小麦钙依赖蛋白激酶基因<i>TaCDPK17</i>的克隆及其抗逆功能初步分析

doi:10.7668/hbnxb.20195063

摘要/Abstract

摘要：

为探究钙依赖蛋白激酶(CDPK)在小麦生长和逆境应答中的作用,从普通小麦中克隆了TaCDPK17基因,并对其进行了序列结构、表达模式和抗逆功能初步分析。结果表明,TaCDPK17基因编码区长1 701 bp,编码566个氨基酸,具有CDPK家族的典型结构特征,包括1个保守的丝氨酸/苏氨酸激酶结构域和4个EF手型结构域。对TaCDPK17与其他12种植物的CDPK17进行进化树分析表明,TaCDPK17与禾本科作物,特别是节节麦、大麦的CDPK17序列有较高同源性。TaCDPK17基因启动子区域富含与激素调控、光照响应,以及非生物应答胁迫相关的顺式调控元件,其中,脱落酸(ABA)响应元件(ABRE)和茉莉酸甲酯响应元件(CGTCA)数量较多。基于实时荧光定量PCR的表达分析结果显示,TaCDPK17受100 μmol/L ABA、100 μmol/L茉莉酸甲酯、20% PEG6000和250 mmol/L NaCl诱导后,表达量有不同程度升高。在2 μmol/L ABA和100 mmol/L NaCl胁迫处理条件下,过表达TaCDPK17的拟南芥种子萌发率显著高于野生型对照。同时,过表达TaCDPK17缓解了ABA或渗透胁迫处理对幼苗根生长的抑制作用。在气孔关闭过程中,过表达TaCDPK17的转基因植物对ABA更敏感,与野生型植物相比,表现出更强的气孔关闭趋势。这些结果表明,TaCDPK17在小麦逆境胁迫和激素信号应答中发挥着重要作用。

关键词: 小麦, 钙依赖蛋白激酶, 基因克隆, 表达分析, 抗逆

Abstract:

To investigate the role of calcium-dependent protein kinase (CDPK) in wheat growth and stress response,the TaCDPK17 gene was cloned from common wheat and its sequence structure,expression pattern,and stress resistance function were preliminarily analyzed.The results showed that the length of the TaCDPK17 gene coding region was 1 701 bp, encoding 566 amino acids and possessing typical structural features of the CDPK family, including one conserved serine/threonine kinase domain and four EF hand shaped domains. Evolutionary tree analysis of TaCDPK17 and CDPK17 from 12 other plants showed that TaCDPK17 had high homology with the CDPK17 sequence of gramineous crops,especially Aegilops tauschii and barley.The promoter region of TaCDPK17 gene contained multiple cis regulatory elements related to hormone signaling pathways,light response.Among them, there are more abscisic acid (ABA) responsive elements (ABRE) and methyl jasmonate responsive elements (CGTCA). The expression analysis based on Real-time Fluorescence Quantitative PCR showed that the expression level of TaCDPK17 increased to varying degrees after induced by 100 μmol/L ABA, 100 μmol/L methyl jasmonate, 20% PEG6000, and 250 mmol/L NaCl. Under stress conditions of 2 μmol/L ABA and 100 mmol/L NaCl, the germination rate of Arabidopsis seeds overexpressing TaCDPK17 was significantly higher than that of the wild type. Meanwhile, overexpression of TaCDPK17 alleviated the inhibitory effects of ABA or osmotic stress treatments on seedling root growth. During stomatal closure, transgenic plants overexpressing TaCDPK17 are more sensitive to ABA and exhibit a stronger stomatal closure trend compared to wild-type plants. These results indicated that TaCDPK17 plays an important role in stress response and hormone signaling in wheat.

Key words: Wheat, Calcium-dependent protein kinase, Gene cloning, Expression analysis, Stress-resistance

甘露, 谢美娟, 卢振华, 李明, 丁博, 邱丽娜, 谢晓东, 王俊斌. 小麦钙依赖蛋白激酶基因TaCDPK17的克隆及其抗逆功能初步分析[J]. 华北农学报, 2024, 39(5): 1-8. doi: 10.7668/hbnxb.20195063.

GAN Lu, XIE Meijuan, LU Zhenhua, LI Ming, DING Bo, QIU Lina, XIE Xiaodong, WANG Junbin. Cloning and Stress-Resistance Functional Analysis of Calcium-Dependent Protein Kinase Gene TaCDPK17 in Wheat[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(5): 1-8. doi: 10.7668/hbnxb.20195063.

http://www.hbnxb.net/CN/Y2024/V39/I5/1

图/表 9

图1 小麦TaCDPK17 PCR扩增

Fig.1 PCR amplification of TaCDPK17 in wheat M.DNA Marker;1,2.TaCDPK17。

图2 TaCDPK17编码的氨基酸序列及保守结构域分析灰色阴影部分为丝氨酸/苏氨酸保守结构域;方框部分为4个EF手型结构域。

Fig.2 Analysis of amino acid sequences and conserved domains encoded by TaCDPK17 The gray shaded area represents the conserved serine/threonine domain;while the boxes represent four EF-hand domains.

图3 TaCDPK17与其他同源CDPK17的系统进化树

Fig.3 Phylogenetic tree of TaCDPK17 and other homologous CDPK17

表1 TaCDPK17基因启动子顺式作用元件

Tab.1 Cis-acting elements of TaCDPK17 gene promoter

元件名称 Element name	序列 Sequence	数量 Amount	功能 Function
CAAT-box	CAAT	53	启动子与增强子区域常见元件
TATA-box	TATA	22	核心启动子元件
ABRE	ACGTG	53	参与ABA响应的顺式作用元件
MBS	CAACTG	21	参与干旱诱导性的MYB结合位点
CAT-box	GCCACT	2	与分生组织表达相关的顺式作用调控元件
ATCT-motif	GGGCGG	31	参与光响应的保守DNA模块的一部分
AuxRR-core	GGTCCAT	2	参与生长素响应的顺式作用调控元件
CGTCA-motif	CGTCA	55	参与茉莉酸甲酯响应的顺式作用元件
GARE-motif	TCTGTTG	1	赤霉素响应元件
LTR	CCGAAA	17	参与低温响应的顺式作用元件

图4 TaCDPK17基因的表达模式不同小写字母表示差异显著(P<0.05,n=3)。图5,7-8同。

Fig.4 Expression profile of TaCDPK17 gene Different lowercase letters indicate significant differences (P<0.05,n=3).The same as Fig.5,7-8.

图5 TaCDPK17转基因拟南芥株系鉴定 A.TaCDPK17转基因拟南芥植株PCR检测:M.DNA Marker,1.野生型,2-6.转基因株系,7.阴性对照;B.转基因拟南芥TaCDPK17基因表达水平。

Fig.5 Identification of TaCDPK17 transgenic Arabidopsis lines A.PCR test of TaCDPK17 transgenic Arabidopsis:M.DNA Marker,1.Wild type,2-6.Transgenic lines,7.Negative control;B.Expression level of TaCDPK17 in Arabidopsis transgenic lines.

图6 不同胁迫条件下野生型和TaCDPK17转基因种子的萌发率

Fig.6 Germination rates of wild-type and TaCDPK17 transgenic seeds under different stress conditions

图7 不同胁迫处理下野生型和TaCDPK17转基因植株的主根长

Fig.7 Main root length of wild-type and TaCDPK17 transgenic plants under different stress conditions

图8 ABA对野生型和TaCDPK17转基因植株气孔开度的影响 A.野生型和TaCDPK17转基因植株响应ABA的代表性气孔开度图像;B.野生型和TaCDPK17转基因植株响应ABA气孔开度分析。

Fig.8 The effect of ABA on stomatal aperture in wild-type and TaCDPK17 transgenic plants A.Representative images of stomatal aperture in response to ABA in wild-type and TaCDPK17 transgenic plants;B.Assays of stomatal aperture in response to ABA in WT and TaCDPK17 transgenic plants.

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小麦钙依赖蛋白激酶基因TaCDPK17的克隆及其抗逆功能初步分析

Cloning and Stress-Resistance Functional Analysis of Calcium-Dependent Protein Kinase Gene TaCDPK17 in Wheat

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小麦钙依赖蛋白激酶基因TaCDPK17的克隆及其抗逆功能初步分析

Cloning and Stress-Resistance Functional Analysis of Calcium-Dependent Protein Kinase Gene TaCDPK17 in Wheat

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