甘薯几丁质酶基因IbChiA启动子的克隆及功能分析

Cloning and Functional Analysis of the Promoter of Chitinase IbChiA from Sweetpotato(Ipomoea batatas)

几丁质酶能够水解真菌细胞壁,抑制真菌的生长与繁殖,水解产物还能诱导植物的系统抗性,在植物抗病过程中发挥重要的调控作用。本实验室前期从甘薯中克隆了几丁质酶基因IbChiA并对其进行了功能验证和表达分析,结果表明黑斑病菌强烈诱导IbChiA基因的表达,且在不同抗性甘薯品种中的表达差异显著。在此基础上,本研究在抗感品种中分别克隆了IbChiA基因的2 034 bp的启动子序列,对比后发现其序列并无差异。PlantCARE在线分析显示,IbChiA启动子中包含核心元件TATA-box和CAAT-box,还有响应真菌诱导的作用元件(W-box)以及植物激素等相关的顺式作用元件。将IbChiA基因的全长启动子及7个启动子5’端缺失片段分别替换植物表达载体pHB的2xCaMV 35S启动子,并在本生烟草叶片中进行瞬时表达。eGFP的荧光及定量表达分析显示在基因上游600 bp及以上都能够正常稳定地发挥启动子的功能,其中在-1 605~1 188 bp的片段内可能存在增强转录的顺式作用元件。本研究为进一步探索甘薯IbChiA基因的调控和表达机制提供了理论依据,并为甘薯抗病基因工程提供了一个理想的候选启动子。

Chitinase plays an important antifungal role in plants by hydrolyzing the β-1,4-glycosidic bond of fungal cell wall chitin into N-acetyl glucosamine oligomers, directly causing rapid lysis of fungal hyphal tips and germinating spores, indirectly inducing plant defense response. Previously, the chitinase IbChiA was cloned from sweetpotato, its antifungal activity and expression pattern were tested, showing that the expression of IbChiA was dramatically enhanced by Ceratocystis fimbriata in resistant cultivars. In this study, the 2 034 bp promoter region of IbChiA were cloned, indicating that there was no difference in the promoter sequence between two cultivars. Online analysis by PlantCARE showed that this IbChiA promoter included a number of known cis-elements, such as TATA-box and CAAT-box, as well as pathogen and phytohormone responsive motifs, etc. The 2 xCaMV 35 S pro moter of plant expression vector pHB were replaced by the full-length IbChiA promoter and its seven 5’-deletion fragments, respectively, which were then transiently expressed in leaves of Nicotiana benthamiana. The fluorescence of eGFP and quantitative analysis of e GFP gene expression revealed that promoter fragments at 600 bp or above in the upstream of IbChiA could play a stable promoter function, and the sequence between-1 605~-1 188 bp may contain some cis-elements which could enhance the transcription levels. These findings laid the foundation for further studying the expression and regulation mechanism of IbChiA, and this promoter would be an ideal candidate for genetic engineering for improving disease resistance of sweet potatoes.