SSMP的结构解析及在拟南芥抗盐过程中的作用

doi:10.3864/j.issn.0578-1752.2015.04.18

摘要/Abstract

摘要： 【目的】解析SSMP（salt-sensitive membrane protein）的结构，明确SSMP的表达特性，预测和分析SSMP在拟南芥逆境胁迫过程中的作用。【方法】利用生物信息学方法，解析SSMP的结构特征；利用Real-time PCR技术分析SSMP的时空表达特性；利用SSMP-GFP融合技术转化拟南芥叶片原生质体，对SSMP进行亚细胞定位；构建SSMP的启动子连接报告基因GUS的表达载体，转化野生型拟南芥，通过染色方法观察报告基因GUS的表达情况，从而进行SSMP的组织定位分析；构建SSMP过表达载体，利用农杆菌侵染花絮法转化拟南芥，qRT-PCR技术验证过表达SSMP拟南芥阳性苗，分析过表达SSMP拟南芥的表型并进行抗逆性分析，明确SSMP的生物学功能；利用电导仪法比较过表达SSMP拟南芥和野生型拟南芥叶片的细胞膜透性，分析SSMP在拟南芥抵抗逆境胁迫过程中的作用。【结果】生物信息学分析表明，SSMP含有START（the lipid/sterol-binding StAR-related lipid transfer protein domains）保守域、共411个氨基酸，具有磷脂酰胆碱的结合位点，预测SSMP可能影响膜的组成。对SSMP的高级结构进行解析，SSMP含有9个反平行的β-折叠和2个α-螺旋，形成1个明显的疏水腔，N端的α-螺旋在疏水腔外，另一个α-螺旋形成疏水腔的盖子结构。Real-time PCR对拟南芥的不同组织及发育时期的SSMP表达量的分析表明，SSMP在拟南芥各组织中均有表达，表达量由高到低的顺序依次为茎生叶、莲座叶、茎、根、花和种子。SSMPpro::GUS转基因拟南芥幼苗的染色结果表明，在正常情况下，GUS主要在下胚轴、整个子叶和真叶的叶脉及表皮毛处表达；50 mmol·L^-1 NaCl处理后，GUS在下胚轴和子叶中表达没有明显的变化，但在真叶中的表达减少；100 mmol·L^-1 NaCl处理后，GUS的表达在下胚轴、子叶和真叶中都明显减少，子叶中GUS主要在叶脉处表达，在真叶中仅在顶端还有表达，这说明SSMP的表达受NaCl抑制。激光共聚焦显微镜488 nm波长下对SSMP-GFP的亚细胞定位进行观察，SSMP主要定位在细胞质膜上。过表达SSMP转基因拟南芥的细胞膜透性增加，不利于植物的抗逆性。进一步的萌发试验对过表达SSMP的转基因拟南芥的萌发率和转绿率进行了统计分析，结果表明，过表达SSMP明显抑制拟南芥种子的萌发。【结论】SSMP是具有磷脂酰胆碱结合位点的共411个氨基酸的蛋白质，含START结构域，主要位于细胞质膜上；在拟南芥各组织中均有表达，尤其在叶片中表达量最高，主要定位在整个子叶和真叶的叶脉及表皮毛着生处；SSMP的表达受盐胁迫的抑制；过表达SSMP的拟南芥细胞膜透性增加，耐盐性降低。

关键词: 拟南芥, SSMP, 结构解析, 表达特性, 耐盐性

Abstract: 【Objective】The objective of this study is to analyze the structure of SSMP (salt-sensitive membrane protein), understand the expressing characteristics of SSMP, predict and analyze the function of SSMP in the process of stress resistance in Arabidopsis thaliana. 【Method】 The structure of SSMP protein was analyzed using bioinforatics method. Real-time PCR was adopted to analyze the temporal and spatial expression of SSMP in Arabidopsis. Preliminary analysis of the subcellular localization of SSMP was performed through the establishment of SSMP-GFP fusion expression vector and protoplast transformation. The tissue localization carrier was established with SSMP promoter connecting GUS reporter gene to analyze the tissue localization of SSMP. To determine the function of SSMP protein, overexpression experiments were performed in Col-0 Arabidopsis. Full-length cDNA was cloned into a binary vector downstream of the SSMP 35S promoter, and the construct was transformed into Col-0 Arabidopsis. Three lines positive seedlings of the transgenic Arabidopsis of over-expression of SSMP were obtained using Real-time PCR analysis. The phenotype and the stress tolerance of the transgenic Arabidopsis over-expression of SSMP were analyzed and so the biological function of SSMP gene could be understand. The cell membrane permeability was compared between wild type and the transgenic Arabidopsis over-expression of SSMP, the data of the cell membrane permeability was detected by the conductance instrument. The function of SSMP in the stress resistance process of Arabidopsis was analyzed.【Result】SSMP is a funtion unknown protein, it has 411 amino acids, containing a START conserved domain(the lipid/sterol-binding StAR-related lipid transfer protein domains). Bioinformatics analysis showed that SSMP has a phosphatidylcholine binding site, START domain could combine with lipids and sterols and transports cholesterol to inner membrane, so SSMP may influence the membrane components and play a role in the process of plant resistance. In this study, the expression level of SSMP in the tissues or organs of Arabidopsis was analyzed by using real-time PCR technique, andthe results showed that the order of the expression level of SSMP in Arabidopsis organs was stem leaf, cauline leaf, stem, root, flower and seed. Under normal circumstances, GUS is mainly expressed in hypocotyls, the cotyledon and true leaf veins and epidermal hairs. After treatment with 50 mmol·L^-1 NaCl, the expression of GUS in hypocotyls and cotyledons showed no significant changes, but the expression of GUS in true leaf were decreased. After treatment with 100 mmol·L^-1 NaCl, the expression of GUS was significantly reduced in the hypocotyl, cotyledon and true leaf, indicating that the expression of SSMP inhibited by NaCl. Preliminary analysis of the subcellular localization of SSMP was performed through the establishment of SSMP-GFP fusion expression vector and protoplast transformation and observed the fluorescence under 488nm wavelength using laser scanning confocal microscope. The result showed that SSMP was mainly localized on cytoplasma membrane. The cell membrane permeability of the transgenic Arabidopsis leaves of over-expression of SSMP was higher than that of the wild type. The expression of SSMP was inhibited and the germination of the seeds of SSMP function-gaining transgenic plants was lower obviously than wild type in the medium containing NaCl. 【Conclusion】SSMP is a protein of 411 amino acids with phosphatidylcholine binding sites, containing a START domain, mainly located on the cytoplasmic membrane, expressed in various tissues of Arabidopsis thaliana plants, especially the highest expression level in leaves. The expression of SSMP mainly localized in the genesis areas of leaf vein and epidermal hair. The expression of SSMP was inhibited by salt stress. The cell membrane permeability increased and the salt tolerance decreased in Arabidopsis overexpressing SSMP.

Key words: Arabidopsis, SSMP, structure analysis, expression characteristics, salt resistance

张海丽，由诗东，张昊，高静，李生辉，张利辉，邢继红，王凤茹，董金皋. SSMP的结构解析及在拟南芥抗盐过程中的作用[J]. 中国农业科学, 2015, 48(4): 804-812.

ZHANG Hai-li, YOU Shi-dong, ZHANG Hao, GAO Jing, LI Sheng-hui, ZHANG Li-hui, XING Ji-hong, WANG Feng-ru, DONG Jin-gao. Structure Analysis of SSMP and Its Function in Salt Tolerance in Arabidopsis thaliana[J]. Scientia Agricultura Sinica, 2015, 48(4): 804-812.

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