Expression and Functional Analysis of Maize Stress Response Gene ZmTPR1

doi:10.15933/j.cnki.1004-3268.2024.01.002

Abstract

Abstract: On the basis of previous transcriptome sequencing of maize under drought‑rehydration treatment，ZmTPR1 （tetratricopeptide repeat 1）was identified in response to drought stress.Bioinformatics analysis was conducted，and expression pattern was analyzed in different tissues and under different stresses. The homologous gene AtTPR1 in Arabidopsis thaliana was knocked out by CRISPR‑Cas9 technology，the phenotype，physiological and biochemical indexes of homozygous mutant plants were analyzed under drought stress，and the function of this gene was preliminarily explored，which would provide genetic resources for breeding drought‑resistant maize varieties.The results showed that ZmTPR1 gene was located on chromosome 3 of maize，encoding 421 amino acids，containing a conserved coiled coil domain，and might be involved in the response of maize to plant hormones，drought and so on.ZmTPR1 gene expressed in all tissues of maize，with the highest expression level in young stems.Drought，high temperature，salt and nitrogen deficiency stresses could induce the expression of ZmTPR1 gene，and the expression level of ZmTPR1 gene was most up‑regulated after drought stress.The expression level of ZmTPR1 gene in drought‑resistant maize inbred line Zheng 36 was significantly higher than that in drought‑sensitive maize inbred line B73 after drought stress. Drought resistance of Arabidopsis thaliana decreased after AtTPR1 gene was knocked out，the growth of Attpr1 mutant was severely inhibited under drought stress，and the leaves were wilted or even dried out. At the same time，the relative water content，chlorophyll content，net photosynthetic rate，activities of peroxidase and superoxide dismutase significantly decreased，and the content of malondialdehyde significantly increased.In summary，ZmTPR1 gene is involved in the response of maize to various abiotic stresses，and plays an important role in drought stress.

Key words: Maize, Tetrapeptide repeat protein, Abiotic stress, Drought resistance, Expression pattern

摘要： 在前期干旱-复水处理玉米转录组测序基础上，发现了一个响应干旱胁迫的基因ZmTPR1（Tetratricopeptide repeat 1），对该基因进行生物信息学分析，并探究其在不同组织及逆境胁迫下的表达模式，利用CRISPR-Cas9技术敲除拟南芥中的同源基因AtTPR1，分析干旱胁迫条件下纯合突变体植株的表型和生理生化指标，初步探究该基因的功能，为培育抗旱玉米品种提供基因资源。结果表明，ZmTPR1 基因位于玉米的第3号染色体，编码421个氨基酸，具有保守的卷曲螺旋结构域，可能参与玉米对植物激素、干旱等胁迫的响应。ZmTPR1 基因在玉米各组织中均表达，在幼茎中的表达量最高；干旱、高温、盐和缺氮胁迫均可诱导ZmTPR1 基因的表达，干旱胁迫后表达量上调最明显；干旱胁迫后ZmTPR1 基因在抗旱玉米自交系郑36中的表达量均极显著高于敏旱玉米自交系B73。敲除AtTPR1 基因后拟南芥抗旱能力下降，Attpr1 突变体在干旱胁迫下生长受到严重抑制，叶片萎蔫甚至干死，同时相对含水量、叶绿素含量、净光合速率及过氧化物酶和超氧化物歧化酶活性极显著降低，丙二醛含量极显著升高。综合来看，ZmTPR1 基因参与玉米对多种非生物胁迫的响应过程，并在响应干旱胁迫中发挥着重要的作用。

关键词: 玉米, 四肽重复蛋白, 非生物胁迫, 抗旱性, 表达模式

CLC Number:

S513

CAO Liru, LIANG Xiaohan, MA Chenchen, YE Feiyu, PANG Yunyun, LI Weiya, ZHANG Xin, LU Xiaomin. Expression and Functional Analysis of Maize Stress Response Gene ZmTPR1[J]. Journal of Henan Agricultural Sciences, 2024, 53(1): 12-21.

曹丽茹, 梁小菡, 马晨晨, 叶飞宇, 庞芸芸, 李伟亚, 张新, 鲁晓民. 玉米逆境胁迫响应基因ZmTPR1 的表达和功能分析[J]. 河南农业科学, 2024, 53(1): 12-21.

References

［1］王保梅. 转录因子ZmNF-YB16在玉米抗旱机制中的作用［D］. 济南：山东大学，2020.
WANG B M. Function mechanism of ZmNF‑YB16 in maize drought stress resistance［D］. Ji’nan：Shandong University，2020.

［2］ CAO L R，LU X M，WANG G R，et al. Transcriptional regulatory networks in response to drought stress and

rewatering in maize（Zea mays L.）［J］. Molecular Genetics and Genomics，2021，296（6）：1203‑1219.

［3］党永富，曹丽茹，鲁晓民，等. 喷施炭吸附聚谷氨酸对干旱胁迫下玉米幼苗生长的缓解效应［J］. 河南农业科学，2021，50（8）：26‑35.

DANG Y F，CAO L R，LU X M，et al. Alleviating effect of spraying carbon‑adsorbed polyglutamic acid on growth of

maize seedling under drought stress［J］. Journal of Henan Agricultural Sciences，2021，50（8）：26‑35.

［4］孙欢欢. 玉米自交系的抗旱性评价和ZmNAC基因的分子克隆［D］. 杨凌：西北农林科技大学，2016.

SUN H H. Drought resistance evaluation of maize inbred lines and molecular cloning of ZmNAC［D］. Yangling：

Northwest A & F University，2016.

［5］WANG X L，WANG H W，LIU S X，et al. Genetic variation in ZmVPP1 contributes to drought tolerance in maize seedlings［J］. Nature Genetics，2016，48（10）：1233‑1241.

［6］ GUO Q Q，LI X，NIU L，et al. Transcription‑associated metabolic adjustments in maize occur during combined drought and cold stress［J］.Plant Physiology，2021，186 （1）：677‑695.

［7］ HERNANDEZ‑GARCIA C M，FINER J J. Identification and validation of promoters and cis‑acting regulatory elements［J］. Plant Science，2014，217‑218：109‑119.

［8］ SONG Y，FENG L，ALYAFEI M A M，et al. Function of chloroplasts in plant stress responses［J］. International Journal of Molecular Sciences，2021，22（24）：13464.

［9］ BOHNE A V，SCHWENKERT S，GRIMM B，et al. Roles of tetratricopeptide repeat proteins in biogenesis of the photosynthetic apparatus［J］. International Review of Cell and Molecular Biology，2016，324：187‑227.

［10］ ALLEN J F，DE PAULA W B M，PUTHIYAVEETIL S，et al. A structural phylogenetic map for chloroplast photosynthesis［J］. Trends in Plant Science，2011，16（12）：645‑655.
［11］SCHEUFLER C，BRINKER A，BOURENKOV G，et al.Structure of TPR domain‑peptide complexes：Critical elements in the assembly of the Hsp70‑Hsp90 multichaperone machine［J］. Cell，2000，101（2）：199‑210.
［12］ROSADO A，SCHAPIRE A L，BRESSAN R A，et al.The Arabidopsis tetratricopeptide repeat‑containing protein TTL1 is required for osmotic stress responses and abscisic acid sensitivity［J］. Plant Physiology，2006，142（3）：1113‑1126.
［13］BHUIYAN N H，FRISO G，POLIAKOV A，et al. MET1 is a thylakoid‑associated TPR protein involved in photosystem Ⅱ super complex formation and repair in Arabidopsis［J］.The Plant Cell，2015，27（1）：262‑285.
［14］CAO L R，MA C C，YE F Y，et al. Genome‑wide identification of NF‑Y gene family in maize（Zea mays L.）and the positive role of ZmNF‑YC12 in drought resistance and recovery ability［J］. Frontiers in Plant Science，2023，14：1159955.
［15］ LIVAK K J，SCHMITTGEN T D. Analysis of relative gene expression data using real‑time quantitative PCR and the 2^-ΔΔCTmethod［J］. Methods，2001，25（4）：402‑408.
［16］TSUTSUI H，HIGASHIYAMA T. pKAMA‑ITACHI vectors for highly efficient CRISPR/Cas9‑mediated gene knockout in Arabidopsis thaliana［J］. Plant and Cell Physiology，2017，58（1）：46‑56.
［17］ MAIN E R，LOWE A R，MOCHRIE S G，et al. A recurring theme in protein engineering：The design，stability and folding of repeat proteins［J］. Current Opinion in Structural Biology，2005，15（4）：464‑471.
［18］ HAMMANI K，BONNARD G，BOUCHOUCHA A，et al. Helical repeats modular proteins are major players for organelle gene expression［J］. Biochimie，2014，100：141‑150.
［19］ RAST A，HEINZ S，NICKELSEN J. Biogenesis of thylakoid membranes［J］. Biochimica et Biophysica Acta，2015，1847（9）：821‑830.
［20］ YANG H X，LI P，ZHANG A H，et al. Tetratricopeptide repeat protein Pyg7 is essential for photosystem Ⅰ assembly by interacting with PsaC in Arabidopsis［J］.The Plant Journal，2017，91（6）：950‑961.
［21］ RENSING S A，ICK J，FAWCETT J A，et al. An ancient genome duplication contributed to the abundance of metabolic genes in the moss Physcomitrella patens［J］.BMC Evolutionary Biology，2007，7：130.
［22］ HU Z H，XU F，GUAN L P，et al. The tetratricopeptide repeat‑containing protein slow green1 is required for chloroplast development in Arabidopsis［J］. Journal of Experimental Botany，2014，65（4）：1111‑1123.

[1]	LI Chuan, ZHANG Panpan, ZHANG Meiwei, NIU Jun, MU Weilin, GUO Hanxiao, QIAO Jiangfang. Screening of miRNAs Related to High Temperature Stress in Maize Pollen and Analysis of Their Target Genes [J]. Journal of Henan Agricultural Sciences, 2024, 53(2): 1-16.
[2]	WANG Peng, LI Duhua, ZHANG Shaohui, YANG Mengning, AN Hongliu, FANG Shouguo, DENG Qingchao, GUO Lingfang, ZHANG Songbai. Characterization of a Novel Hypovirus RsHV4 in the Phytopathogenic Fungus Rhizoctonia solani [J]. Journal of Henan Agricultural Sciences, 2024, 53(2): 92-100.
[3]	YANG Mingda, ZHANG Suyu, LI Shuai, ZHENG Dongfang, YANG Shenjiao, GUAN Xiaokang, WANG Tongchao. Effects of Different Sowing and Irrigation Methods on Seedling Emergence，Dry Matter Accumulation，Transport and Yield of Summer Maize [J]. Journal of Henan Agricultural Sciences, 2024, 53(1): 22-31.
[4]	LU Xiaomin, GUO Shulei, ZHANG Xin, WEI Liangming, ZHANG Qianjin, CAO Liru, LIU Haijing, DENG Yazhou, ZHANG Zhen, WANG Zhenhua. Analysis of Yield Composition of Stem Lodging Resistance Related Traits of Maize under Different Densities [J]. Journal of Henan Agricultural Sciences, 2023, 52(9): 44-55.
[5]	. Cloning and Expression Analysis of RsCHS1 Gene in Red Radish（Raphanus sativus L.) [J]. Journal of Henan Agricultural Sciences, 2023, 52(9): 122-132.
[6]	XU Lina, AN Zhiliang, CHEN Shilin, ZHANG Huaisheng, JIN Xining, WANG Pingxi. Effect of Planting Density on Stem Strength and Ear Characters of Maize [J]. Journal of Henan Agricultural Sciences, 2023, 52(8): 11-17.
[7]	BAO Congcong, ZHANG Baolin, NIU Panting, PAN Lijie. Extraction and Application of Maize Leaf Inclination Angle Using Digital Images [J]. Journal of Henan Agricultural Sciences, 2023, 52(8): 142-154.
[8]	HUANG Hai, QU Xiaojie, LIU Jinhai, PENG Zanwen. Expression Patterns of Genes of Four Transcription Factor Families in Different Ploidy Rice under Salt⁃Alkali Stress [J]. Journal of Henan Agricultural Sciences, 2023, 52(6): 22-33.
[9]	WEI Quanquan, GU Xiaofeng, GOU Jiulan, ZHANG Meng, RAO Yong, XIAO Huagui. Effect of Nitrogen Fertilizer Combined with Organic Materials on Microbial Community in Winter Rapeseed⁃Maize Rotation in Yellow Soil [J]. Journal of Henan Agricultural Sciences, 2023, 52(6): 41-50.
[10]	WANG Yakun, LI Hongping, XU Zhenzhen, LIU Tianxue. Effects of Combined Stress of High Temperature and Drought on Photosynthetic Physiology of Maize [J]. Journal of Henan Agricultural Sciences, 2023, 52(5): 17-23.
[11]	DOU Dandan, SUN Jianjun, GUO Yuxi, WANG Dexin, GUO Xinhai, DING Chaoming. Genetic Diversity and Population Structure Analyses of Maize Inbred Lines Based on DUS Test Traits in Huang‐Huai‐Hai Region [J]. Journal of Henan Agricultural Sciences, 2023, 52(5): 24-32.
[12]	ZHAO Haijun, SHI Jiaqing, WANG Bin, GUO Yiyang, HU Xiaoli, HAN Zanping. Comprehensive Evaluation of Grain and Its Quality Traits of 150 Maize Inbred Lines [J]. Journal of Henan Agricultural Sciences, 2023, 52(5): 33-39.
[13]	FENG Kang, YANG Yuanyuan, LIU Qiong, CHENG Lan, HAO Haohao, WANG Pingping, JIN Weihuan, GUO Hongxiang. Cloning and Expression Analysis of NtCCX2 Gene in Tobacco [J]. Journal of Henan Agricultural Sciences, 2023, 52(5): 52-60.
[14]	HAO Hongyan, SANG Huitong, LÜ Shanhua, FAN Yinglun, LI Haiyun. New Research Progress of bZIP Transcription Factors in Improving Plant Stress Resistance [J]. Journal of Henan Agricultural Sciences, 2023, 52(4): 1-8.
[15]	WEI Liangming, CAO Liru, YE Feiyu, ZHANG Qianjin, ZHANG Xin, WANG Zhenhua, PANG Yunyun, MA Chenchen, JIN Songcan, LU Xiaomin. Analysis of Mechanical Grain Harvest Quality Related Indexes of Maize Parental Inbred Lines and Hybrids [J]. Journal of Henan Agricultural Sciences, 2023, 52(4): 31-41.