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中草药 Chinese Traditional and Herbal Drugs 第45 卷第20 期2014 年10 月・2968・ ? 药材与资源 ? 白木香AsNINJA1基因全长cDNA克隆及其在愈伤组织中的茉莉酸甲酯诱导表达 廖永翠

1 ,徐艳红

1 ,张争1,

2 ,隋春1,梁 良1,

3 ,魏建和 1, 2* 1.

中国医学科学院 北京协和医学院药用植物研究所,北京

100193 2. 中国医学科学院 北京协和医学院药用植物研究所海南分所, 海南省南药资源保护与开发重点实验室, 海南 万宁

571533 3. 山东中医药大学药学院,山东 济南

250012 摘要:目的 克隆国产沉香基原植物白木香 Aquilaria sinensis 互作蛋白(jasmonate Zim-domain,JAZ)基因(NINJA) ,为深 入研究其在沉香倍半萜合成途径中的功能奠定基础.方法 以白木香总 RNA 为模板,采用 RACE 法和 RT-PCR 方法,克隆白 木香 NINJA 基因 cDNA 全长,并进行生物信息学的分析;

采用实时荧光定量(qRT-PCR)方法分析经茉莉酸甲酯(MeJA)处 理的白木香愈伤组织中 AsNINJA1 基因的表达模式.结果 获得白木香 NINJA 基因全长 cDNA,命名为 AsNINJA1.序列分析 表明,该基因的序列全长为

1 982 bp,包含一个

1 221 bp 的开放阅读框,编码

406 个氨基酸,蛋白质相对分子质量为

43 697, 等电点为 6.02.qRT-PCR 结果显示,经MeJA 处理

4 h 后AsNINJA1 基因相对表达量最高,是对照(未经 MeJA 处理的白木香 愈伤组织)的近

100 倍,随后显著下降.结论 获得 AsNINJA1 基因全长 cDNA 序列,该基因对 MeJA 诱导极为敏感,且能在 伤害早期响应. 关键词:白木香;

AsNINJA1 基因;

cDNA 快速末端扩增;

序列分析;

表达分析;

茉莉酸甲酯 中图分类号:R282.12 文献标志码:A 文章编号:0253-2670(2014)20 -

2968 -

06 DOI: 10.7501/j.issn.0253-2670.2014.20.019 Full-length cDNA cloning of AsNINJA1 gene in Aquilaria sinensis and MeJA-induced expression in callus LIAO Yong-cui1 , XU Yan-hong1 , ZHANG Zheng1,

2 , SUI Chun1 , LIANG Liang1,

3 , WEI Jian-he1,

2 1. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences &

Peking Union Medical College, Beijing 100193, China 2. Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine Hainan Branch Institute of Medicinal Plant, Chinese Academy of Medical Sciences &

Peking Union Medical College, Wanning 571533, China 3. College of Pharmacy, Shandong Traditional Chinese Medicine University, Jinan 250012, China Abstract: Objective To clone the full-length cDNA of interacting protein of JAZ (NINJA) gene in Aquilaria sinensis, and to provide the basic information for further study on gene function in sesquiterpenes biosynthesis pathway. Methods With the total RNA as template, the full-length cDNA of NINJA in A. sinensis was cloned through RACE technique and RT-PCR method. The bioinformatics of cloing NINJA gene was analyzed as well. The expression mode of this gene was with MeJA treatment in A. sinensis callus detected by qRT-PCR method. Results The full-length cDNA(1

982 bp) of NINJA gene in A. sinensis, named as AsNINJA1 was obtained with an open reading frame of

1 221 bp and encoding

406 amino acids. The relative molecular mass ofAsNINJA1 protein calculated was

43 697, and the isoelectric point was 6.02. The qRT-PCR results indicated that MeJA treatment could stimulate the increase of mRNA expression of AsNINJA1;

There was a sharp rise at

4 h with nearly

100 times higher than the control (without MeJA treatment), then dropped significantly. Conclusion The full-length cDNAsequence of AsNINJA1 gene is obtained;