荷花<i>NnWRKY</i>22基因的克隆与表达分析

王婉茹; 刘莹; 刘红利; 贺丹; 刘艺平; 孔德政

doi:10.19303/j.issn.1008-0384.2022.004.009

荷花NnWRKY22基因的克隆与表达分析

doi: 10.19303/j.issn.1008-0384.2022.004.009

王婉茹^1,,
刘莹¹,
刘红利^{1, 2},
贺丹^{1, 2},
刘艺平^{1, 2, ,},
孔德政^{1, 2}

1.
河南农业大学风景园林与艺术学院，河南　郑州　450002
2.
河南省优质花卉蔬菜种苗工程研究中心，河南　郑州　450002

基金项目: 国家自然科学基金项目（31600568）；河南省高等学校重点研究项目（21A220003）；河南农业大学科技创新基金项目（KJCX2017C01）

详细信息

作者简介:
王婉茹（1998-），女，硕士，研究方向：风景园林植物资源应用（E-mail：wanruwork@163.com）

通讯作者:
刘艺平（1977-），女，副教授，研究方向：风景园林植物资源应用（E-mail：Lyp_163@163.com）

中图分类号: S 682.32
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出版历程
- 收稿日期: 2021-12-07
- 修回日期: 2022-03-08
- 网络出版日期: 2022-04-24
- 刊出日期: 2022-04-28

Cloning and Expression of NnWRKY22 in Lotus

WANG Wanru^1
,,
LIU Ying¹,
LIU Hongli^{1, 2},
HE Dan^{1, 2},
LIU Yiping^{1, 2
, ,},
KONG Dezheng^{1, 2}

1.
Landscape Architecture and Art College, Henan Agricultural University, Zhengzhou, Henan　450002, China
2.
Henan Provincial Engineering Research Center of Quality Flowers and Vegetables Seedling, Zhengzhou, Henan　450002, China

摘要

摘要: 目的克隆荷花（Nelumbo nucifera）NnWRKY22基因，分析该基因在荷花根、茎、叶、花中的表达模式及不同天数铜胁迫处理下的表达情况，为进一步研究该基因功能和荷花抗铜胁迫机制奠定基础。方法采用RT-PCR方法从荷花cDNA中克隆得到NnWRKY22基因，利用Prot Param等工具对NnWRKY22氨基酸序列进行分析，利用qRT-PCR方法分析NnWRKY22组织表达特异性以及在铜胁迫下表达量的变化。结果成功克隆得到荷花NnWRKY22基因，该基因ORF全长573 bp，编码190个氨基酸，含有1个保守的WRKY结构域。该蛋白理论分子量、等电点、不稳定系数和亲水性指数分别为：21.33 kDa、 9.03、71.93和−0.692，属于不稳定的亲水性蛋白。进化树分析表明荷花与洛矶山耧斗菜（Aquilegia coerulea）和博落回（Macleaya cordata）亲缘关系较近。qPCR结果表明，NnWRKY22在荷花的根、茎、叶、花中均有表达，但具有组织表达特异性，在根中表达量最高，花中表达量最低。NnWRKY22基因在铜胁迫下表达增强，胁迫7 d表达量达到最高值。结论克隆了荷花NnWRKY22基因，该基因在荷花中特异性表达，在根中表达量最高。NnWRKY22在铜胁迫下表达增强，推测该基因在荷花抗铜胁迫中发挥重要作用。
- 荷花 /
- NnWRKY22基因 /
- 表达分析 /
- 抗铜性
Abstract: Objective NnWRKY22 in Nelumbo nucifera was cloned and its expressions in various organs under copper stress analyzed. Method RT-PCR was used to clone the gene from cDNA of lotus leaves, and bioinformatics of the sequence obtained using ProtParam. The gene expressions in roots, stems, leaves, and flowers under an imposed copper stress for varied durations were detected by qRT-PCR. Result The successfully cloned NnWRKY22 had an ORF of 573 bp and encoded 190 amino acid residues. The unstable hydrophilic protein had a sequence containing a conserved WRKY domain and a molecular weight of 21.33 kDa, an isoelectric point of pH 9.03, an instability index of 71.93, and hydrophilicity of −0.692. The phylogenetic analyses revealed a close genetic relationship of the gene with those of Aquilegia coerulea and Macleaya cordata. The qRT-PCR results showed the organ-specific expressions of the gene in the tissues which were highest in the roots and lowest in the flowers. They were significantly upregulated and peaked in 7 d by the copper treatment. Conclusion NnWRKY22 was successfully isolated from lotus and cloned. The gene was most highly expressed in the lotus roots and significantly upregulated by the presence of copper. It presumably played an important role in the response of the lotus plants to copper stress.
- Lotus /
- NnWRKY22 gene /
- expression analysis /
- copper resistance

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图 1 NnWRKY22基因PCR扩增结果

注：M：Marker DL2 000；WRKY22：目的条带。

Figure 1. PCR amplification of NnWRKY22 gene

Note: M: Marker DL2 000; WRKY22: target band.

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图 2 NnWRKY22基因核苷酸序列及其推导的氨基酸序列

注：*为终止密码子；红色框部分为 WRKYGQK 基序。

Figure 2. Nucleotide sequence and deduced amino acids of NnWRKY22

Note: * indicates stop codon; sequence of WRKYGQK motif is highlighted in red.

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图 3 NnWRKY22蛋白保守结构域分析

Figure 3. The analysis of conserved domain for NnWRKY22 protein

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图 4 NnWRKY22磷酸化位点预测

Figure 4. Predicted phosphorylation site of NnWRKY22

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图 5 NnWRKY22与其他植物同源WRKY蛋白的系统进化树

Figure 5. Phylogenetic tree on NnWRKY22 and WRKY proteins of other plants

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图 6 NnWRKY22在荷花不同组织中的相对表达量

注：* 与 ** 分别表示差异显著（P<0.05)或极显著(P<0.01)。

Figure 6. Relative expressions of NnWRKY22 in lotus tissuesThe same as Fig.7.

Note: *and * * indicate significant difference (P < 0.05) or extremely significant difference (P < 0.01).图7同。

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图 7 铜胁迫下NnWRKY22的相对表达量

Figure 7. Relative expressions of NnWRKY22 under copper stress

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表 1 引物序列

Table 1. Primers

引物名称 Primer name	引物序列（5′-3′） Primer sequence（5′-3′）
NnWRKY22-F	ATGGAAGTCGACTGGGATCTACAA
NnWRKY22-R	CTAGAGACTAAAGACCCACCTGGGA
qRT-NnWRKY22-F	AATCGCGGACGGCTATTGAA
qRT-NnWRKY22-R	CTTCTCTTGGATCGAGGGGC
18Sr RNA-F 18Sr RNA-R	CTACCTACAACTCCATCAT CTCATACGGTCAGCAATA

下载: 导出CSV

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