茶陵野生稻冷响应基因OrCrGHl的表达与克隆

Expression and Cloning of OrCrGHl, a Novel Cold-responsive Gene in Chaling Common Wild Rice

摘要

为了发现新的水稻耐逆基因, 采用水稻基因芯片分析了茶陵野生稻及籼型栽培稻培矮64S苗期全基因组在冷胁迫下的表达水平, 从茶陵野生稻中筛选出一个强烈响应低温的耐冷候选基因, 暂命名为OrCrGHl (Oryza rufipogon cold responsive glucan hydrolase-like)。用实时定量PCR方法对其表达水平进行了进一步的分析, 所得结果与基因芯片分析结果基本吻合。以茶陵野生稻为材料, 用逆转录PCR (reverse transcription PCR, RT-PCR)方法获得了包含其完整开放阅读框(open reading frame, ORF)的cDNA克隆。根据其ORF序列进行预测, 该基因编码一个包含558个氨基酸残基的糖基水解酶相似蛋白质, 其理论分子质量为59.252 kD, pI为5.81。经蛋白质相似性比对, 其编码蛋白与日本晴第7号染色体上的LOC4343492基因编码的蛋白质(XP_015647631.1, glucan endo-1,3-beta-glucosidase GII isoform X2)的氨基酸序列完全一致, 与籼稻93-11预测蛋白质(EEC82204.1, hypothetical protein OsI_26347)的相似性为99.28%。对其可能的启动子区域序列进行分析, 发现了多个可能与逆境或逆境激素响应有关的顺式作用调控元件。以上结果提示, 该基因为一新的野生稻耐冷候选基因, 进一步的研究正在进行。

Abstract

In order to identify novel cold-tolerance genes in rice, genome-wide gene expression profiling of Chaling common wild rice (CWR) and the cultivar indica rice Peiai 64S subjected to cold stress was perfor-med at the seedling stage using the GeneChip Rice Genome Array. A gene, designated OrCrGHl (Oryza rufi-pogon cold responsive glucan hydrolase-like), was screened from Chaling CWR as a cold-tolerance candi-date, which was highly responsive to cold stress in Chaling CWR but not in Peiai 64S. The expression pro-file of OrCrGHl obtained by the microarray analysis was confirmed by real-time quantitative PCR, suggesting that OrCrGHl is a cold-responsive gene in Chaling CWR. Its full open reading frame (ORF)-containing cD-NA clone was obtained by reverse transcription PCR (RT-PCR). Its sequence analysis showed that it encodes a glucan hydrolase-like protein of 558 amino acid residues, with a molecular weight of 59.252 kD and pI value of 5.81. The deduced amino acid sequence was completely identical with that of the glucan endo-1,3-beta-glucosidase GII isoform X2 of japonica rice Nipponbare, and was 99.28% identical with that of hypo-thetical protein OsI_26347 of indica rice 93-11. Analysis of the putative promoter region revealed several cis-acing regulatory elements that may be related to stress or stress hormone responses. The results indicated that OrCrGHl is a novel candidate gene associated with cold tolerance in wild rice. Further study is in progress to understand more about the gene.

关键词

野生稻 / 冷胁迫 / 基因芯片 / 实时定量PCR / 基因克隆

Key words

Oryza rufipogon / cold stress / microarray / real-time quantitative PCR / gene cloning