烟草BTB/POZ蛋白家族的鉴定及其在抗马铃薯Y病毒中的作用

doi:10.3785/j.issn.1008-9209.2023.10.081

浙江大学学报（农业与生命科学版）

2024, Vol. 50

Issue (2): 295-307 DOI: 10.3785/j.issn.1008-9209.2023.10.081

研究论文

烟草BTB/POZ蛋白家族的鉴定及其在抗马铃薯Y病毒中的作用

张劭文¹(

),赵天伦^1,^2,³,肖钦之⁴(

),祝水金^1,^2,³,陈进红^1,^2,³(

)

^1.浙江大学农业与生物技术学院，浙江杭州 310058
^2.浙江大学海南研究院，海南三亚 572025
^3.浙江省作物种质资源重点实验室，浙江杭州 310058
^4.湖南省烟草公司永州市公司，湖南永州 425000

Identification of BTB/POZ protein family in Nicotiana tabacum and its role in resistance to potato virus Y (PVY)

Shaowen ZHANG¹(

),Tianlun ZHAO^1,^2,³,Qinzhi XIAO⁴(

),Shuijin ZHU^1,^2,³,Jinhong CHEN^1,^2,³(

)

^1.College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang, China
^2.Hainan Institute of Zhejiang University, Sanya 572025, Hainan, China
^3.Zhejiang Key Laboratory of Crop Germplasm Resources, Hangzhou 310058, Zhejiang, China
^4.Yongzhou Branch of Hunan Tobacco Company, Yongzhou 425000, Hunan, China

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摘要：

马铃薯Y病毒（potato virus Y, PVY）侵染烟草（Nicotiana tabacum）会引起烟草脉带病，造成烟叶品质下降。broad complex, tramtrack, and bric-à-brac/pox virus and zinc finger（BTB/POZ）是动植物体内广泛存在的蛋白质家族，在植物生长和发育的各个环节都起着非常重要的作用。本研究鉴定到90个烟草BTB/POZ家族蛋白，对应71个基因。在这些蛋白质的序列中，识别到9种保守基序（motif），并根据其出现顺序将BTB/POZ家族成员划分至6个亚家族。结构域分析表明，BTB/POZ同一亚家族成员的结构域具有一致性。对BTB/POZ蛋白的三维构象进行预测发现，所有亚家族成员结构都以α螺旋为主，同一亚家族成员的三维构象相似。烟草品种K326和突变体M867（抗PVY）的BTB/POZ家族基因表达模式分析显示，叶片接种PVY后，NtBTB2M、NtBTB2K、NtBTB2L、NtBTB6E基因的表达丰度显著增加，这可能与烟草M867对PVY的抗性较强有关。顺式作用元件分析显示，NtBTB2K、NtBTB2L、NtBTB2M和NtBTB6E基因的上游2 000 bp区域内含有若干逆境响应相关元件，包括水杨酸响应元件、MYB结合位点等，可能和这些基因的上调表达有关。本研究为BTB/POZ蛋白的功能研究提供了理论依据，为烟草抗病育种提供了一定参考。

关键词： 烟草; BTB/POZ家族; 保守基序; 顺式作用元件

Abstract:

The infection of Nicotiana tabacum with potato virus Y (PVY) can cause tobacco vein disease, leading to a decrease in the tobacco leaf quality. The broad complex, tramtrack, and bric-à-brac/pox virus and zinc finger (BTB/POZ) family exists widely in plants and animals. It plays a very important role in various stages of plant growth and development. In this study, 90 proteins in the BTB/POZ family of N. tabacum were identified, corresponding to 71 genes. Among these protein sequences, nine conserved motifs were identified, and the members of the BTB/POZ family were divided into six subfamilies according to the order of their appearance. Domain analysis showed that the domains of members of the same subfamily in BTB/POZ are consistent. The three-dimensional (3D) conformation of the BTB/POZ protein was predicted based on the amino acid sequence. All the members of the subfamily are mainly structured with α helixes, and the 3D conformations of members of the same subfamily are similar. Analysis of the BTB/POZ family gene expression patterns of N. tabacum K326 and mutant M867 (anti-PVY) showed that the expression levels of NtBTB2K, NtBTB2L, NtBTB2M and NtBTB6E increased significantly after inoculation with PVY, which may be related to the strong resistance of N. tabacum M867 to PVY. Cis-acting element analysis showed that the 2 000 bp upstream region of NtBTB2K, NtBTB2L, NtBTB2M and NtBTB6E contained several stress responsive elements, including TCA-element and MYB-binding site, which may be related to the upregulated expression of these genes. This study provides a theoretical basis for the study of BTB/POZ protein function, and provides a reference for disease resistance of tobacco breeding.

Key words: Nicotiana tabacum BTB/POZ family conserved motif cis-acting element

收稿日期: 2023-10-08 出版日期: 2024-04-25

CLC:

S330

基金资助: 国有企业委托项目（K横20200424）

通讯作者: 肖钦之,陈进红 E-mail: 22116179@zju.edu.cn;xwiao@126.com;jinhongchen@zju.edu.cn

作者简介: 张劭文（https://orcid.org/0009-0001-2804-7375），E-mail：22116179@zju.edu.cn

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引用本文:

张劭文,赵天伦,肖钦之,祝水金,陈进红. 烟草BTB/POZ蛋白家族的鉴定及其在抗马铃薯Y病毒中的作用[J]. 浙江大学学报（农业与生命科学版）, 2024, 50(2): 295-307.

Shaowen ZHANG,Tianlun ZHAO,Qinzhi XIAO,Shuijin ZHU,Jinhong CHEN. Identification of BTB/POZ protein family in Nicotiana tabacum and its role in resistance to potato virus Y (PVY). Journal of Zhejiang University (Agriculture and Life Sciences), 2024, 50(2): 295-307.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2023.10.081 或 https://www.zjujournals.com/agr/CN/Y2024/V50/I2/295

基因名称

Gene name

引物序列（5 $′$ →3 $′$ ）

Primer sequence (5 $′$ →3 $′$ )

NtActin-7

F: TCAGGAAGGACCTCTACGGT

R: ATGTGCTAAGGGATGCGAGG

NtBTB2L

F: ACTTTGCCACCGATTTTAATG

R: TAGAATGTGCTGGAATGCG

NtBTB2K/NtBTB2M

F: ATGCACTGAAGGATGCACCA

R: ATAAGGAGCTGGACGCCTTG

NtBTB6E

F: ATGGAGGCCAGCAGCAGC

R: TCATGTGGAAGCAACCATTTCC

表1 qRT-PCR引物序列

图1 拟南芥、番茄和烟草中BTB/POZ家族成员的进化树

表2 烟草BTB/POZ家族中鉴定到的9种保守基序

图2 烟草BTB/POZ家族中9种基序的保守程度每个位点上字母堆积的总高度表示基序在该位置上的保守程度。字母堆中单个字母的高度表示这一位点上该氨基酸的相对频率。

图3 烟草BTB/POZ家族中保守基序的分布

图4 烟草BTB/POZ家族中结构域的分布

图5 烟草BTB/POZ家族基因表达模式聚类热图K326_PVY、M867_PVY分别表示接种PVY的烟草K326和突变体M867。

图6 BTB/POZ家族差异基因的相对表达量短栅上不同小写字母表示在P＜0.05水平差异有统计学意义。

图7 烟草BTB/POZ蛋白的三维构象预测红色部分指示该蛋白质的N端，蓝紫色部分指示蛋白质的C端。在部分三维构象几乎完全一致的蛋白质中，仅选取第1个蛋白质进行三维构象展示。

表3 烟草中高表达BTB/POZ家族基因的顺式作用元件

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