三七几丁质酶基因PnCHI1的克隆及表达特性分析

Cloning and expression analysis of a chitinase gene PnCHI1 from Panax notoginseng

几丁质酶(chitinase,EC3.2.1.14)广泛存在于多种生物体中,催化水解几丁质(chitin),在抗真菌防卫反应体系中具有重要作用,同时几丁质酶还调控植物的生长和发育,参与细胞的程序性死亡(programmed cell death,PCD)。该实验基于三七Panax notoginseng中编码几丁质酶的EST序列设计引物,采用快速扩增cDNA末端技术,克隆得到1个新的几丁质酶基因的全长cDNA序列,命名为PnCHI1。PnCHI1的cDNA全长为1 022 bp,含有822 bp的开放阅读框,26 bp 5'-非翻译区以及174bp的3'-非翻译区,编码具有273个氨基酸的蛋白质。该基因编码的蛋白属于糖苷水解酶19家族成员,进化树分析表明PnCHI1编码的氨基酸序列属于IV类几丁质酶。qRT-PCR分析结果显示,植物信号分子水杨酸、茉莉酸甲酯、H_2O_2和乙烯处理可均明显诱导三七根中PnCHI1的转录水平;三七叶片接种人参链格孢Altemaria panax后,PnCHI1的表达量迅速升高,呈波动上升趋势;外源茉莉酸甲酯预处理三七根部,PnCHI1的表达水平上升,接种茄腐镰刀菌Fusarium solani后,PnCHI1的转录水平急剧上升,接种后4 h达到最高转录水平;而无菌水预处理三七受茄腐镰刀菌侵染过程中,PnCHI1的表达量逐渐上升,至48h达到最大值。上述结果表明PnCHI1参与三七对根腐病菌以及黑斑病菌的防卫反应。

Chitinases（EC3.2. 1.14）, which are present in various organisms, catalyze the hydrolytic cleavage of chitin and play a vital role in plant defense mechanisms against fungal pathogens. In addition, the chitinases are well known to regulate plant growth and development and are involved in programmed cell death（PCD）. A chitinase expressed sequence tag（EST） was isolated from Panax notoginseng, and the full-length cDNA of this EST was cloned with the method of rapid amplification of cDNA ends and named as PnCHI1. PnCHI1 was 1 022 bp in length and contained an intact open reading frame（ORF） of 822 bp, a 26 bp 5＇-untranslated region （UTR）, and a 174 bp 3＇-UTR. The predicted protein of PnCHI1 with 273 amino acid residues belongs to glycoside hydrolase family 19 and fell into the class Ⅳ of chitinases through phylogenetic analysis. QRT-PCR analysis showed that the expression of PnCHI1 was induced by methyl jasmonate, ethylene, H2O2, and salicylic acid. PnCHI1 was quickly induced after inoculation with Alternaria panax. Moreover, the expression level of PnCHI1 was increased after pretreatment with methyl jasmonate, and then the transcription level of PnCHllwas sharp increased after inoculation with Fusarium solani, and the highest transcription level was achieved at 4 h post inoculation. But the expression level of PnCHI1 in the sterile water pretreated P. notoginseng was increased gradually after inoculation with F. solani, and the highest expression level was achieved at 48 h post inoculation. All the results of present study indicated that PnCHI1 was involved in defense response of P. notoginseng against the F. solani and A. panax.