玉米<i>ZmMPI</i>基因克隆及其过表达转基因株系检测

doi:10.7668/hbnxb.20194774

摘要/Abstract

摘要：

盐碱胁迫已成为制约我国农业生产的重要因素之一,探索农作物耐盐分子机理,对作物育种具有重要的理论价值和实际应用价值。旨在克隆玉米中的ZmMPI基因,转化玉米植株。首先通过qRT-PCR对盐碱溶液处理下植株中的ZmMPI表达量变化进行分析,之后利用DNAMAN软件对ZmMPI蛋白序列进行多重比较分析同时利用MEGA 7.0软件构建系统发生树,并利用一系列软件对ZmMPI进行生物信息学分析。最后利用分子克隆技术,成功克隆ZmMPI基因的编码序列,构建植物过表达载体并利用农杆菌介导的遗传转化方法转化玉米自交系B104,在基因组水平、转录水平以及蛋白质水平对过表达转基因植株鉴定,分析其表达量变化。结果表明,ZmMPI基因的表达量受盐碱胁迫后整体呈现出先上升后下调的趋势;ZmMPI蛋白序列比较结果相似率为64.15%,系统发生树显示,ZmMPI与玉米(小颖大刍草亚种) ABA34115.1同源性最高,该蛋白含有一个蛋白结构域Potato_inhibit,有α螺旋结构、无规则卷曲结构和β转角结构,偏疏水性,预测潜在的磷酸化位点有10个;对获得的49个转化事件鉴定结果显示,其中有13个过表达转基因株系中的ZmMPI基因在基因组水平能正常表达,有10个过表达转基因株系中的ZmMPI基因能正常转录、翻译。最终获得10个能够正常转录翻译的过表达转基因株系,为进一步探究ZmMPI基因响应盐碱胁迫的分子机制奠定基础。

关键词: 玉米, ZmMPI, 盐碱胁迫, 转基因植株, 鉴定

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

姚梦瑶, 李娟, 刘志刚, 蔡大润, 李晓荣, 李波, 杨洋, 王子轩, 王勇攀, 陈勋基, 耿洪伟, 陈果. 玉米ZmMPI基因克隆及其过表达转基因株系检测[J]. 华北农学报, 2024, 39(4): 1-9. doi: 10.7668/hbnxb.20194774.

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.

http://www.hbnxb.net/CN/Y2024/V39/I4/1

图/表 8

表1 转基因植株鉴定引物

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

图1 盐碱胁迫处理下ZmMPI基因表达不同小写字母表示处理间有显著性差异(P<0.05)。

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

图2 ZmMPI蛋白氨基酸序列比对及系统进化树 A.ZmMPI蛋白多序列比对;B.ZmMPI蛋白系统进化树。

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

图3 ZmMPI蛋白氨基酸序列分析 A.ZmMPI蛋白保守结构域预测;B.ZmMPI蛋白的三级结构预测;C.ZmMPI蛋白的亲/疏水性预测;D.ZmMPI蛋白的磷酸化位点分析。

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.

图4 植物表达载体构建 M.DL2000 Marker。A.部分玉米叶片RNA提取胶图;B.ZmMPI目的基因扩增;C.重组质粒pCAMBIA3301-UBI-ZmMPI菌液PCR检测:+.重组质粒阳性对照,-.阴性对照;D.pCAMBIA3301-UBI-ZmMPI重组质粒植物表达载体示意图。

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.

图5 部分转基因株系基因组水平鉴定 M.DL2000 Marker;+.质粒阳性对照;WT.野生型;1—10.不同样品。A.筛选标记基因BlpR的PCR检测结果;B.目的基因ZmMPI的PCR检测结果。

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.

图6 部分转基因株系转录水平鉴定 M.DL2000 Marker;WT.野生型;-.阴性对照;1—10.不同样品。A.内参基因ZmGAPDH的PCR结果;B.目的基因Flag的PCR结果。

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.

图7 部分转基因株系蛋白水平鉴定 M.蛋白Marker;WT.野生型;1—10.不同样品。A.内参Anti-Actin条带结果;B.Anti-Flag条带结果。

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.

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玉米ZmMPI基因克隆及其过表达转基因株系检测

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