| 摘要: |
| 【目的】 克隆川杨几丁质酶基因cDNA全长序列，分析其在病原菌侵染过程中不同时段的表达特征。【方法】 以受落叶松 杨栅锈菌(Melampsora larici populina)单孢子堆菌系Sb052侵染后的川杨叶片为试材，采用RACE法克隆川杨几丁质酶基因cDNA序列，对其进行生物信息学和实时荧光定量表达分析。【结果】 克隆到1条川杨几丁质酶基因序列，将其命名为PsChiⅠ(GenBank 登录号：KC416180)。该序列全长1 145 bp，包含1段960 bp的开放阅读框(ORF)，38 bp的5′非编码区和147 bp的3′非编码区。其编码蛋白含有319个氨基酸，具有2条糖苷水解酶19家族特征序列，理论等电点为5.03，为ClassⅠb型酸性几丁质酶，属于几丁质酶第19家族。序列比对和系统进化树结果表明，PsChiⅠ与毛果杨(Populus trichocarpa)几丁质酶基因相似性最高，且亲缘关系最近。实时荧光定量PCR结果表明，菌系Sb052侵染杨树叶片后PsChiⅠ基因在各个时段都有表达，但以侵染后12和48 h时的表达量相对较高。【结论】 获得了几丁质酶基因全长序列，推测其参与了寄主川杨抵抗真菌的防御机制。 |
| 关键词: 杨树 几丁质酶基因 RACE技术 实时荧光定量PCR |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金项目（30872027） |
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| Cloning and expression of PsChiⅠ gene from poplar |
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WANG Qiong,CAO Zhi-min
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| Abstract: |
| 【Objective】This research aimed to clone the full length cDNA of chitinase gene from Populus szechuanica and analyze its expression characteristics during pathogenesis process.【Method】The cDNA of chitinase gene (GenBank accession:KC416180) from P.szechuanica leaves infected by Melampsora larici populina Sb052 was cloned using RACE technology,and analyzed by bioinformatics method and real time quantitative PCR.【Result】Full length of PsChiⅠ gene (GenBank accession number:KC416180 ) with 1 145 bp in length was obtained.It contained a 38 bp 5′ untanslated region (5′ UTR),a 147 bp 3′ UTR,and a 960 bp open reading frame (ORF).The predicted protein encoded 319 amino acid residues and contained two sequences belonging to the 19th family signature sequences of chitinases.Its theoretical isoelectric point was 5.03.The gene obtained from P.szechuanica belonged to ClassⅠb acidic chitinase,a member of the 19th chitinase family.Sequence alignment and phylogenetic tree revealed that the deduced amino acid sequence of PsChiⅠ had the highest similarity and closest phylogenetic relationship with P.trichocarpa.QRT PCR analysis showed that PsChiⅠ gene expressed in all pathogenesis processes and the highest relative expression levels occurred at 12 hpi and 48 hpi.【Conclusion】A full length of chitinase gene was obtained,and its role in poplar against fungal infection was analyzed. |
| Key words: poplar chitinase gene RACE technology real-time PCR |
