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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。
Objective To clone the JAS gene (NINJA) from Aquilaria sinensis, a native Agrocybe aegerita plant, and lay a foundation for further studies on its function in the biosynthesis pathway. Methods The full length cDNA of NINJA gene was cloned by RACE and RT-PCR methods using bioinformatics method. The total RNA was extracted from the leaves of Mumex jamesonii, and analyzed by real-time fluorescence quantitative (qRT-PCR) (MeJA) treatment of the white woody calli AsNINJA1 gene expression patterns. Results The full length cDNA of NINJA gene was obtained and named AsNINJA1. Sequence analysis showed that the full length cDNA of this gene was 1 982 bp in length and contained a 1 221 bp open reading frame encoding a protein of 406 amino acids with a relative molecular mass of 43 697 and an isoelectric point of 6.02. The results of qRT-PCR showed that the expression of AsNINJA1 gene was the highest in 4 h after treatment with MeJA, which was nearly 100 times of the control (without MeJA treatment) and then decreased significantly. Conclusion The full-length cDNA sequence of AsNINJA1 gene was obtained. The gene was extremely sensitive to MeJA induction and could respond to injury early.