陆地棉耐碱基因GHZAT12的克隆、表达及生物信息学分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-1516

摘要/Abstract

摘要：

一些锌指蛋白转录因子家族成员在调节植物生长、发育和非生物胁迫中发挥重要作用。本研究旨在了解棉花C2H2转录因子家族中的成员GhZAT12在应对抵御逆境胁迫方面的作用。生物信息学分析结果显示,GhZAT12基因的编码区序列全长为474 bp,编码157个氨基酸,蛋白质分子量为17.074 kD。GhZAT12蛋白不含跨膜结构域,是一种非分泌型亲水蛋白,有7个磷酸化位点,没有信号肽。GhZAT12包含两个C2H2型锌指结构域,在其N端含有保守的L-box,C末端含有一个EAR-Motif。系统发育树分析表明GhZAT12与可可（XP_017982131.1）中的锌指蛋白亲缘关系较近。从棉花中克隆获得GhZAT12基因CDS序列,并构建了亚细胞定位载体,利用烟草瞬时表达系统对GhZAT12-GFP融合蛋白进行了亚细胞定位,结果表明,GhZAT12蛋白定位于细胞核中。qRT-PCR分析结果表明,陆地棉中GhZAT12的相对表达量在根中最高,其次是叶,在茎中表达量最低。碱胁迫处理后不同时间内,GhZAT12基因的表达量先升高后下降,推测GhZAT12基因可能在碱胁迫中发挥着重要的作用。

关键词: GhZAT12, 棉花, 生物信息学分析, 亚细胞定位, qRT-PCR, 碱胁迫

Abstract:

Some members of zinc finger protein（ZFP）transcription factor family play important roles in regulating plant growth,development,and response to abiotic stress. In this study,we aim to determine the role of GhZAT12,a C2H2-type transcription factor,in responding to adversity and stress. Bioinformatics analysis of the protein showed that the gene had the full length of 474 bp in coding region,encoding 157 amino acids with a predicted molecular mass of 17.074 kD. GhZAT12 protein did not contain a transmembrane domain. It was a non-secreted hydrophilic protein with 7 phosphorylation sites and no signal peptide. GhZAT12 contained two C2H2-type zinc finger domains,with a conserved L-box at the N-terminus,and a EAR-Motif at the C-terminus. Phylogenetic tree analysis demonstrated that GhZAT12 was closely related to the zinc finger protein in cocoa（XP_017982131.1）. The CDS sequence of GhZAT12 gene was cloned from cotton,and a subcellular localization vector was constructed. The GhZAT12-GFP fusion protein was observed for subcellular localization using the tobacco transient expression system. The results indicated that the GhZAT12 protein was localized in the nucleus. qRT-PCR analysis results showed that the relative expression of GhZAT12 in the root of Gossypium hirsutum was the highest,followed by the leaves and the lowest in the stem. At different times after alkaline stress,the expression of GhZAT12 gene generally increased first and then decreased,suggesting that GhZAT12 gene may play an important role in alkaline stress.

Key words: GhZAT12, Gossypium hirsutum, bioinformatics analysis, subcellular localization, qRT-PCR, alkaline stress

范亚朋, 芮存, 张悦新, 陈修贵, 陆许可, 王帅, 张红, 徐楠, 王晶, 陈超, 叶武威. 陆地棉耐碱基因GHZAT12的克隆、表达及生物信息学分析[J]. 生物技术通报, 2021, 37(8): 121-130.

FAN Ya-peng, RUI Cun, ZHANG Yue-xin, CHEN Xiu-gui, LU Xu-ke, WANG Shuai, ZHANG Hong, XU Nan, WANG Jing, CHEN Chao, YE Wu-wei. Cloning,Expression and Preliminary Bioinformatics Analysis of the Alkaline Tolerant Gene GhZAT12 in Gossypium hirsutum[J]. Biotechnology Bulletin, 2021, 37(8): 121-130.

图/表 12

表1 实验所用引物

Table 1 Primer pairs used in the experiments

引物名称Primer name	引物序列Primers sequence（5'-3'）	描述Description
GhZAT12	F：ATGAAGAGAGGCAGAGATATTGATG	GhZAT12基因引物 Primer pairs for GhZAT12 gene
GhZAT12	R：CTATATAAAACAATGAACAACCGGA	GhZAT12基因引物 Primer pairs for GhZAT12 gene
GFP-GhZAT12	F：AGAACACGGGGGACTCTAGAATGAAGAGAGGCAGAGATATTG	GhZAT12亚细胞定位载体引物 Primer pairs for subcellular localization vector
GFP-GhZAT12	R：TAGTCAGGCGCGCCGGTACCTATAAAACAATGAACAACCGG
Actin（AY305733）	F：ATCCTCCGTCTTGACCTTG	内参基因引物 Primer pairs for internal reference gene
Actin（AY305733）	R：TGTCCGTCAGGCAACTCAT	内参基因引物 Primer pairs for internal reference gene
QGhZAT12	F：CTGCTCTGAATGACGGGTTG	GhZAT12荧光定量引物 Primer pairs for qRT-PCR of GhZAT12
QGhZAT12	R：CCGGAGTTGGTGTCACTTTC	GhZAT12荧光定量引物 Primer pairs for qRT-PCR of GhZAT12

图 1 GhZAT12的PCR扩增产物的琼脂糖凝胶电泳分析 M：DL2000 marker. 1-4：GhZAT12的PCR扩增产物

Fig.1 Agarose gel electrophoretic analysis of PCR product of GhZAT12 M：DL2000 marker. 1-4：PCR amplification products of GhZAT12

图2 GhZAT12蛋白同源氨基酸序列多重比对 XP_002314198.3杨树;XP_002284111.1 葡萄;XP_017982131.1可可;XP_008654280.1玉米;NP_200790.1拟南芥;NP_001237020.2大豆;XP_021628881.1木薯;XP_002533110.1蓖麻;XP_021887642.1番木瓜;XP_004147699.1黄瓜;XP_006345939.1马铃薯;XP_012471399.1雷蒙德氏棉;XP_017618875.1亚洲棉;KAB2038530.1海岛棉;XP_016740578.1陆地棉

Fig.2 Multiple sequence alignment of homologous amino acids in GhZAT12 protein XP_002314198.3 Populus trichocarpa, XP_002284111.1 Vitis vinifera, XP_017982131.1 Theobroma cacao, XP_008654280.1 Zea mays, NP_200790.1 Arabidopsis thaliana, NP_001237020.2 Glycine max, XP_021628881.1 Manihot esculenta, XP_002533110.1 Ricinus communis, XP_021887642.1 Carica papaya, XP_004147699.1 Cucumis sativus, XP_006345939.1 Solanum tuberosum, XP_012471399.1 Gossypium raimondii, XP_017618875.1 Gossypium arboretum, KAB2038530.1 Gossypium barbadense, XP_016740578.1 Gossypium hirsutum

图3 不同物种中ZAT12同源蛋白的系统进化树参数设为Neighbor-Joining,boostrap value=1 000。系统树各分支上数字为 Bootstrap1000 时循环检验的置信度

Fig.3 Phylogenetic tree of ZAT12 homologous proteins in different species Parameters are set to be neighbor-joining,boostrap value=1 000. The numbers on the tree branches represent bootstrap confidence values as “Bootstrap” is 1000

图4 GhZAT12蛋白亲/疏水性分析

Fig.4 Hydrophilic/hydrophobic analysis of GhZAT12 protein

图5 GhZAT12蛋白的信号肽分析

Fig.5 Signal peptide analysis of GhZAT12 protein

图6 陆地棉GhZAT12蛋白磷酸化位点分析

Fig.6 Phosphorylation site analysis of GhZAT12 protein of G. hirsutum L.

图7 陆地棉GhZAT12蛋白跨膜螺旋预测

Fig.7 Transmembrane helical projections of GhZAT12 protein of G. hirsutum L.

图8 GhZAT12蛋白的二级结构预测

Fig.8 Secondary structure prediction of GhZAT12 protein

图9 GhZAT12蛋白的三级结构预测

Fig.9 Tertiary structure prediction of GhZAT12 protein

图10 GhZAT12在烟草叶片中的亚细胞定位 a、d：分别为GFP空载和GhZAT12-GFP荧光图;b、e：为明场图;c、f：为荧光和明场的叠加图;标尺为50 μm

Fig.10 Subcellular localization of GhZAT12 in tobacco leaves a,d：Confocal section of tobacco leave expressing GFP and GhZAT12-GFP,respectively. b,e：Bright field. c,f：Fluorescence and bright field merged image. Bar=50 μm

图11 棉花GhZAT12基因在不同组织不同碱胁迫时间的相对表达量

Fig.11 Relative expressions of GhZAT12 gene in different tissues and different times of alkaline stress in G. hirsutum L.

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