| 苹果MdMAP70-1克隆及异源过表达番茄的抗旱性分析 |
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| 引用本文：李 敏，韩丽娜，梁国平，陈佰鸿，毛 娟.苹果MdMAP70-1克隆及异源过表达番茄的抗旱性分析[J].西北植物学报,2023,43(2):181~189 |
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| 基金项目:甘肃省教育厅“双一流”重大科研项目(GSSYLXM-02)；甘肃省高等学校产业支撑引导项目(2019C-11)；甘肃农业大学“伏羲杰出人才培育计划”(Gaufx-03J02) |
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| 中文摘要:植物应答非生物胁迫与微管的动态变化有着密切的联系，而微管蛋白的结构和功能通过微管结合蛋白(microtubule-associated proteins, MAPs)进行调控。MAP70-1是一种微管结合蛋白，为了揭示MAP70-1响应干旱胁迫的分子机制，该研究以苹果砧木M26为材料，采用PCR方法，克隆MdMAP70-1基因(登录号XM_008390949)，并进行生物信息学分析；采用叶盘法转化‘黄珍珠’番茄，并进行抗旱性分析。结果显示：(1)成功克隆得到苹果MdMAP70-1，其开放阅读框(ORF)为1 860 bp，编码619个氨基酸，相对分子量为68.77 kD，等电点8.64；多序列比对和系统进化分析表明,苹果MdMAP70-1具有MAP70家族蛋白的保守结构域，与葡萄VvMAP70-1蛋白(Vitis vinifera VIT_201s0011g05790)的亲缘关系较近。(2)用农杆菌介导的叶盘法转化野生型番茄，经抗性筛选获得12株阳性植株, PCR检测获得分别含有目的条带(1 860 bp和680 bp)的8个过表达株系，证明MdMAP70-1基因已转入番茄中；qRT-PCR检测并选择表达量显著高于野生型的3个转基因番茄株系OE-1、OE-2和OE-3。(3)T3代转基因番茄干旱胁迫抗性分析表明，干旱胁迫0 d和5 d，过表达番茄株系叶片的相对含水量(RWC)、脯氨酸(Pro)含量、可溶性蛋白(SP)含量及抗氧化酶活性等与野生型(WT)对照无差异；胁迫10 d 和15 d时，过表达MdMAP70-1转基因番茄植株的RWC、Pro含量、SP含量及抗氧化酶活性显著高于WT，而相对电导率(REC)和MDA含量较WT降低。研究表明，番茄中过表达苹果MdMAP70-1基因能够提高番茄体内RWC、Pro、SP含量以及SOD、POD和CAT活性，并降低REC和MDA含量，增强番茄的抗旱性；该研究结果为利用MdMAP70-1基因提高植株的耐旱性研究以及微管结合蛋白的功能研究奠定了基础。 |
| 中文关键词:苹果 MdMAP70-1 番茄 干旱胁迫 抗氧化酶 |
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| Cloning of MdMAP70-1 Gene from Apple Rootstock M26 and Analysis with Drought Resistance of Heterologous Overexpression of MdMAP70-1 Gene in Tomato |
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| Abstract:Plant responses to abiotic stress are closely linked to the dynamics of microtubules, and the structure and function of tubulins are regulated by microtubule-associated proteins (MAPs). MAP70-1 is a microtubule binding protein, which plays a vital role in plant growth and development and stress. In order to reveal the molecular mechanism of MAP70-1 response to drought stress, we used the rootstock M26 of apple (Malus domestica Borkh.) as material, cloned the MdMAP70-1 gene (GenBank No. XM_008390949) by PCR and performed bioinformatics analysis. The ‘yellow pearl’ tomato was transformed by Agrobacterium-mediated leaf disc method and its drought resistance was analyzed. The results showed that: (1) MdMAP70-1 was successfully cloned and its open reading frame (ORF) was 1 860 bp and encoded 691 amino acids，the relative molecular weight was 68.77 kD and the isoelectric point (pI) 8.64. Comparison of amino acid sequences and phylogenetic analysis indicated that MdMAP70-1 contained a conserved domain of MAP70 family and had a closest relationship with VvMAP70-1 (Vitis vinifera VIT_201s0011g05790). (2) Wild tomato was transformed by Agrobacterium-mediated leaf disc method, 12 positive plants were obtained by resistance screening, and 8 overexpressed lines containing target bands (1 860 bp and 680 bp) were obtained by PCR, which proved that the MdMAP70-1 gene had been transferred into tomato. qRT-PCR detected and selected three transgenic lines OE-1, OE-2 and OE-3 with significantly higher expression levels than WT (wild type). (3) Analysis of drought stress resistance of T3 generation transgenic tomatoes showed that there were no differences in the relative water content (RWC), proline (Pro) content, content of soluble protein (SP), malondialdehyde (MDA) content, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities of overexpressed tomato leaves under drought stress 0 and 5 days compared with control (WT). After 10 and 15 days of stress, compared with WT, the RWC, Pro content, SP content and antioxidant enzyme activities of the overexpressed MdMAP70-1 transgenic tomatoes were significantly higher than those of WT, while the relative conductivity (REC) and MDA contents were decreased. The study have shown that overexpression of MdMAP70-1 gene in tomato can increase the contents of RWC, Pro and SP, activities of SOD, POD and CAT in tomato, reduce the contents of REC and MDA, and enhance the drought resistance of tomato. The results of the study laid a foundation for the use of MdMAP70-1 gene to improve the drought tolerance of plants and the function of microtubule-associated proteins. |
| keywords:apple MdMAP70-1 tomato drought stress antioxidant enzymes |
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