硅酸钠诱导的马铃薯抗黑痣病<i>StWRKY11</i>基因克隆及生物信息学分析

doi:10.7668/hbnxb.20192773

摘要/Abstract

摘要：

为探讨硅酸钠增强马铃薯黑痣病抗性的分子机制,将硅酸钠诱导马铃薯转录组中表达量较高的基因StWRKY11进行克隆和生物信息学分析。从马铃薯中提取总RNA,并利用RT-PCR方法扩增该基因并克隆,通过生物信息学相关软件对其进行结构预测及分析。结果表明,从马铃薯大西洋中克隆了阅读框为1 005 bp的StWRKY11基因,编码334个氨基酸,表达蛋白分子式为C₃₀₁₃H₅₀₂₃N₁₀₀₅O₁₂₆₀S₁₉₉,分子质量为81.867 94 ku,理论等电点(pI)为5.09,原子总数为10 500。表达蛋白含有一个典型的WRKYGQK保守结构域,锌指结构为CX₅CX₂₃HXH,属于第二类Ⅱd亚家族;表达的为疏水稳定性蛋白,无跨膜区和信号肽结构域,二级结构元件是α-螺旋、延伸链、β-折叠和无规卷曲,其中无规则卷曲比例最高,达61.68%,共有29个磷酸化位点,可能定位于细胞核内。启动子上游具有与抗性胁迫响应相关的顺式调控元件及与生长发育和激素响应顺式作用调控元件。该基因与马铃薯StWRKY5基因的亲缘关系较近,编码蛋白的氨基酸同源性达到95%。

关键词: 马铃薯, StWRK11, 基因克隆, 生物信息学

Abstract:

In order to study the molecular mechanism of sodium silicate enhancing risistance of potato to Rhizoctonia solani,a gene StWRKY11 with high expression level in potato transcriptome induced by sodium silicate was cloned and its bioinformatics was analysed.Total RNA was extracted from potato,amplified by RT-PCR and cloned.Through bioinformatics related software,the structure prediction and prediction analysis were carried out.The results showed that StWRKY11 gene with a open reading frame of 1 005 bp was cloned from potato Atlantic,encoding 334 amino acids.The molecular formula of the expressed protein was C₃₀₁₃H₅₀₂₃N₁₀₀₅O₁₂₆₀S₁₉₉,the molecular weight was 81.867 94 ku,the theoretical isoelectric point(pI)was 5.09,and the total number of atoms was 10 500.The expressed protein contained a typical WRKYGQK conserved domain,and the zinc finger structure was CX₅CX₂₃HXH,belonging to the second Ⅱ d subfamily.The secondary structural elements were α-helix,extended chain,β-folding and random coiling,among which the proportion of random curl was the highest,up to 61.68%.There were 29 phosphorylation sites in total,which might be located in the nucleus.There were cis-regulatory elements upstream of the promoter that might related to resistance stress response and cis-regulatory elements related to growth and development and hormone response.The gene was closely related to potato StWRKY5 gene,and the amino acid homology of the coding protein reached 95%.

Key words: Potato, StWRKY11, Gene cloning, Bioinformatics

张冬梅, 冯亚艳, 修志君, 杨春芳, 杜美娥, 李得宙, 张笑宇. 硅酸钠诱导的马铃薯抗黑痣病StWRKY11基因克隆及生物信息学分析[J]. 华北农学报, 2022, 37(5): 194-203. doi: 10.7668/hbnxb.20192773.

ZHANG Dongmei, FENG Yayan, XIU Zhijun, YANG Chunfang, DU Meie, LI Dezhou, ZHANG Xiaoyu. Cloning and Bioinformatics Analysis of Resistant Gene StWRKY11 Induced by Sodium Silicate in Potato by Rhizoctonia solani[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 194-203. doi: 10.7668/hbnxb.20192773.

http://www.hbnxb.net/CN/Y2022/V37/I5/194

图/表 11

表1 不同物种的WRKY蛋白质氨基酸序列

Tab.1 Amino acid sequences of WRKY proteins in different species

物种 Species	登录号 GenBank ID
辣椒 Capsicum annuum	AHD24521.1
茄子 Solanum melongena	AHI90951.1
枸杞 Lycium barbarum	ANW07460.1
天仙子 Hyoscyamus albus	BAN58138.1
番茄 Solanum lycopersic	NP001306910.1
烟草 Nicotiana tabacum	XP016497266.1
矮牵牛 Ipomoea nil	XP019174085.1
拟南芥 Arabidopsis thaliana	NP182248.1
马铃薯 Solanum tuberosum	XP006358112.1
	NP001274847.1

图1 马铃薯StWRKY11基因扩增 M.DL2000 Marker;1—2.StWRKY11扩增片段。

Fig.1 StWRKY11 gene amplification in potato M.DL2000 Marker;1—2.StWRKY11 amplified fragments.

图2 StWRKY11基因转化DH5α大肠杆菌的菌液PCR验证 M.DL2000 Marker;1—3.StWRKY11基因克隆扩增片段。

Fig.2 Conversion of StWRKY11 gene into DH5α Escherichia coli by PCR M.DL2000 Marker;1—3. StWRKY11 gene clone amplified fragment.

图3 StWRKY11基因的碱基序列及其表达蛋白质氨基酸序列

Fig.3 Sequence of StWRKY11 gene and amino acid sequence of its expressed protein

图4 StWRKY11基因表达蛋白质亲水性/疏水性分析

Fig.4 Hydrophilic/hydrophobic analysis of StWRKY11 gene expressed proteins

图5 StWRKY11基因表达蛋白质跨膜区分析

Fig.5 Transmembrane region analysis of StWRKY11 gene expression protein

图6 StWRKY11基因表达蛋白质的二级结构预测蓝色.α螺旋;红色.延伸链;绿色.β-折叠;黄色.无规则卷曲。

Fig.6 Secondary structure prediction of StWRKY11 gene expressed protein Blue.α-helix;Red.Extended chain;Green.β-folding;Yellow.Random coiling.

图7 StWRKY11基因表达蛋白三级结构预测

Fig.7 Prediction of tertiary structure of StWRKY11 gene expressed protein

表2 StWRKY11基因启动子区的顺式作用元件分析

Tab.2 Analysis of cis-acting elements in the promoter region of StWRKY11 gene

顺式元件 Cis-elements	序列 Sequence	元素的数量/个 Numbers of element	功能 Function
CAAT区 CAAT-box	CAAT	10	转录起始及增强元件
TATA区 TATA-box	TTATTT/TATAAAT	2	转录起始的关键调控位点
铜响应元件CURECORECR	GTAC	4	参与铜和氧的反应
MYB顺式元件 MYB2AT	TAACTG/AACGG	5	MYB 结合位点
DNA结合蛋白调控元件 DOFCOREZM	AAAG	12	DNA结合蛋白
转录激活元件 ARR1	GGATT/AGATT/CGATT/TGATT	17	转录因子参与基因表达的激活
CGCG区 CGCG-box	ACGCG/CCGCG/GCGCG	2	Ca²⁺钙调蛋白
W-区 W-box	TTGAC/TGAC	10	抗性胁迫响应调控元件
GATA区 GATA-box	GATA	9	光信号响应元件
光调控顺式元件 GT1CONSENSUS	CGTCA	11	光信号响应元件
ARE调控元件 ARE	AAACCA	1	缺氧诱导必需元件
叶肉特异基因表达调控元件CACTFTPPCA1	TACT/CACT	14	参与叶肉特异性表达
花粉调控元件 GTGANTG10	GTGA/AGAAA	12	花粉启动子元件
脱水顺式元件 CBFHV	ACCGAC/ATCGACGCCGAC/GTCGAC	4	脱水响应元件
赤霉素调控元件 PYRIMIDINEBOXOSRAMY1A	CCTTTT	1	赤霉素应答响应元件
茉莉酸调控元件 ASF1MOTIFCAMV	TGACG	2	茉莉酸信号响应调控元件
脱落酸响应元件 ABRE	ACGT/ACGTG	7	脱落酸响应元件

图8 StWRKY11基因表达蛋白质与其他物种的WRKY蛋白质氨基酸序列比对

Fig.8 The amino acid sequence of StWRKY11 gene expressed protein was compared with that of WRKY protein of other species

图9 StWRKY11基因表达蛋白质与其他物种的WRKY蛋白质的系统进化树

Fig.9 The StWRKY11 gene expresses proteins in a phylogenetic tree with WRKY proteins of other species

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硅酸钠诱导的马铃薯抗黑痣病StWRKY11基因克隆及生物信息学分析

Cloning and Bioinformatics Analysis of Resistant Gene StWRKY11 Induced by Sodium Silicate in Potato by Rhizoctonia solani

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硅酸钠诱导的马铃薯抗黑痣病StWRKY11基因克隆及生物信息学分析

Cloning and Bioinformatics Analysis of Resistant Gene StWRKY11 Induced by Sodium Silicate in Potato by Rhizoctonia solani

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