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
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摘要
【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.
【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.
引文
[1]GARNIER M,BOVE J M.Tranmission of the organism associated with citrus greening disease from sweet orange to periwinkle by dodder[J].Phytopathology,1983,73(10):1358-1363.
[2]BOVéJ M.Huanglongbing:A destructive,newly-emerging,century-old disease of citrus[J].Journal of Plant Pathology,2006,88(1):7-37.
[3]BOVéJ M.Heat-tolerant Asian HLB meets heat-sensitive African HLB in the Arabian Peninsula!Why?[J].Journal of Citrus Pathology,2014,1(1):1-78.
[4]ALSCHER R G,ERTURK N,HEATH L S.Role of superoxide dismutases(SODs)in controlling oxidative stress in plant[J].Journal of Experimental Botany,2002,53(372):1331-1341.
[5]ALLEN R D,WEBB R P,SCHAKE S A.Use of transgenic plants to study antioxidant defenses[J].Free Radical Biology and Medicine,1997,23(3):473-479.
[6]SUMIO K,KOZI A.Characteristic amino acid sequences of chloroplast and cytosol isozymes of Cu/Zn superoxide dismutase in spinach,rice and horsetail[J].Plant and Cell Physiolohy,1990,31(1):99-112.
[7]KERNODLE S P,SCANDALIOS J G.A comparison of the structure and function of the highly homologous maize antioxidant Cu/Zn superoxide dismutase genes SOD4 and SOD4A[J].Genetics,1996,144(1):317-328.
[8]FAIZE M,BURGOS L,FAIZE L,et al.Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress[J].Journal of Experimental Botany,2011,62(8):2599-2613.
[9]胡根海,喻树迅,范术丽,等.编码棉花胞质铜锌超氧物歧化酶基因的克隆与表达分析[J].中国农业科学,2007,40(8):1602-1609.
[10]李玉坤,王学敏,高洪文,等.东方山羊豆Cu/Zn SOD基因的克隆及表达分析[J].遗传,2012,34(1):95-101.
[11]纪砚耘,化文平,王喆之.丹参铜锌超氧化物歧化酶(Cu/Zn-SOD)基因的克隆与生物信息学分析[J].陕西师范大学学报(自然科学版),2011,39(3):56-61.
[12]曾秀存,刘自刚,史鹏辉,等.白菜型冬油菜铜锌超氧化物歧化酶(Cu/Zn-SOD)基因的克隆及其在低温条件下的表达[J].作物学报,2014,40(4):636-643.
[13]肖翠,严佳文,龙桂友,等.柑橘内参基因的稳定性评价[J].果树学报,2012,29(6):978-984.
[14]李亚,贾奥琳,鲍敏丽,等.长春花感染黄龙病菌多酚氧化酶基因的表达及活性分析[J].华南农业大学学报,2017,38(2):63-68.
[15]HU L X,HUANG Z H,LIU S Q,et al.Growth response and gene expression in antioxidant-related enzymes in two bermudagrass genotypes differing in salt tolerance[J].Journal of the american society for horticultural science,2012,137:134-143.
[16]MACHO A P,LOZANO-DURAN R,ZIPFEL C.Importance of tyrosine phosphorylation in receptor kinase complexes[J].Trends in Plant Science.2015,20(5):269-272.
[17]MCKERSIE B D,MURNAGHAN J,JONES K S,et al.Iron-superoxide dismutase expression in transgenic alfalfa increases winter survival without a detectable increase in photosynthetic oxidative stress tolerance[J].Plant Physiol,2000,122(4):1427-1438.
[18]GóMEZ J M,JIMéANEZ A,OLMOS E.Location and effects of long term NaCl stress on superoxide dismutase and ascorbate peroxidase isoenzymes of pea(Pisum sativum cv.Puget)chloroplasts[J].Journal of Experimental Botany,2004,55(394):119-130.
[19]NEGI N P,SHRIVASTAVA D C,SHARMA V,et al.Overexpression of Cu Zn SOD from Arachis hypogaea alleviates salinity and drought stress in tobacco[J].Plant Cell Reports,2015,34(7):1109-1126.
[20]巨延虎,杨旸,孔令广,等.过表达嗜热毛壳菌Cu/Zn-SOD基因提高转基因水稻对3种病害的抗性[J].植物病理学报,2015,45(3):297-306.
[21]杨鸯鸯,李云,丁勇,等.甘蓝型油菜Cu/Zn SOD和FeSOD基因的克隆及菌核病菌诱导表达[J].作物学报,2009,35(1):71-78.
[22]XU J,DUAN X G,YANG J,et al.Enhanced reactive oxygen species scavenging by overproduction of superoxide dismutase and catalase delays postharvest physiological deterioration of cassava storage roots[J].Plant Physiology,2013,161(3):1517-1528.
[23]ALBRECHT U,BOWMAN K D.Transcriptional response of susceptible and tolerant citrus to infection with Candidatus Liberibacter asiaticus[J].Plant Science,2012,185-186(4):118-130.
[24]FAN J,CHEN C,YU Q,et al.Comparative transcriptional and anatomical analyses of tolerant rough lemon and susceptible sweet orange in response to‘Candidatus Liberibacter asiaticus’infection[J].Molecular Plant Microbe Interaction,2012,25(11):1396-1407.
[25]XU M R,LI Y,ZHENG Z,et al.Transcriptional analyses of mandarins SERIOUSLY infected by‘Candidatus Liberibacter asiaticus’[J].PLoS One,2015,10(7):e0133652.
[2]BOVéJ M.Huanglongbing:A destructive,newly-emerging,century-old disease of citrus[J].Journal of Plant Pathology,2006,88(1):7-37.
[3]BOVéJ M.Heat-tolerant Asian HLB meets heat-sensitive African HLB in the Arabian Peninsula!Why?[J].Journal of Citrus Pathology,2014,1(1):1-78.
[4]ALSCHER R G,ERTURK N,HEATH L S.Role of superoxide dismutases(SODs)in controlling oxidative stress in plant[J].Journal of Experimental Botany,2002,53(372):1331-1341.
[5]ALLEN R D,WEBB R P,SCHAKE S A.Use of transgenic plants to study antioxidant defenses[J].Free Radical Biology and Medicine,1997,23(3):473-479.
[6]SUMIO K,KOZI A.Characteristic amino acid sequences of chloroplast and cytosol isozymes of Cu/Zn superoxide dismutase in spinach,rice and horsetail[J].Plant and Cell Physiolohy,1990,31(1):99-112.
[7]KERNODLE S P,SCANDALIOS J G.A comparison of the structure and function of the highly homologous maize antioxidant Cu/Zn superoxide dismutase genes SOD4 and SOD4A[J].Genetics,1996,144(1):317-328.
[8]FAIZE M,BURGOS L,FAIZE L,et al.Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress[J].Journal of Experimental Botany,2011,62(8):2599-2613.
[9]胡根海,喻树迅,范术丽,等.编码棉花胞质铜锌超氧物歧化酶基因的克隆与表达分析[J].中国农业科学,2007,40(8):1602-1609.
[10]李玉坤,王学敏,高洪文,等.东方山羊豆Cu/Zn SOD基因的克隆及表达分析[J].遗传,2012,34(1):95-101.
[11]纪砚耘,化文平,王喆之.丹参铜锌超氧化物歧化酶(Cu/Zn-SOD)基因的克隆与生物信息学分析[J].陕西师范大学学报(自然科学版),2011,39(3):56-61.
[12]曾秀存,刘自刚,史鹏辉,等.白菜型冬油菜铜锌超氧化物歧化酶(Cu/Zn-SOD)基因的克隆及其在低温条件下的表达[J].作物学报,2014,40(4):636-643.
[13]肖翠,严佳文,龙桂友,等.柑橘内参基因的稳定性评价[J].果树学报,2012,29(6):978-984.
[14]李亚,贾奥琳,鲍敏丽,等.长春花感染黄龙病菌多酚氧化酶基因的表达及活性分析[J].华南农业大学学报,2017,38(2):63-68.
[15]HU L X,HUANG Z H,LIU S Q,et al.Growth response and gene expression in antioxidant-related enzymes in two bermudagrass genotypes differing in salt tolerance[J].Journal of the american society for horticultural science,2012,137:134-143.
[16]MACHO A P,LOZANO-DURAN R,ZIPFEL C.Importance of tyrosine phosphorylation in receptor kinase complexes[J].Trends in Plant Science.2015,20(5):269-272.
[17]MCKERSIE B D,MURNAGHAN J,JONES K S,et al.Iron-superoxide dismutase expression in transgenic alfalfa increases winter survival without a detectable increase in photosynthetic oxidative stress tolerance[J].Plant Physiol,2000,122(4):1427-1438.
[18]GóMEZ J M,JIMéANEZ A,OLMOS E.Location and effects of long term NaCl stress on superoxide dismutase and ascorbate peroxidase isoenzymes of pea(Pisum sativum cv.Puget)chloroplasts[J].Journal of Experimental Botany,2004,55(394):119-130.
[19]NEGI N P,SHRIVASTAVA D C,SHARMA V,et al.Overexpression of Cu Zn SOD from Arachis hypogaea alleviates salinity and drought stress in tobacco[J].Plant Cell Reports,2015,34(7):1109-1126.
[20]巨延虎,杨旸,孔令广,等.过表达嗜热毛壳菌Cu/Zn-SOD基因提高转基因水稻对3种病害的抗性[J].植物病理学报,2015,45(3):297-306.
[21]杨鸯鸯,李云,丁勇,等.甘蓝型油菜Cu/Zn SOD和FeSOD基因的克隆及菌核病菌诱导表达[J].作物学报,2009,35(1):71-78.
[22]XU J,DUAN X G,YANG J,et al.Enhanced reactive oxygen species scavenging by overproduction of superoxide dismutase and catalase delays postharvest physiological deterioration of cassava storage roots[J].Plant Physiology,2013,161(3):1517-1528.
[23]ALBRECHT U,BOWMAN K D.Transcriptional response of susceptible and tolerant citrus to infection with Candidatus Liberibacter asiaticus[J].Plant Science,2012,185-186(4):118-130.
[24]FAN J,CHEN C,YU Q,et al.Comparative transcriptional and anatomical analyses of tolerant rough lemon and susceptible sweet orange in response to‘Candidatus Liberibacter asiaticus’infection[J].Molecular Plant Microbe Interaction,2012,25(11):1396-1407.
[25]XU M R,LI Y,ZHENG Z,et al.Transcriptional analyses of mandarins SERIOUSLY infected by‘Candidatus Liberibacter asiaticus’[J].PLoS One,2015,10(7):e0133652.