论文部分内容阅读
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。
Chitinase (EC3.2.1.14) is widely found in many organisms and catalyzes the hydrolysis of chitin, which plays an important role in the antifungal defense reaction system. Chitinase also regulates plants Of the growth and development, involved in programmed cell death (PCD). Based on the EST sequence encoding chitinase in Panax notoginseng from Panax notoginseng, primers were designed and the full-length cDNA sequence of a new chitinase gene was cloned by means of rapid amplification of cDNA ends and named PnCHI1. The full-length cDNA of PnCHI1 was 1 022 bp with an open reading frame of 822 bp, a 26 bp 5'-untranslated region and a 174 bp 3'-untranslated region encoding a 273 amino acid protein. The protein encoded by this gene belonged to the 19 family members of glycoside hydrolase. Phylogenetic tree analysis showed that the amino acid sequence encoded by PnCHI1 belonged to class IV chitinase. The result of qRT-PCR analysis showed that plant signal molecules salicylic acid, methyl jasmonate, H 2 O 2 and ethylene could obviously induce the transcription of PnCHI1 in Panax notoginseng. After inoculation with Altemaria panax of Panax notoginseng, PnCHI1 The expression of PnCHI1 increased rapidly and reached the peak at 4 h after inoculation with Fusarium solani. The expression of PnCHI1 was up-regulated after pre-treatment with methyl jasmonate PnCHI1 expression reached the maximum at 48h. However, during the infection of Fusarium solani with Pseudomonas solanacearum, the expression level of PnCHI1 gradually increased. The above results indicate that PnCHI1 is involved in the defense response of P. notoginseng against Alternaria solani and Alternaria.