<i>CsZFP</i>2<italic/>基因的克隆及其在烟草中的抗旱功能分析

李婉雪; 姚新转; 张宝会; 刘洋

doi:10.19303/j.issn.1008-0384.2022.004.005

CsZFP2基因的克隆及其在烟草中的抗旱功能分析

doi: 10.19303/j.issn.1008-0384.2022.004.005

1.
贵州大学茶学院, 贵州贵阳　550025
2.
贵州大学生命科学学院/农业生物工程研究院/山地植物资源保护与保护种质创新教育部重点实验室/山地生态与农业生物工程协同创新中心, 贵州贵阳　550025
3.
贵州大学烟草学院，贵州贵阳　550025

基金项目: 贵州省茶产业技术创新中心项目（黔科中引地[2017]4005）；贵州省优秀青年科技人才培养项目（黔科合平台人才[2019]5651）；中国烟草总公司贵州省公司重点研发项目（中烟黔科[2021]1号2021XM04）；贵州省科技计划项目（黔科合支撑[2021]一般111）

详细信息

作者简介:
李婉雪（1997−），女，硕士研究生，研究方向：农艺与种业（E-mail：1114652676@qq.com）

通讯作者:
刘洋 (1978−)，女，副教授，博士研究生，研究方向：转基因生物农药（E-mail：652493683@qq.com）

中图分类号: Q 784
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- 被引次数: 0
出版历程
- 收稿日期: 2022-01-07
- 修回日期: 2022-03-11
- 网络出版日期: 2022-06-19
- 刊出日期: 2022-04-28

Connection between Tea ZFP2 and Drought Resistance of Tobacco Plants

1.
College of Tea Science, Guizhou University, Guiyang, Guizhou550025, China
2.
.College of Life Sciences，Guizhou University/Institute of Agro-bioengineering/Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education)/ Collaborative Innovation Center for Mountain Ecology & Agro-Bioengineering（CICMEAB）, Guiyang, Guizhou 550025, China
3.
College of Tobaco Science, Guizhou University, Guiyang, Guizhou550025, China

摘要

摘要: 目的对茶树ZFP2基因应答干旱胁迫表达进行分析，为进一步深入研究茶树ZFP2基因在植物生长发育与抵御干旱胁迫中的作用机制提供科学数据。方法本实验室在前期工作中已通过酵母单杂筛选出一个与F3’5’H基因启动子区域互作的锌指蛋白，利用生物信息学的方法对茶树ZPF2的蛋白结构进行预测和分析，通过农杆菌介导法将CsZFP2基因转入烟草，在模拟干旱胁迫条件下进行功能验证。结果茶树（Camellia sinensis，XM_028257969.1）锌指蛋白的蛋白结构域与烟草（Nicotiana tabacum，XP_016463943.1）的锌指蛋白相似性为68.57%。茶树锌指蛋白基因内含子数为13个，蛋白长度为1 233 a，蛋白分子量为141 204.45，等电点为5.9，等电点Pi＜7表明茶树锌指蛋白富含酸性氨基酸；CsZPF2编码蛋白质主要二级结构为α螺旋且该蛋白是由α-螺旋和β-转角组成无规则卷曲的三维空间结构； CsZPF2编码蛋白质亚细胞定位在细胞核且氨基酸序列小于零部分多于50%，为亲水性蛋白；在自然干旱胁迫28 d后野生型烟草与转基因烟草底部老叶均明显发黄，但野生型烟草底部老叶基本完全失绿，呈枯萎状态，而转基因烟草底部叶片呈轻度萎蔫状态，大部分仍然呈现绿色。结论 CsZFP2基因能够响应干旱胁迫，并在干旱胁迫应答中起到作用，过表达CsZFP2的转基因植株比野生型植株更具抗旱能力。
- 烟草 /
- 干旱胁迫 /
- 锌指蛋白 /
- 抗旱
Abstract: Objective Expression of tea ZFP2 in tobacco plants in response to drought stress was studied to elucidate the molecular mechanism. Method A zinc finger protein (ZFP) was previously found in our laboratory to interact with the promoter region of F3'5'H gene. Using bioinformatics methods, the structure of ZPF2 from Camellia sinensis was predicted and analyzed. Then, CsZFP2 was cloned and transferred into tobacco by an Agrobacterium-mediated method for functional verification under a simulated drought stress. Result The domain of the ZFP from C. sinensis XM_028257969.1 was 68.57% homologous to that from Nicotiana tabacum XP_016463943.1. There were 13 introns in CsZFP2 with a length of 1,233a, a molecular weight of 141 204.45, an isoelectric point of pH 5.9, and an isoelectric point Pi＜7 indicating high content of acidic amino acids. The gene encoded mainly two proteins with an α-helix primary structure and a random coil 3D structure composed of α-helix and β-turn. The hydrophilic protein was subcellularly located in the nucleus with more than 50% of the amino acids having a sequence less than zero. Under natural drought for 28 d, the old leaves at the bottom of both wild-type and transgenic tobacco plants were turning yellow. However, those leaves on the wild-type plants became basically chlorotic and withered, but those on the transgenic plants wilted only slightly and largely remained green in color. Conclusion CsZFP2 affected the response of tobacco plants to drought stress. The transgenic plants with overexpressed CsZFP2 were more resistant to the stress than the wild-type counterparts.
- Tobacco /
- drought stress /
- zinc finger protein /
- drought resistance

HTML全文

图 1 锌指蛋白转录因子的过表达载体

Figure 1. Overexpression vector of ZFP transcription factor

下载: 全尺寸图片幻灯片

图 2 CsZPF2编码蛋白质的二级结构预测

注:竖条纹图和峰图，代表每个位点的预测结果信息，相同的颜色代表相同的结构。

Figure 2. Predicted secondary structure of CsZPF protein

Note:The vertical fringe pattern and peak pattern represent the prediction result information of each locus, and the same color represents the same structure.

下载: 全尺寸图片幻灯片

图 3 茶树ZPF2编码蛋白质的三级结构预测

Figure 3. Predicted tertiary structure of CsZPF protein

下载: 全尺寸图片幻灯片

图 4 CsZPF2编码蛋白质序列的亲疏水性分析

Figure 4. Hydrophilicity and hydrophobicity of CsZPF2 protein sequence

下载: 全尺寸图片幻灯片

图 5 茶树锌指蛋白与其他物种的蛋白质序列同源比对

Figure 5. Homologous analysis on ZPF protein sequences of various species

下载: 全尺寸图片幻灯片

图 6 茶树锌指蛋白与其他物种锌指蛋白基因的系统进化分析

Figure 6. Phylogenetic relationship between ZFP of C. sinensis and other species

下载: 全尺寸图片幻灯片

图 7 干旱胁迫对烟草植株生长的影响

注：A：移栽烟草 B：干旱胁迫0 d C：干旱胁迫28 d。

Figure 7. Effect of drought stress on growth of tobacco plants

Note: A: Transplanted tobacco; B: 0 d under drought stress; C: 28 d under drought stress.

下载: 全尺寸图片幻灯片

表 1 11个物种锌指蛋白基因的特征

Table 1. Characteristics of ZFP genes in 11 species

基因名称 Gene Name	缩写 Abbrev	基因 Gene				蛋白 Protein
基因名称 Gene Name	缩写 Abbrev	染色体 Chr.No	开始 Start	终止 End	内含子数 No. of Intron	蛋白长度 Length	等电点 pI	蛋白分子量 MW (Da)
猕猴桃 Actinidia chinensis var. Chinensis	PSR84926.1	8	21348050	21361229	12	1202	5.62	142287.43
灰山杜鹃 Rhododendron griersonianum	KAG5536119.1	29	6694780	6711730	14	1256	5.81	135123.75
甜樱桃 Prunus avium	XP_021810862.1	Unknow	101840	109887	13	1250	5.90	141072.2
水蜜桃 Prunus persica	ONI09200.1	5	17316836	17317953	11	1204	5.87	135936.84
杏花 Prunus armeniaca	CAB4311713.1	ContigORA5G	16168668	16176202	13	1250	5.90	141162.37
笋瓜 Cucurbita maxima	XP_023002699.1	Unknow	5837622	5847224	13	1256	5.83	141740.74
茶树 Camellia sinensis	XM_028257969.1	Unknow	748792	762882	13	1233	5.9	141204.45
烟草 Nicotiana tabacum	XP_016463943.1	Unknow	11729	22582	13	1234	5.82	141724.7
梅花 Prunus mume	XP_008240245.1	7	1633036	16340978	13	1250	5.87	139916.92
南瓜 Cucurbita moschata	XP_022930579.1	Unknow	1396522	1400153	13	1256	5.97	139899.6
月季 Rosa chinensis	XP_024174941.1	7	5794370	5802666	13	1238	5.94	139029.55
注: Contig: 未挂载到染色体上的序列片段。 Note: Contig: Sequence fragments not attached to the chromosom.

下载: 导出CSV

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