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

doi:10.7668/hbnxb.20195063

Abstract

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

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.

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Figures/Tables 9

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

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.

Fig.3 Phylogenetic tree of TaCDPK17 and other homologous CDPK17

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

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.

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.

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

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

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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