芝麻肌醇半乳糖苷合成酶基因SiGolS6的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2020.17.002

摘要/Abstract

摘要：

【目的】SiGolS6是芝麻肌醇半乳糖苷合成酶家族成员,在植物抗旱过程中可能发挥重要作用。研究SiGolS6在植物干旱胁迫抗性中的功能,以期为芝麻抗旱遗传改良提供理论基础和基因资源。【方法】利用反转录PCR（reverse transcription PCR,RT-PCR）方法从芝麻中克隆获得肌醇半乳糖苷合成酶基因SiGolS6。使用InterProScan、ClustalX2和MEGA5.2等生物信息学软件对序列进行分析。通过对转SiGolS6拟南芥材料的表型分析和生理指标测定,研究SiGolS6在植物抗旱性中的功能及作用机理。【结果】克隆获得SiGolS6的全长CDS序列为921 bp,编码306个氨基酸,其蛋白质分子量为35.07 kD,等电点为4.75。序列分析显示SiGolS6蛋白含有糖基转移酶保守结构域（IPR002495）,属于糖基转移酶超家族。与其他物种GolS蛋白构建的系统进化树中,SiGolS6与马铃薯中的同源基因相似度较高。利用潮霉素筛选和PCR鉴定,获得6个独立的转基因拟南芥株系;qRT-PCR分析筛选出表达量较高的3个转基因株系（OE-2、OE-3和OE-4）用于后续试验分析,检测发现这三个转基因株系的棉子糖含量显著高于野生型植株。干旱胁迫下,转基因株系萎蔫程度较野生型轻,干旱胁迫28 d和复水恢复生长5 d后,转基因株系的鲜重均显著高于野生型,而正常供水条件下无明显差异;干旱胁迫28 d后复水生长5 d,50%的转基因植株基本恢复,而野生型植株存活率不到10%。干旱胁迫21 d,转基因植株的相对电导率,活性氧积累和相对MDA含量均显著低于野生型,而相对SOD、POD活性则显著高于野生型。【结论】在拟南芥中超量表达芝麻SiGolS6,能够提高植物在干旱胁迫中的耐受性。

关键词: 芝麻, 肌醇半乳糖苷合成酶, 转基因拟南芥, 耐旱性, 功能分析

Abstract:

【Objective】SiGolS6 is a member of galactinol synthase family in sesame, which may play important role in drought stress resistance. Studying the function of SiGolS6 in plant drought stress resistance, so as to provide theoretical basis and gene resources for drought resistance improvement of sesame. 【Method】The galactinol synthase gene, SiGolS6, was isolated from sesame through reverse transcription PCR (RT-PCR). The sequence was analyzed by bioinformatics tools such as InterProScan, ClustalX2 and MEGA5.2. Then the function of SiGolS6 in plant drought resistance was characterized by phenotype analysis and physiological index measurement of SiGolS6 transgenic Arabidopsis thaliana. 【Result】The total length of the CDS sequence of SiGolS6 was 921 bp, encoding a polypeptide of 306 amino acids. The molecular weight of SiGolS6 is 35.07 kD and its isoelectric point is 4.7. Sequence analysis showed that SiGolS6 protein contained conserved glycosyl transferase domain (IPR002495), belonging to the glycosyl transferase superfamily. Based on phylogenetic tree constructed with GolS proteins from sesame and other species, SiGolS6 was highly similar to the homologous genes in potato. Six independent transgenic Arabidopsis thaliana lines were identified by hygromycin screening and PCR. Three transgenic lines (OE-2, OE-3, and OE-4) with high expression levels were identified by qRT-PCR, and used for subsequent experiment. Raffinose content in those transgenic plants was higher than that in wild-type (WT) plants. Under drought stress, the wilting degree of transgenic lines was less than that of WT lines. After 28 d of drought stress and 5 d of recovery, the fresh weight of transgenic lines was significantly higher than that of WT, but there was no significant difference under normal conditions. After 5 d of recovery, 50% of transgenic plants recovered, while the survival rate of WT was less than 10%. After 21 d of drought stress, the relative electrical conductivity, ROS accumulation and MDA content of transgenic plants were significantly lower than those of WT, while the relative activities of SOD and POD were significantly higher than those of WT.【Conclusion】Overexpression of SiGolS6 in Arabidopsis thaliana could improve the drought tolerance of transgenic plant.

Key words: sesame, galactinol synthase, transgenic Arabidopsis, drought tolerance, functional analysis

刘爱丽,魏梦园,黎冬华,周瑢,张秀荣,游均. 芝麻肌醇半乳糖苷合成酶基因SiGolS6的克隆及功能分析[J]. 中国农业科学, 2020, 53(17): 3432-3442.

LIU AiLi,WEI MengYuan,LI DongHua,ZHOU Rong,ZHANG XiuRong,YOU Jun. Cloning and Function Analysis of Sesame Galactinol Synthase Gene SiGolS6 in Arabidopsis[J]. Scientia Agricultura Sinica, 2020, 53(17): 3432-3442.

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)
SiGolS6q-F	CCAATCCCCGCAACCTATAAC
SiGolS6q-R	TGCTCTTTGTCGGTGTACTTC
Actinq-F	CCCGCTATGTATGTCGCCA
Actinq-R	AACCCTCGTAGATTGGCACAG
GolS6FL-F	GGGGTACCATGGTTCCTGAAATTATCATTCC
GolS6FL-R	CGGGATCCTCAACCGGAAACTTGATCAATTG