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作物学报 ›› 2021, Vol. 47 ›› Issue (11): 2184-2198.doi: 10.3724/SP.J.1006.2021.04240

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

烟草非特异性脂质转移蛋白基因家族的鉴定与分析

李鹏1(), 刘彻1, 宋皓1, 姚盼盼1, 苏沛霖1, 魏跃伟1, 杨永霞1,*(), 李青常2,*()   

  1. 1河南农业大学烟草学院, 河南郑州 450002
    2中国烟草总公司郑州烟草研究院, 河南郑州 450001
  • 收稿日期:2020-11-05 接受日期:2021-04-26 出版日期:2021-11-12 网络出版日期:2021-05-13
  • 通讯作者: 杨永霞,李青常
  • 作者简介:E-mail: lpeng1995@126.com
  • 基金资助:
    河南省科技攻关计划(农业领域)项目(182102110315);河南省科技攻关计划(农业领域)项目(192102110002);河南省科技攻关计划(农业领域)项目(192102110121);河南省大学生创新创业训练计划项目(S202010466013);河南农业大学本科实验室开放创新项目(KF1908)

Identification and analysis of non-specific lipid transfer protein family in tobacco

LI Peng1(), LIU Che1, SONG Hao1, YAO Pan-Pan1, SU Pei-Lin1, WEI Yao-Wei1, YANG Yong-Xia1,*(), LI Qing-Chang2,*()   

  1. 1Tobacco College, Henan Agricultural University, Zhengzhou 450002, Henan, China
    2Zhengzhou Tobacco Research Institute of CNTC, Zhengzhou 450001, Henan, China
  • Received:2020-11-05 Accepted:2021-04-26 Published:2021-11-12 Published online:2021-05-13
  • Contact: YANG Yong-Xia,LI Qing-Chang
  • Supported by:
    Henan Province Science and Technology Research Plan (Agricultural Field) Project(182102110315);Henan Province Science and Technology Research Plan (Agricultural Field) Project(192102110002);Henan Province Science and Technology Research Plan (Agricultural Field) Project(192102110121);Henan University Student Innovation and Entrepreneurship Training Program(S202010466013);Open Innovation Project of Undergraduate Laboratory of Henan Agricultural University(KF1908)

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摘要：

植物非特异性脂质转移蛋白(non-specific lipid transfer proteins, nsLTPs)可以在体外转移脂质, 调节植物的生长发育以及对环境非生物和生物胁迫作出反应等。本研究从烟草栽培品种K326 (Nicotiana tabacum L.)基因组中鉴定出74个nsLTPs基因, 对其系统发育关系、基因结构、保守基序、染色体定位、基因重复、启动子顺式作用元件、3D结构和激素与非生物胁迫处理下的表达模式进行了分析。结果表明, 根据8个半胱氨酸之间的间隔和序列相似性将其分为I、II、III、IV、V、VII、VIII和XIII八种类型, 相同类型的NtLTPs具有相似的内含子-外显子基因结构和保守基序模式, motif 2和motif 3是NtLTPs基因家族的特征基序。在进化过程中, 片段重复是NtLTPs基因家族扩展的主要原因, 干旱处理后的RNA-seq分析发现, 在进化过程中, 不同基因重复事件发生后功能分化模式存在差异。启动子分析表明, 它们含有多种光反应、激素和非生物胁迫响应顺式作用元件。进一步采用qRT-PCR分析发现, NtLTPs家族基因在烟草植株的不同组织和器官中具有不同的表达模式, 可以响应干旱、盐等非生物胁迫以及IAA、GA、SA等激素处理。研究结果为深入分析NtLTPs家族基因的功能提供了理论参考, 并为进一步的分子育种提供了理论基础。

关键词: 烟草, nsLTPs, 基因家族, 生物信息学, 基因表达

Abstract:

Plant non-specific lipid transfer proteins (nsLTPs) can transfer lipids in vitro, regulate plant growth and development, and respond to environmental abiotic and biotic stresses. In this study, 74 nsLTPs genes were identified from the genome of Nicotiana tabacum variety K326, and we analyzed multiple characteristics of these genes, including phylogeny, gene structures, conserved motifs, protein domains, chromosome locations, cis-elements in the promoter sequences, 3D structure, and the expression patterns under different hormones and abiotic stresses. The results revealed that nsLTPs in tobacco could be divided into eight types, including type I, II, III, IV, V, VII, VIII, and XIII, according to the interval and sequence similarity between the eight cysteines. The same types of NtLTPs had similar intron-exon patterns and conserved motifs, motif 2 and motif 3 were the characteristic motifs of NtLTPs family. In the process of evolution, fragment duplication dominated the expansion of the NtLTPs family. RNA-seq analysis after drought treatment revealed that the functional differentiation patterns of repeat gene pairs were diverse during evolution period. Promoter analysis showed that they contained a variety of cis-acting elements in response to light response, hormones, and abiotic stress. Furthermore, qRT-PCR demonstrated that NtLTPs family genes had different expression patterns in different tissues and organs of tobacco plants, which could respond to abiotic stresses such as drought, salt, and hormone treatments (IAA, GA, and SA etc.). These results provide a theoretical reference for the in-depth analysis of the functions of NtLTPs family genes and molecular breeding.

Key words: tobacco, nsLTPs, gene family, bioinformatics, gene expression

表1

引物列表"

引物名称Primer name 引物序列Primer sequence (5′-3′)
L25-F CCCCTCACCACAGAGTCTGC
L25-R AAGGGTGTTGTTGTCCTCAATCTT
Nitab4.5_0000125g0010-F CGGATCGCCGGAGTGTTTGC
Nitab4.5_0000125g0010-R GCCACATTTGCCAGGGAGGG
Nitab4.5_0004362g0040-F AAGCCGATTTGCGTTGTATG
Nitab4.5_0004362g0040-R CTTAGGCAGTTTCATAGCAG

表2

烟草nsLTPs八个半胱氨酸基序(ECM)的多样性"

类型
Type		 数量
Number of members		 ECM
I 38 C X9,24 C X12-16 CC X19,20,22 CXC X21-23 C X10-15,25 C
II 6 C X7 C X13 CC X8 CXC X23 C X6,10 C
III 1 C X9 C X14 CC X9 CXC X12 C X6 C
IV 10 C X9,10 C X14-16 CC X9,12 CXC X24 C X7-10 C
V 1 C X14 C X14 CC X11 CXC X24 C X10 C
VII 5 C X9 C X14,16 CC X12 CXC X25,27 C X9 C
VIII 7 C X6 C X12,14 CC X12 CXC X25,27 C X8 C
XIII 6 C X9 C X14 CC X12 CXC X26,30 C X8 C

图1

烟草nsLTPs蛋白的分子量和等电点的分布"

图2

烟草中nsLTPs家族的系统发育关系"

图3

烟草nsLTPs蛋白的保守基序(中间部分)和nsLTPs基因的基因结构(右侧部分)"

图4

烟草nsLTPs的保守半胱氨酸结构域"

图5

烟草nsLTPs基因在染色体上的分布"

图6

烟草中nsLTPs基因的基因重复"

图7

重复的NtLTPs基因之间的Ka和Ka/Ks值的频率分布"

图8

烟草中9个nsLTPs蛋白的3D结构"

图9

烟草nsLTPs启动子的顺式作用元件分析"

图10

干旱处理下烟草品种K326的差异基因热图 使用每个样本的FPKM数值log2标准化后绘制差异基因表达量热图。CK表示未进行干旱处理, D表示干旱处理, 均包含3个重复。红色和蓝色分别表示表达量高和低。"

图11

Nitab4.5_0000125g0010 (I型)和Nitab4.5_0004362g0040基因(IV型)在烟草不同组织中的表达水平 以根中的相对表达量作为对照, 红色和绿色表示相对表达量高和低。R: 根; ST: 茎; L: 下部叶; ML: 中部叶; UL: 上部叶; F: 花。"

图12

Nitab4.5_0000125g0010 (黑色)和Nitab4.5_ 0004362g0040基因(灰色)在不同处理下的表达模式 用2-Δ∆CT方法计算相对基因表达量。将0 h的相对表达量设置为1。误差线表示3个生物学重复的标准偏差。37℃: 高温处理; NaCl: 盐处理; Mannitol: 甘露醇处理; GA: 赤霉素处理; IAA: 生长素处理; MeJA: 茉莉酸甲酯处理; ABA: 脱落酸处理; SA: 水杨酸处理。*表示在0.05水平上差异显著。"

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