葡萄耐铝毒基因MATE的克隆与表达分析

Cloning and Expression Analysis of the Aluminum-Toxicity-Tolerance Gene MATE in Grape

【目的】铝毒胁迫是酸性土壤中抑制葡萄生长的主要因素之一,为了探明葡萄MATE基因的结构特征和表达量与其耐铝毒能力的关系。【方法】以耐铝性强的品种‘红地球’和耐铝性弱的品种‘贝达’的叶片cDNA为模板,采用PCR和TA克隆技术获得VvMATE和VrlMATE两个基因序列,对其进行生物信息学和铝胁迫下的表达分析,并测定铝胁迫下的丙二醛含量及抗氧化酶活性,对两个品种该基因的表达和生理指标的变化与耐铝毒能力的关系进行比较。【结果】克隆获得了‘红地球’VvMATE基因和‘贝达’VrlMATE基因,二者序列全长均为1794 bp,均编码597个氨基酸;二者的分子质量分别为68.28 ku和68.82 ku,理论等电点分别为10.15和9.92;二者的二级结构组分占比由高到低均为无规则卷曲、α-螺旋和折叠延伸链,三级空间结构由α-螺旋、β-折叠片和β-转角等组成;系统进化树分析发现,VvMATE和VrlMATE的同源性为91%,二者与大麦HvMATE、小麦TaMATE、高粱SbMATE和水稻OsMATE的同源性均在70%以上;此外,二者的MATE基因表达量在铝胁迫下均先上升后下降,其中耐铝性强的‘红地球’的表达量和抗氧化酶活性的上升幅度均高于耐铝性弱的‘贝达’。【结论】铝胁迫下,葡萄MATE基因的表达量与其耐铝毒能力呈正相关,本研究为进一步探明葡萄耐铝毒的分子调控机制提供参考。

[Objective]Aluminum(Al)toxicity stress is one of the main factors to inhibit grape growth in acidic soil.The relationship between the structure and expression of grape MATE gene and its ability to resist aluminum toxicity was investigated.[Method]The cDNA from the leaves of‘Red Globe’with strong Al resistance and‘Beta’with weak Al resistance were used as the template,two gene sequences VvMATE and Vrl⁃MATE were obtained by PCR and TA cloning technology,and then bioinformatics analysis and expression analysis under Al stress were carried out.The malondialdehyde content and antioxidant enzyme activities were measured under Al stress.The relationship between the expression of these genes,the changes in physiological indi-ces and Al resistance were compared between the two cultivars.[Result]The‘Red Globe’VvMATE gene and the‘Beta’VrlMATE gene were cloned,the total length of the two sequences were 1794 bp,encoding 597 amino acids;the relative molecular weights were 68.28 ku and 68.82 ku,and the theoretical isoelectric points were 10.15 and 9.92,respectively;the secondary structure was in the order of irregular curl>α-helix>folding extension chain,and the third spatial structure was composed ofα-helix,β-folded sheet andβ-angle.Phylogenetic tree analysis found that the homology of VvMATE and VrlMATE was 91%,and the homology of them with barley HvMATE,wheat TaMATE,sorghum SbMATE and rice OsMATE was more than 70%.In addition,under Al stress,the expression of MATE genes in both of them increased first and then decreased,and the expression of MATE gene and antioxidant enzymes in‘Red Globe’with strong Al tolerance were higher than those in‘Beda’with weak Al tolerance[.Conclusion]Under Al stress,the expression of grape MATE gene was positively correlated with its ability to resist aluminum toxicity.This study provides a reference for the further investigation of the molecular regulation mechanism of Al resistance in grape.