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

过氧化物酶(POD)广泛存在于植物各种器官及其不同发育阶段,在植物生长发育及应对逆境胁迫中起重要作用。本研究基于前期转录组数据,从黑穗病菌侵染2 d的甘蔗抗黑穗病品种崖城05-179中分离到ScPOD02基因的cDNA(GenBank登录号为KU593507)及基因组DNA(GenBank登录号为KU593508)序列。其cDNA全长为1434 bp,ORF区为1047 bp,编码348个氨基酸,而基因组DNA全长为1558 bp,含2个外显子和1个内含子。系统发育树显示,ScPOD02与水稻Os Prx11(GenBank登录号为gi|55700889)属于同一个进化分支,推测ScPOD02属于酸性胞外分泌/细胞壁类型的I.1型过氧化物酶家族。将该基因克隆到原核表达载体p ET 32a上,转化大肠杆菌BL21,经异丙基-β-d-硫代半乳糖苷诱导成功获得了大小约60 k D的融合蛋白,且该重组菌在聚乙二醇胁迫下的长势较对照快,表明其耐受干旱胁迫的能力更强。实时荧光定量PCR(q RT-PCR)分析表明,除ROC22和YZ03-103外,ScPOD02基因在甘蔗抗病品种(YZ03-258、YZ01-1413、YT96-86和LC05-136)中受黑穗病菌诱导上调表达,但在中感(GT02-467和FN39)和感病(FN40)品种中的基因表达量基本维持不变或略有降低;此外,ScPOD02积极应答水杨酸、脱落酸、聚乙二醇及氯化钠的胁迫。通过农杆菌介导法在本氏烟叶片上瞬时表达ScPOD02,结果显示出较深的DAB染色,且过表达后本氏烟中的目标基因(ScPOD02)及过敏性反应(HR)标记基因(Nt HSR201和Nt HSR203)和乙烯合成依赖基因(Nt EFE26和Nt Accdeaminase)均上调表达。以上研究结果显示,ScPOD02具有参与甘蔗免疫应答及抗旱和抗盐害的潜在功能。

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 c DNA（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 c DNA 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 Os Prx11（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 p ET 32 a and then transformed into Escherichia coli BL21. An approximately 60 k D fusion protein was obtained by isopropyl-β-d-thiogalactoside induction. Compared with the control, the growth of recombinant BL21 cells was enhanced under the stress of polyethylene glycol, indicating its high tolerance to drought stress. q RT-PCR analysis showed that the transcripts of ScPOD02 were up-regulated in sugarcane smut-resistant cultivars（YZ03-258, YZ01-1413, YT96-86, and LC05-136） by S. 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 of ScPOD02 in Nicotiana benthamiana was performed using Agrobacterium mediated method. A deeper DAB staining color in N. benthamiana leaves was observed after overexpressing ScPOD02. Furthermore, the target gene ScPOD02 and the N. benthamiana hypersensitive reaction（HR） marker genes（Nt HSR201 and Nt HSR203） and ethylene synthesis dependent genes（Nt EFE26 and Nt Accdeaminase） 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.