Cloning of ZmMPI Gene in Maize and Detection of Overexpression Mutant Lines

doi:10.7668/hbnxb.20194774

Abstract

Abstract:

Salt-alkali stress has become one of the important factors restricting agricultural production in my country.Exploring the molecular mechanism of salt tolerance of crops has important theoretical and practical value for crop breeding.The purpose of this study is to clone the ZmMPI gene in corn and transform corn plants.First,qRT-PCR was used to analyze the ZmMPI expression changes in plants treated with saline-alkali solutions.Then DNAMAN software was used to perform multiple comparison analysis of the ZmMPI protein sequence.MEGA 7.0 software was used to construct a phylogenetic tree,and a series of software were used to analyze the ZmMPI protein sequence.ZmMPI performed bioinformatics analysis.Finally,molecular cloning technology was used to successfully clone the coding sequence of the ZmMPI gene,construct a plant overexpression vector,and use Agrobacterium-mediated genetic transformation method to transform the corn inbred line B104.The overexpression transgenic plants were transformed at the genome level,transcription level and protein level.Identify and analyze changes in expression levels.The results showed that the expression level of the ZmMPI gene showed an overall trend of first increasing and then decreasing after being subjected to salt-alkali stress;the ZmMPI protein sequence comparison result showed a similarity rate of 64.15%,and the phylogenetic tree showed that ZmMPI had the highest homology with Zea mays subsp.parviglumis ABA34115.1.The protein contained a protein domain Potato_inhibit,which had an α-helix,a random coil and a β-turn.It was relatively hydrophobic and had 10 predicted Potential phosphorylation sites;the identification results of the 49 transformation events obtained showed that the ZmMPI gene in 13 over-expressed transgenic lines could be expressed normally at the genome level,and the ZmMPI gene in 10 over-expressed transgenic lines could be transcribed and translated normally.Finally,10 overexpression transgenic lines capable of normal transcription and translation were obtained,laying the foundation for further exploring the molecular mechanism of ZmMPI gene in response to salt-alkali stress.

Key words: Zea mays, ZmMPI, Salt-alkali stress, Transgenic plants, Authenticate

YAO Mengyao, LI Juan, LIU Zhigang, CAI Darun, LI Xiaorong, LI Bo, YANG Yang, WANG Zixuan, WANG Yongpan, CHEN Xunji, GENG Hongwei, CHEN Guo. Cloning of ZmMPI Gene in Maize and Detection of Overexpression Mutant Lines[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 1-9. doi: 10.7668/hbnxb.20194774.

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Figures/Tables 8

Tab.1 Transgenic plant identification primer

引物名称	引物序列(5'—3')	用途
Primer name	Primer sequence(5'—3')	Use
ZmSeqMPI-F	ATGAGCTCCACGGAGTGCG	用于扩增ZmMPI基因
ZmSeqMPI-R	TCAGCCGATGTGGGGCGTCT
BlpR-F	GCACCATCGTCAACCACTACAT	用于检测BlpR基因
BlpR-R	AGCTGCCAGAAACCCACGTCAT
3301-UBI-F	TCATACGCTATTTATTTGCTTGG	pCAMBIA3301表达载体UBI启动子区域的正向引物
ZmSeqMPI-Flag-F	ATTATCGCCCTAACCGTGTC	用于检测FLAG基因
ZmSeqMPI-Flag-R	GTCGTGATCCTTATAGTCTCCA
Zm_GAPDH-F	GGGTGAGGCTGGTGCTGAGTATGT	内参基因
Zm_GAPDH-R	TTAGCAAGGGGAGCAAGGCAGTT
ZmSeqMPI-QF	TGAGGTGGTCGGGCTGAG	ZmMPI实时荧光定量PCR引物
ZmSeqMPI-QR	ATGCGGACACGGTTAGGG

Fig.1 The expression of ZmMPI gene under saline-alkali stress There were significant differences between different lowercase letters(P<0.05).

Fig.2 ZmMPI protein amino acid sequence alignment and phylogenetic tree A.Multiple sequence alignment of ZmMPI protein;B.ZmMPI protein phylogenetic tree.

Fig.3 Amino acid sequence analysis of ZmMPI protein A.Prediction of conserved structural domains of ZmMPI protein;B.Prediction of tertiary structure of ZmMPI protein;C.Prediction of hydrophilicity/hydrophobicity of ZmMPI protein;D.Analysis of phosphorylation sites of ZmMPI protein.

Fig.4 Plant expression vector construction M.DL2000 Marker.A.RNA extraction gelatin diagram of some maize leaves;B.Amplification of ZmMPI target gene;C.PCR detection of recombinant plasmid pCAMBIA3301-UBI-ZmMPI bacterial solution:+.Positive control of recombinant plasmid,-.The negative control;D.Plant expression vector of pCAMBIA3301-UBI-ZmMPI recombinant plasmid.

Fig.5 Identification of some transgenic lines at genome level M.DL2000 Marker;+.Plasmid positive control;WT.Wild type;1—10.Different samples.A.PCR detection results of screening marker gene BlpR,respectively;B.The PCR results of the target gene ZmMPI.

Fig.6 Identification of some transgenic lines at transcription level M.DL2000 Marker;WT.Wild type;-.Negative control;1—10.Different samples.A.The PCR results of the internal reference gene ZmGAPDH were respectively;B.The PCR results of the target gene Flag.

Fig.7 Identification of some transgenic lines at protein level M.Protein Marker;WT.Wild type;1—10.Different samples.A.The result of the inner reference Anti-Actin band;B.The result of the Anti-Flag band.

References 23

[1]	Chatre L, Biard D S, Sarasin A, Ricchetti M. Reversal of mitochondrial defects with CSB-dependent serine protease inhibitors in patient cells of the progeroid Cockayne syndrome[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(22):E2910—E2919.doi:10.1073/pnas.1422264112.
[2]	Malefo M B, Mathibela E O, Crampton B G, Makgopa M E. Investigating the role of Bowman-Birk serine protease inhibitor in Arabidopsis plants under drought stress[J]. Plant Physiology and Biochemistry, 2020, 149:286—293.doi:10.1016/j.plaphy.2020.02.007. pmid: 32097847
[3]	Law R H P, Zhang Q W, McGowan S, Buckle A M, Silverman G A, Wong W, Rosado C J, Langendorf C G, Pike R N, Bird P I, Whisstock J C. An overview of the serpin superfamily[J]. Genome Biology, 2006, 7(5):216.doi:10.1186/gb-2006-7-5-216. pmid: 16737556
[4]	Laskowski M Jr, Kato I. Protein inhibitors of proteinases[J]. Annual Review of Biochemistry, 1980, 49:593—626.doi:10.1146/annurev.bi.49.070180.003113. pmid: 6996568
[5]	Janciauskiene S, Moraga F, Lindgren S. C-terminal fragment of alpha1-antitrypsin activates human monocytes to a pro-inflammatory state through interactions with the CD36 scavenger receptor and LDL receptor[J]. Atherosclerosis, 2001, 158(1):41—51.doi:10.1016/s0021-9150(00)00767-x. pmid: 11500173
[6]	Shan L, Li C L, Chen F, Zhao S Y, Xia G M. A Bowman-Birk type protease inhibitor is involved in the tolerance to salt stress in wheat[J]. Plant,Cell & Environment, 2008, 31(8):1128—1137.doi:10.1111/j.1365-3040.2008.01825.x.
[7]	Shakeel M, Xu X X, De Mandal S, Jin F L. Role of serine protease inhibitors in insect-host-pathogen interactions[J]. Archives of Insect Biochemistry and Physiology, 2019, 102(3):e21556.doi:10.1002/arch.21556.
[8]	胡聪. 两种杀虫蛋白Bt与CpTI的原核表达及纯化[D]. 兰州: 兰州理工大学, 2023.doi:10.27206/d.cnki.ggsgu.2023.001242.
	Hu C. Prokaryotic expression and purification of insecticidal proteins Bt and CpTI[D]. Lanzhou: Lanzhou University of Technology, 2023.
[9]	张重阳. 水稻Bowman-Birk类型胰蛋白酶抑制剂介导的水稻与稻瘟菌互作机制研究[D]. 北京: 中国农业科学院, 2020.doi:10.27630/d.cnki.gznky.2020.000170.
	Zhang C Y. The molecular mechanism of A rice bowman-birk trypsin inhibitor mediating the interaction between rice and magnaporthe oryzae[D]. Beijing: Chinese Academy of Agricultural Sciences, 2020.
[10]	Chen X J, Chen G, Li J P, Hao X Y, Tuerxun Z, Chang X C, Gao S Q, Huang Q S. A maize calcineurin B-like interacting protein kinase ZmCIPK42 confers salt stress tolerance[J]. Physiologia Plantarum, 2021, 171(1):161—172.doi:10.1111/ppl.13244.
[11]	李春雷. 热带玉米种质CML493抗大斑病基因的精细定位与克隆研究[D]. 长春: 吉林农业大学, 2021.doi:10.27163/d.cnki.gjlnu.2021.000052.
	Li C L. Fine mapping and cloning of resistance gene to northern corn leaf blight in tropical maize germplasm CML493[D]. Changchun: Jilin Agricultural University, 2021.
[12]	陈尘. 丹参茉莉素信号通路关键成员COI1基因功能研究[D]. 西安: 陕西师范大学, 2017.
	Chen C. Study on the function of COI1 gene,a key member of Jasmonate signaling pathway in Salvia miltiorrhiza[D]. Xi'an: Shaanxi Normal University, 2017.
[13]	刘新园. 拟南芥及玉米生长发育关键基因AtSLO8、GRP23、ZmPPR21和ZmPPR26功能研究[D]. 济南: 山东大学, 2022.doi:10.27272/d.cnki.gshdu.2022.006797.
	Liu X Y. Functional analysis of key genes AtSLO8, GRP23, ZmPPR21,and ZmPPR26 in growth and development of Arabidopsis and maize[D]. Jinan: Shandong University, 2022.
[14]	韩丽娟. 里氏木霉纤维素外切酶Cel7A的表达调控及其酶催化的研究[D]. 济南: 山东大学, 2022.doi:10.27272/d.cnki.gshdu.2022.001193.
	Han L J. Study on transcript-level regulation of cellobiohydrolase Cel7A and its enzymatic catalysis in trichoderma reesei[D]. Jinan: Shandong University, 2022.
[15]	张啸天. 玉米ZmRING-93耐旱功能初步分析[D]. 荆州: 长江大学, 2023.doi:10.26981/d.cnki.gjhsc.2023.001314.
	Zhang X T. Preliminary analysis of drought tolerance function of ZmRING-93 in maize[D]. Jingzhou: Yangtze University, 2023.
[16]	任珍静. 转mAM79基因玉米的获得及草甘膦抗性分析[D]. 北京: 中国农业科学院, 2015.doi:10.7666/d.Y278673.
	Ren Z J. Obtainment and glyphosate tolerance analysis of mAM79 transgenic maize[D]. Beijing: Chinese Academy of Agricultural Sciences, 2015.
[17]	房超伟. ZmACOS5和ZmMs13/ABCG2a调控玉米花药和花粉发育的机理解析[D]. 北京: 北京科技大学, 2023.doi:10.26945/d.cnki.gbjku.2023.000377.
	Fang C W. ZmACOS5 and ZmMs13/ABCG2a regulate the anther and pollen development in maize[D]. Beijing: University of Science and Technology Beijing, 2023.
[18]	谭燕华. 转植酸酶基因玉米比较蛋白质组学研究[D]. 海口: 海南大学, 2017.
	Tan Y H. Comparative proteomics of phytase-transgenic maize and its non-transgenic isogenic variety[D]. Haikou: Hainan University, 2017.
[19]	郑宾. 密植夏玉米穗位叶光环境差异对其光合性能的影响及蛋白质组学分析[D]. 泰安: 山东农业大学, 2022.doi:10.27277/d.cnki.gsdnu.2022.000030.
	Zheng B. Effects of difference in ear leaf light environment of densely planted summer maize on its photosynthetic performance and proteomics analysis[D]. Taian: Shandong Agricultural University, 2022.
[20]	颜统利, 何雨, 玛丽亚, 文爱秀, 钱峰, 周琬敏, 蒋玉蓉, 戎均康. 外源钙对盐胁迫下不同衰老类型小麦幼苗生长生理的缓解效应[J]. 浙江农林大学学报, 2023, 40(5):991—998.doi:10.11833/j.issn.2095-0756.20220677.
	Yan T L, He Y, Ma L Y, Wen A X, Qian F, Zhou W M, Jiang Y R, Rong J K. Alleviative effect of exogenous calcium ions on growth physiology of different senescence types of wheat seedlings under salt stress[J]. Journal of Zhejiang A&F University, 2023, 40(5):991—998.
[21]	张斌. 大豆GmPP2C89基因在盐胁迫中的功能分析[J]. 华北农学报, 2022, 37(4): 20—27. doi: 10.7668/hbnxb.20193021.
	Zhang B. Functional analysis of soybean GmPP2C89 gene under salt stress[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(4): 20—27.
[22]	黄旭升, 周雅莉, 史先飞, 高宇, 董书言, 蔡桂萍, 李润植, 王计平. 紫苏bZIP转录因子全基因组鉴定及对非生物胁迫的响应分析[J]. 植物生理学报, 2023, 59(7):1383—1397.doi:10.13592/j.cnki.ppj.100454.
	Huang X S, Zhou Y L, Shi X F, Gao Y, Dong S Y, Cai G P, Li R Z, Wang J P. Genome-wide characterization and abiotic stress response analysis of bZIP transcription factors in Perilla frutescens[J]. Plant Physiology Journal, 2023, 59(7):1383—1397.
[23]	李腾. 特色油料作物油莎豆硬脂酰-ACP脱氢酶基因CeSAD的鉴定及功能分析[D]. 太谷: 山西农业大学, 2022.doi:10.27285/d.cnki.gsxnu.2022.000118.
	Li T. Identification and functional analysis of stearoyl-ACP dehydrogenase gene CeSAD from special oil crops of Cyperus esculentus[D]. Taigu: Shanxi Agricultural University, 2022.

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