfa Ca²⁺ ATPases. Hence, to screening the stress responsive Ca²⁺ ATPases, we used the Hidden Markov Model to search the alfalfa proteome database, and identified a total of 16 Ca²⁺ ATPase family genes in alfalfa genome. Chromosomal location and synteny analysis revealed that alfalfa Ca²⁺ ATPase family members located on 7 chromosomes, and possessed 1 pairs of duplicated genes. By analyzing the topological structure of the phylogenetic tree, we found that alfalfa Ca²⁺ ATPase family was clustered into ACA and ECA subfamilies. The ACA subfamily was further divided into four groups (Group I–IV), and members in each group displayed high similarity in terms of intron–exon organization pattern. Furthermore, we also suggested that alfalfa Ca²⁺ ATPase family protein sequences were highly conserved, including 4 conserved functional domains, 10 transmenbrane domains, and 11 conserved motifs. By using the RNA-seq data, we also investigated the expression profiles of alfalfa Ca²⁺ ATPase genes under different environmental stresses, and found that the expression of alfalfa Ca²⁺ ATPase genes, especially MtACA8 and MtACA9, was induced by cold and freezing stresses. By using quantitative real-time PCR analysis, we further confirmed that MtACA8 and MtACA9 expression was up-regulated by both cold and freezing stresses. Taken together, results presented in this study will facilitate further research about the biological function of alfalfa Ca²⁺ ATPases in plant responses to environmental stress.

Key words: alfalfa; Ca²⁺ ATPase; gene family analysis; stress responses; expression analyses