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作物学报 ›› 2017, Vol. 43 ›› Issue (04): 510-521.doi: 10.3724/SP.J.1006.2017.00510

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗过氧化物酶基因ScPOD02的克隆与功能鉴定

苏亚春,王竹青,李竹,刘峰,许莉萍,阙友雄,戴明剑,陈允浩   

  1. 福建农林大学 / 农业部福建甘蔗生物学与遗传育种重点实验室 / 国家甘蔗产业技术研发中心, 福建福州 350002
  • 收稿日期:2016-07-04 修回日期:2016-11-02 出版日期:2017-04-12 网络出版日期:2016-12-14
  • 通讯作者: 许莉萍, E-mail: xlpmail@126.com
  • 基金资助:

    本研究由福建省杰出青年科学基金项目(2015J06006和2015J05055)和国家现代农业产业技术体系建设专项(CARS-20)项目资助。

Molecular Cloning and Functional Identification of Peroxidase Gene ScPOD02 in Sugarcane

SU Ya-Chun,WANG Zhu-Qing,LI Zhu,LIU Feng,XU Li-Ping*,QUE You-Xiong,DAI Ming-Jian,Chen Yun-Hao   

  1. Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture / Fujian Agriculture and Forestry University / Sugarcane Research & Development Center, China Agricultural Technology System, Fuzhou 350002, China
  • Received:2016-07-04 Revised:2016-11-02 Published:2017-04-12 Published online:2016-12-14
  • Contact: Xu liping, E-mail: xlpmail@126.com
  • Supported by:

    This work was supported by Natural Science Foundation of Fujian Province, China (2015J06006 and 2015J05055) and the China Agriculture Research System (CARS-20).

摘要:

过氧化物酶(PODs)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OsPrx11 (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pET 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 kD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 (qRT-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(NtHSR201和NtHSR203)和乙烯合成依赖基因(NtEFE26和NtAccdeaminase)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。

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

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 Acc. No. KU593507) and genomic DNA (GenBank No. 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 Acc. No. 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 prokaryotic expression vector of pET 32a and then transformed into Escherichia 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 enhanced under the stress of polyethylene glycol, indicating its high tolerance to drought stress. RT-qPCR 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 (NtHSR201 and NtHSR203) and ethylene synthesis dependent genes (NtEFE26 and NtAccdeaminase) 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.

Key words: Sugarcane, ScPOD02, Gene cloning, Biotic and abiotic stresses, Gene expression level, Prokaryotic expression analysis, Transient expression analysis

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