长春花铜锌超氧化物岐化酶基因克隆及其在黄龙病菌侵染后的表达

Cloning and Expression Analysis of Copper and Zinc Superoxide Dismutase(Cu/Zn-SOD) Gene from Periwinkle(Catharanthus roseus) Following Infection by 'Candidatus liberibacter asiaticus'

【目的】克隆长春花(Catharanthus roseus)铜锌超氧化物歧化酶(Cu/Zn superoxide dismutase, Cu/Zn-SOD)基因,研究黄龙病菌("CandidatusLiberibacterasiaticus")侵染后长春花Cu/Zn-SOD基因(Cr Cu/Zn-SOD)的表达。【方法】根据不同作物Cu/Zn-SOD基因序列同源性筛选出CrCu/Zn-SOD基因引物,扩增得Cr Cu/Zn-SOD基因片段,用SMART-RACE方法扩增出Cr Cu/Zn-SOD基因的3′和5′末端,用q RT-PCR分析嫁接后不同时间长春花叶、主茎和根中Cr Cu/Zn-SOD基因的m RNA转录水平。【结果】拼接后获得Cr Cu/Zn-SOD基因全长796 bp(Genbank登录号KP864639),该基因与其他物种的Cu/Zn-SOD基因同源性达86%,编码1个亲水性稳定蛋白,无跨膜结构域及N末端无信号肽。染病长春花叶、茎和根中,CrCu/Zn-SOD的表达在侵染25 d前均上调,30～45 d时表达量持续下降,45 d时叶、茎和根中表达量分别下调1.8倍、2.97倍和2.9倍。【结论】长春花Cu/Zn-SOD序列与其他物种同源性较高,Cr Cu/Zn-SOD表达受黄龙病菌侵染诱导,表达量变化与长春花抗黄龙病菌侵染能力密切相关。

【Objectives】To obtain the full-length of Cu/Zn-SOD gene from Catharanthus roseus(CrCu/Zn-SOD) and study the expression profile of CrCu/Zn-SOD in C. roseus infected by "CandidatusLiberibacter asiaticus"(Ca Las). 【Method】The fragment of CrCu/Zn-SOD was obtained using PCR technique by designing specific primers according to the homology of Cu/Zn-SOD genes from different species. By rapid amplification of cDNA ends(RACE), the 3′ and 5′ ends of cDNA sequence of CrCu/Zn-SOD gene were amplified from C. roseus. Quantitative Real-time PCR(qRT-PCR) was adopted to analyze the expression of CrCu/Zn-SOD gene in the leaves, center stems, and roots of C. roseus infected by Ca Las in different days after inoculation(DAI). 【Result】Nucleotide sequence analysis showed that the total length of cDNA sequence of CrCu/Zn-SOD is 796 bp. Bioinformatics analyses showed that amino sequence similarity was 86% in contrast with the amino sequence of other plants, and the predicted Cr Cu/Zn-SOD protein was a hydrophilic protein without signal-peptide and transmembrane region. The results showed that the expression level of Cr Cu/ZnSOD was up-regulated before 25 DAI and decreased with the DAI. The expression of CrCu/ZnSOD were down-regulated 1.8, 2.97 and 2.9 fold at 45 DAI in the leaves, center stems, and roots, respectively. 【Conclusion】The CrCu/Zn-SOD protein has a higher homology with Cu/Zn-SOD of other species. The results revealed that the CrCu/Zn-SOD gene was induced by infection of Ca Las, and the expression level could be related to the resistance ability of host periwinkle against Ca Las invasion.