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几丁质酶(Chitinase,EC 3.2.2.14)参与植物的生长发育及防卫反应。本研究借助电子克隆技术,以甘蔗(Saccharum spp.)抗黑穗病基因型崖城05-179针刺接种黑穗病菌(Sporisorium scitamineum)48 h的蔗芽c DNA为实验材料,克隆获得一个甘蔗几丁质酶c DNA序列(Gen Bank登录号:KP165001),命名为Sc ChiⅣ1。生物信息学分析结果显示,该核苷酸序列全长为1 037 bp,包含1个长为825 bp的完整开放读码框,编码274个氨基酸,理论等电点为8.29。该基因推导的编码蛋白属于糖苷水解酶19家族成员,含信号肽和几丁质结合区,胞外定位的概率为0.820,推测Sc ChiⅣ1是一种胞外分泌蛋白,同时兼具溶菌酶活性。聚类结果显示,Sc ChiⅣ1归属于ClassⅣ几丁质酶基因。荧光定量PCR分析显示,甘蔗接种黑穗病菌后的96 h内,抗、感基因型中Sc ChiⅣ1表达量均高于对照,且在亲和互作中Sc ChiⅣ1转录本上升应答快(6 h),并在接种黑穗病菌后96 h持续更长,其在甘蔗不同黑穗病抗/感基因型材料(崖城05-179/柳城03-182)与黑穗病菌互作中的表达模式存在差异,但总体结果表明Sc ChiⅣ1受黑穗病菌胁迫诱导表达。本研究为后续甘蔗几丁质酶功能鉴定及甘蔗抗黑穗病基因工程研究奠定基础。
Chitinase (EC 3.2.2.14) is involved in plant growth and defense response. In this study, we cloned c DNA from sugarcane (Saccharum spp.) Cl. Yac-cheung 05-179, which was inoculated with Sporisorium scitamineum for 48 h, to obtain a sugarcane Chitinase c DNA sequence (Gen Bank accession number: KP165001), named Sc Chi IV1. Bioinformatics analysis showed that the full length of the nucleotide sequence was 1 037 bp, including a complete open reading frame of 825 bp, encoding 274 amino acids with a theoretical isoelectric point of 8.29. The deduced protein encoded by this gene belongs to the 19 family members of glycoside hydrolase, which contains signal peptide and chitin binding region. The probability of extracellular localization is 0.820. It is speculated that Sc ChiⅣ1 is an extracellular secreted protein with both lysozyme activity. Cluster analysis showed that Sc ChiⅣ1 belongs to Class Ⅳ chitinase gene. Fluorescence quantitative PCR analysis showed that Sc ChiⅣ1 expression level in resistant and susceptible genotypes was higher than that in control within 96 hours after inoculation with Saccharomyces cerevisiae, and Sc ChiⅣ1 transcripts increased rapidly in affinity interaction (6 h) , And persisted longer after 96 h inoculation with S. japonicum, which existed in the interaction between resistant and susceptible genotypes of sugarcane (Yacheng 05-179 / Liucheng 03-182) However, the overall results showed that Sc Chi IV was induced by Staphylococcus aureus. This study laid the foundation for the subsequent functional identification of sugarcane chitinase and genetic engineering of Sugarcane smut resistance.