紫花苜蓿β-淀粉酶基因家族成员鉴定及其在盐碱胁迫下的表达分析

Identification and expression analysis of β-amylase gene family members in alfalfa under saline-alkali stress

摘要:

β-淀粉酶(beta-amylase,BAM)作为水解淀粉的关键酶，在植物生长发育和抵抗非生物胁迫方面发挥着重要作用。为了挖掘紫花苜蓿(Medicago sativa L.)抗盐碱相关的BAM基因，本研究对紫花苜蓿BAM基因进行全基因组鉴定，对BAM基因家族成员的理化性质、系统进化、基因结构、保守基序、二级结构、启动子顺式作用元件、染色体定位和基因复制关系进行分析，利用转录组和实时荧光定量PCR (quantitative real-time PCR,qRT-PCR)分析BAM家族在盐碱胁迫下的表达模式。结果表明，在紫花苜蓿基因组中鉴定出54个BAM基因，根据系统进化树将其分为8个亚族，除了第1、7亚族成员之间差异较大外，其余同一亚族成员之间具有相似的基因结构。保守基序分析显示，所有MsBAM蛋白均具有典型的糖基水解结构域。染色体定位分析表明，β-淀粉酶基因家族成员不均匀地分布在27条染色体上。基因的片段重复导致了紫花苜蓿BAM基因数目增多。BAM基因启动子中含有大量与植物激素和抗逆性相关的响应元件。转录组和qRT-PCR分析表明，大部分MsBAM基因响应盐碱胁迫，MsBAM6等28个基因在盐碱胁迫1 d和7 d时均上调表达，MsBAM9等5个基因上调2倍以上。另外，在盐碱胁迫下，紫花苜蓿中的β-淀粉酶活性显著增强，可溶性糖含量显著增加，表明BAM基因在紫花苜蓿抵御盐碱胁迫方面具有重要的作用。以上结果为进一步研究紫花苜蓿BAM基因抵抗盐碱胁迫的功能奠定了基础。

关键词:紫花苜蓿;β-淀粉酶;基因家族;盐碱胁迫

Abstract:

Beta-amylases (BAMs), key enzymes in starch hydrolysis, play an important role in plant growth, development, and resistance to abiotic stress. To mine the saline-alkali tolerance-related BAM genes in alfalfa (Medicago sativa L.), we identified MsBAM genes in the whole genome. The physicochemical properties, phylogeny, gene structures, conserved motifs, secondary structures, promoter cis-acting elements, chromosome localization, and gene replication relationships of BAM gene family members were analyzed. RNA-seq and quantitative real-time PCR (qRT-PCR) were employed to analyze the expression patterns of BAM family members under saline-alkali stress. The results showed that 54 BAM genes were identified in the genome, which were classified into 8 subgroups according to the phylogenetic tree. The members of the same subgroup had similar gene structures except that those of subgroups 1 and 7 had large differences. Conserved motif analysis showed that all MsBAM proteins had a typical glycohydrolysis domain. The chromosome localization analysis showed that MsBAM gene family members were unevenly distributed on 27 chromosomes. The duplication of gene segments led to the increase in BAM gene number in alfalfa. The promoters of BAM genes contained a large number of elements in response to plant hormones and stress. Transcriptome data and qRT-PCR results showed that the expression levels of most MsBAM genes were up-regulated in response to saline-alkali stress. Under the saline-alkali stress, the expression levels of 28 genes, including MsBAM6, were up-regulated on days 1 and 7, and those of 5 genes, including MsBAM9, were up-regulated by over 2 folds. In addition, under salt-alkali stress, BAM activity and soluble sugar content were significantly increased. These results indicate that BAM genes play a key role in alfalfa in response to saline-alkali stress, laying a foundation for further research in this field.

Key words:alfalfa (Medicago sativaL.);β-amylase;gene family;saline-alkali stress