摘要：

为深入研究CIPK基因家族在应对非生物胁迫应答等过程中的分子机制,探究非生物胁迫下草地早熟禾CIPK32基因的作用。以草地早熟禾午夜Ⅱ号为试验材料,应用RT-PCR技术克隆CIPK32基因,分析该基因在不同组织部位及不同非生物胁迫条件下的表达规律。并通过NCBI-CDD、SWISS-MODEL等在线软件对编码氨基酸序列进行蛋白结构、同源比对等生物信息学分析。结果表明:草地早熟禾CIPK32基因序列ORF为1 320 bp,共编码439个氨基酸。预测CIPK32蛋白相对分子质量为50.28 ku,等电点6.82,蛋白质分子式为C₂₂₄₉H₃₅₇₄N₆₀₈O₆₆₅S₁₆,属于不稳定亲水性蛋白。草地早熟禾CIPK32含有典型STKc_SnRK3和CIPK_C结构域,与山羊草(XP_020198391.1)、黑麦草(XP_047091407.1)、大麦(XP_044980943.1)编码氨基酸同源性较高,并含有酰胺化位置、N-糖基化位点、N-肉豆蔻酰化位点等多个结合位点。草地早熟禾CIPK32基因的表达水平呈现组织特异性,叶部>茎部>根部。干旱胁迫下该基因呈现先上调后下降的趋势,10% PEG6000处理16 h为最高值,是0 h的6.2倍;随着NaCl浓度的升高显著促进该基因的表达,200 mmol/L NaCl是0 mmol/L NaCl的6.9倍;低浓度NaNO₃及低浓度KH₂PO₄(0.1 mmol/L KH₂PO₄)环境均可促进CIPK32基因的表达。综上所述,草地早熟禾CIPK32基因具有组织特异性,干旱、盐、氮素和磷素胁迫均能诱导CIPK32基因的表达。CIPK基因家族在非生物胁迫中具有潜在的作用。

关键词: 草地早熟禾, 蛋白激酶, CIPK32, 基因克隆, 表达分析

Abstract:

In order to further study the molecular mechanism of CIPK gene family in response to abiotic stress and explore the role of CIPK32 gene in Poa pratensis L. under abiotic stress. The CIPK32 gene was cloned from Poa pratensis L. midnight Ⅱ by RT-PCR, and its expression in different tissues and under different abiotic stress conditions was analyzed. On-line softwares such as NCBI-CDD and SWISS-MODEL were used to analyze the protein structure, homology alignment and other bioinformatics. The results showed that the sequence ORF of CIPK32 gene of Poa pratensis L. was 1 320 bp, encoding 439 amino acids. It is predicted that the relative molecular weight of CIPK32 protein was 50.28 ku, the isoelectric point was 6.82, and the protein molecular formula was C₂₂₄₉H₃₅₇₄N₆₀₈O₆₆₅S₁₆, which belonged to unstable hydrophilic protein. CIPK32 of Poa pratensis L. contained typical STKc_SnRK3 and CIPK_C domains, and had high homology with amino acids encoded by Aegilops tauschii (XP_020198391.1), Lolium rigidum(XP_047091407.1) and Hordeum vulgare(XP_044980943.1). It also contained many binding sites such as Amidation site, N-glycosylation site, N-myristoylation site and so on. The expression level of CIPK32 gene in Poa pratensis L. was tissue-specific, and the order was leaf>stem>root. Under drought stress, the gene was up-regulated first and then decreased, and the highest value was reached when 10% PEG6000 was treated for 16 h, which was 6.2 times that of 0 h; with the increase of NaCl concentration, the expression of the gene was significantly promoted, and 200 mmol/L NaCl was 6.9 times that of 0 mmol/L NaCl. The expression of CIPK32 gene could be promoted by low concentration of NaNO₃ and low concentration of KH₂PO₄(0.1 mmol/L KH₂PO₄). To sum up, the CIPK32 gene of Poa pratensis L. has tissue specificity, and the expression of CIPK32 gene can be induced by drought, salt, nitrogen and phosphorus stress. CIPK gene family plays a potential role in abiotic stress.

Key words: Poa pratensis L., Protein kinase, CIPK32, Gene cloning, Expression analysis