梨PbChi Ⅱ基因的克隆及荧光定量表达分析

Cloning and Quantitative Expression Analysis of PbChi Ⅱ Gene in Pyrus bretschneideri 'Yali'

以鸭梨为试材,采用qRT-PCR技术克隆鸭梨果实几丁质酶基因PbChi Ⅱ,并对几丁质酶氨基酸序列进行聚类分析;同时采用荧光定量PCR的方法对鸭梨植株不同器官及水杨酸(salicylic acid,SA)和梨轮纹病菌(Botryosphaeria berengeriana de.Not.f.sp.piricola(Nose)Koganecawa et.Sokwwa)处理后几丁质酶基因的表达量进行了分析,以期为该基因的进一步研究与利用提供参考依据。结果表明:PbChi Ⅱ基因长度为969bp,核苷酸序列及推导的氨基酸序列与沙梨的同源性均达到100%,该基因属于第Ⅱ类几丁质酶基因;PbChi Ⅱ在根和果实中表达量较大。在鸭梨果实中,SA和病原菌可诱导该基因表达,且表达量在48h达到最高,初步推测PbChi Ⅱ可能参与SA介导的植物抗病防卫反应的信号通路及轮纹病菌引起的防卫反应。

The CDS of PbChiⅡ was cloned from ‘Yali’ using qRT-PCR method,phylogenetic tree was clustered by MEGA 6.0 based on amino acid of PbChiⅡ and known chitinase gene of other plants.At the same time,gene expression levels were analyzed by real-time quantitative PCR method for ‘Yali’ in different organs and the expression after treatment with SA（salicylic acid）and Botryosphaeria berengeriana de.Not.f.sp.piricola （Nose） Koganecawa et.Sokwwa.The results showed that cDNA lengths of PbChiⅡ gene was 969 bp,the nucleotide sequence and the induced amino acids had 100% homology with Pyrus pyrifolia,this gene belonged to Ⅱ chitinase gene.The expression of PbChiⅡ in roots and fruits were higher,the expression of the gene could be induced by SA and pathogen.In addition,the expression was enhanced and up to the peak at 48 hours after treatment with SA and pathogen.It was speculated that PbChiⅡ might be involved in SA-mediated basic signaling pathway of plant defense responses and the defense responses mediated by B.berengeriana.f.sp.Piricola.