作物学报2017，Vol.43Issue(4)：510-521,12.DOI:10.3724/SP.J.1006.2017.00510
甘蔗过氧化物酶基因ScPOD02的克隆与功能鉴定
Molecular Cloning and Functional Identification of Peroxidase Gene ScPOD02 in Sugarcane
摘要
Abstract
Peroxidases (PODs), widely existing in various plant organs and different development stages, play a vital role in plant growth and development, and also respond to adverse stresses. Based on the previous transcriptome data, we isolated a cDNA (GenBank accession number KU593507) and genomic DNA (GenBank accession number KU593508) sequences of ScPOD02 from smut resistant genotype Yacheng 05-179 infected by Sporisorium scitamineum for two days. Sequence analysis showed that the full length cDNA of ScPOD02 is 1434 bp with an ORF of 1047 bp in length, encoding 348 amino acids. Its genomic DNA length is 1558 bp containing two exons and one intron. Phylogenetic tree analysis demonstrated that ScPOD02 and rice OsPrx11 (GenBank accession number gi|55700889) were clustered into the same evolutionary branch, suggesting that ScPOD02 belongs to one of the acidic exocytosis/cell wall type of class I.1 peroxidase family members. ScPOD02 was further ligated with a prokary- otic expression vector of pET 32a and then transformed intoEscherichia coli BL21. An approximately 60 kD fusion protein was obtained by isopropyl-β-d-thiogalactoside induction. Compared with the control, the growth of recombinant BL21 cells was en-hanced under the stress of polyethylene glycol, indicating its high tolerance to drought stress. qRT-PCR analysis showed that the tran-scripts ofScPOD02 were up-regulated in sugarcane smut-resistant cultivars (YZ03-258, YZ01-1413, YT96-86, and LC05-136) byS. scitamineum except for ROC22 and YZ03-103, but remained or slightly decreased in the middle-susceptible (FN39 and GT02-467) and susceptible (FN40) cultivars. In addition,ScPOD02 positively responded to salicylic acid, abscisic acid, polyethylene glycol and sodium chloride stresses. The transient expression ofScPOD02 in Nicotiana benthamiana was performed usingAgrobacterium medi-ated method. A deeper DAB staining color inN. benthamiana leaves was observed after overexpressing ScPOD02. Furthermore, the target geneScPOD02 and theN. benthamiana hypersensitive reaction (HR) marker genes (NtHSR201andNtHSR203) and ethylene synthesis dependent genes (NtEFE26 andNtAccdeaminase) were all up-regulated. These reach a conclusion that the ScPOD02 has potential roles in the immune response and in protecting sugarcane from drought and salt stresses.关键词
甘蔗:ScPOD02/基因克隆/生物和非生物胁迫/基因表达量/原核表达分析/瞬时表达分析Key words
Sugarcane/ScPOD02/Gene cloning/Biotic and abiotic stresses/Gene expression level/Prokaryotic expression analysis/Transient expression analysis引用本文复制引用
苏亚春,王竹青,李竹,刘峰,许莉萍,阙友雄,戴明剑,陈允浩..甘蔗过氧化物酶基因ScPOD02的克隆与功能鉴定[J].作物学报,2017,43(4):510-521,12.基金项目
本研究由福建省杰出青年科学基金项目(2015J06006), 福建农林大学杰出青年基金项目(xjq201630)和国家现代农业产业技术体系建设专项(CARS-20)资助. This study was supported by the Fujian Natural Science Funds for Distinguished Young Scholar (2015J06006), the Research Funds for Dis-tinguished Young Scientists in Fujian Agriculture and Forestry University (xjq201630), and the China Agriculture Research System (CARS-20). （2015J06006）
