DOI: 10.3724/SP.J.1006.2017.00510

Acta Agronomica Sinica (作物学报) 2017/43:4 PP.510-521

Molecular Cloning and Functional Identification of Peroxidase Gene ScPOD02 in Sugarcane

Peroxidases (PODs), widely existing in various plant organs and different development stages, play a vital role in plant growth and development, 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 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. qRT-PCR analysis showed that the transcripts of ScPOD02 were up-regulated in sugarcane smut-resistant cultivars (YZ03-258, YZ01-1413, YT96-86, 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

ReleaseDate:2017-12-07 14:01:49

[1] 陈如凯, 许莉萍, 林彦铨, 邓祖湖, 张木清, 罗俊, 张华, 高三基, 徐景升, 徐良年, 阙友雄, 陈平华, 袁照年, 林国栋. 现代甘蔗遗传育种. 北京:中国农业出版社, 2011. pp 1-3 Chen R K, Xu L P, Lin Y Q, Deng Z H, Zhang M Q, Luo J, Zhang H, Gao S J, Xu J S, Xu N L, Que Y X, Chen P H, Yuan Z N, Lin G D. Modern Sugarcane Genetic Breeding. Beijing:China Agriculture Press, 2011. pp 1-3(in Chinese)

[2] 昝逢刚, 应雄美, 吴才文, 赵培方, 陈学宽, 马丽, 苏火生, 刘家勇. 98份甘蔗种质资源遗传多样性的AFLP分析. 中国农业科学, 2015, 48:1002-1010 Zan F G, Ying X M, Wu C W, Zhao P F, Chen X K, Ma L, Su H S, Liu J Y. Genetic diversity analysis of 98 collections of sugarcane germplasm with AFLP markers. Sci Agric Sin, 2015, 48:1002-1010(in Chinese with English abstract)

[3] Ye J, Que Y X, Li Y R, Xu L P. Evaluating sugarcane productivity in China over different periods using data envelopment analysis and the malmquist index. Sugar Tech, 2016:1-10

[4] 许莉萍, 陈如凯. 甘蔗黑穗病及其抗病育种的现状与展望. 福建农业学报, 2000, 15(2):26-31 Xu L P, Chen R K. Current status and prospects of smut and smut resistance breeding in sugarcane. Fujian J Agric Sci, 2000, 15(2):26-31(in Chinese with English abstract)

[5] Que Y X, Xu L P, Wu Q B, Liu Y F, Ling H, Liu Y H, Zhang Y Y, Guo J L, Su Y C, Chen J B, Wang S S, Zhang C G. Genome sequencing of Sporisorium scitamineum provides insights into the pathogenic mechanisms of sugarcane smut. BMC Genomics, 2014, 15:996

[6] Sundar A R, Barnabas E L, Malathi P, Viswanathan R. A Mini-review on Smut Disease of Sugarcane Caused by Sporisorium scitamineum. Croatia:InTech, 2012. pp 109-128

[7] Scortecci K C, Creste S, Calsa T J, Xavier M A, Landell M G A, Figueira A, Benedito V A. Challenges, opportunities and recent advances in sugarcane breeding. Plant Breed, 2012:267-296

[8] Blokhina O, Virolainen E, Fagerstedt K V. Antioxidants, oxidative damage and oxygen deprivation stress:a review. Ann Bot, 2003, 91:179-194

[9] Tognolli M, Penel C, Greppin H, Simon P. Analysis and expression of the class Ⅲ peroxidase large gene family in Arabidopsis thaliana. Gene, 2002, 288:129-138

[10] Hiraga S, Yamamoto K, Ito H, Sasaki K, Matsui H, Honma M, Nagamura Y, Sasaki T, Ohashi Y. Diverse expression profiles of 21 rice peroxidase genes. Febs Lett, 2000, 471:245-250

[11] Hiraga S, Sasaki K, Ito H, Ohashi Y, Matsui H. A large family of class Ⅲ plant peroxidases. Plant Cell Physiol, 2001, 42:462-468

[12] 许莉萍, 王建南, 陈如凯. 甘蔗对黑穗病的生化反应及其与抗病性的关系. 甘蔗, 1994, 1(3):13-16 Xu L P, Wang J N, Chen R K. Biochemical reaction of sugarcane to smut and its relation to resistance. Sugarcane, 1994, 1(3):13-16(in Chinese)

[13] 邢慧清. 高粱丝黑穗病群体生理指标测定与抗病基因分子标记分析. 沈阳师范大学硕士学位论文, 辽宁沈阳, 2013 Xing H Q. Physiological Index Determination of Sorghum Head Smut Group and Molecular Markers Analysis of Disease Resistance Genes. MS Thesis of Shenyang Normal University, Shenyang, China, 2013(in Chinese with English abstract)

[14] 温琪汾, 刘润堂, 王纶, 王星玉, 师颖. 谷子种质资源抗黑穗病鉴定与过氧化物酶研究. 植物遗传资源学报, 2006, 7:349-351 Wen Q F, Liu R T, Wang L, Wang X Y, Shi Y. Screening of foxtail millet germplasm for resistance to grain smut and related peroxidase study. J Plant Gene Resour, 2006, 7:349-351(in Chinese with English abstract)

[15] Passardi F, Longet D, Penel C, Dunand C. The class Ⅲ peroxidase multigenic family in rice and its evolution in land plants. Phytochemistry, 2004, 65:1879-1893

[16] Cesarino I, Araújo P, Leme A F P, Creste S, Mazzafera P. Suspension cell culture as a tool for the characterization of class Ⅲ peroxidases in sugarcane. Plant Physiol Biochem, 2013, 62:1-10

[17] Cesarino I, Araujo P, Mayer J L S, Leme A F P, Mazzafera P. Enzymatic activity and proteomic profile of class Ⅲ peroxidases during sugarcane stem development. Plant Physiol Biochem, 2012, 55:66-76

[18] Cesarino I, Araujo P, Leme A F P, Creste S, Mazzafera P. Suspension cell culture as a tool for the characterization of class Ⅲ peroxidases in sugarcane. Plant Physiol Biochem, 2013, 62:1-10

[19] 胡小文, 姚艳丽, 邢淑莲, 徐磊, 刘洋. 割手密过氧化物酶基因(SsPOD-1) cDNA克隆与功能分析. 热带作物学报, 2015, 36:1290-1296 Hu X W, Yao Y L, Xing S L, Lei L, Liu Y. Isolation and characterization of peroxidases gene (SsPOD-1) cDNA in Saccharum spontaneum L. Chin J Trop Crop, 2015, 36:1290-1296(in Chinese with English abstract)

[20] 胡小文, 姚艳丽, 刑淑莲, 徐磊, 刘洋. 甘蔗、斑茅及割手密Ⅲ型过氧化物酶基因克隆及比较分析. 分子植物育种, 2015, 13:2340-2347 Hu X W, Yao Y L, Xing S L, Xu L, Liu Y. Isolation and comparative analysis of class Ⅲ peroxidases gene DNA on Saccharum officinarum, Saccharum spontaneum and Saccharum arundinaceum. Mol Plant Breed, 2015, 13:2340-2347(in Chinese with English abstract)

[21] LaO M, Arencibia A D, Carmona E R, Acevedo R, Rodríguez E, León O, Santana I. Differential expression analysis by cDNA-AFLP of Saccharum spp. after inoculation with the host pathogen Sporisorium scitamineum. Plant Cell Rep, 2008, 27:1103-1111

[22] 许莉萍, 陈如凯, 潘大仁, 林胜杰. 甘蔗分离群体的分析及抗感黑穗病池的构建. 福建农业大学学报, 2001, 30:153-157 Xu L P, Chen R K, Pan D R, Lin S J. Analysis of sugarcane segregating population and construction of pools resistant or sensitive to smut. J Fujian Agric Univ, 2001, 30:153-157(in Chinese with English abstract)

[23] Su Y C, Wang Z Q, Xu L P, Peng Q, Liu F, Li Z, Que Y. Early selection for smut resistance in sugarcane using pathogen proliferation and changes in physiological and biochemical indices. Front Plant Sci, 2016, 7:1133

[24] Su Y C, Guo J L, Ling H, Chen S S, Wang S S, Xu L P, Allan A C, Que Y X. Isolation of a novel peroxisomal catalase gene from sugarcane, which is responsive to biotic and abiotic stresses. PLoS One, 2014, 9:e84426

[25] 姚伟, 余爱丽, 徐景升, 周会, 张木清, 陈如凯. 甘蔗基因组DNA简单和快速提取方法. 农业生物技术学报, 2005, 13:121-122 Yao W, Yu A L, Xu J S, Zhou H, Zhang M Q, Chen R K. A simple and quick method for extracting sugarcane genomic DNA. J Agric Biotechnol, 2005, 13:121-122(in Chinese)

[26] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 2001, 25:402-408

[27] Su Y C, Xu L P, Xue B T, Wu Q B, Guo J L, Wu L G, Que Y X. Molecular cloning and characterization of two pathogene-sis-related β-1,3-glucanase genes ScGluA1 and ScGluD1 from sugarcane infected by Sporisorium scitamineum. Plant Cell Rep, 2013, 32:1503-1519

[28] Su Y C, Xu L P, Fu Z W, Yang Y T, Guo J L, Wang S S, Que Y X. ScChi, encoding an acidic class Ⅲ chitinase of sugarcane, confers positive responses to biotic and abiotic stresses in sugarcane. Int J Mol Sci, 2014, 15:2738-2760

[29] Grivet L, Arruda P. Sugarcane genomics:depicting the complex genome of an important tropical crop. Curr Opin Plant Biol, 2002, 5:122-127

[30] 阙友雄, 杨志霞, 许莉萍, 陈如凯. 对甘蔗受黑穗病菌侵染后差异表达基因的分离与鉴定. 作物学报, 2009, 35:452-458 Que Y X, Yang Z X, Xu L P, Chen R K. Isolation and identification of differentially expressed genes in sugarcane infected by Ustilago scitaminea. Acta Agron Sin, 2009, 35:452-458(in Chinese with English abstract)

[31] Ren L L, Liu Y J, Liu H J, Qian T T, Qi L W, Wang X R, Zeng Q Y. Subcellular relocalization and positive selection play key roles in the retention of duplicate genes of populus class Ⅲ peroxidase family. Plant Cell, 2014, 26:2404-2419

[32] Blokhina O, Virolainen E, Fagerstedt K V. Antioxidants, oxidative damage and oxygen deprivation stress:a review. Ann Bot, 2003, 91:179-194

[33] DíAz J, Bernal A, Pomar F, Merino F. Induction of shikimate dehydrogenase and peroxidase in pepper (Capsicum annuum L.) seedlings in response to copper stress and its relation to lignification. Plant Sci, 2001, 161:179-188

[34] Esh A, Guirgis A, El-kholi M, El-Absawy E, Nasr M, Hassanien E. The activity of pathogenesis related proteins in smut resistant and susceptible sugarcane (GT54-9) mutants induced by gamma radiation. Adv Plants Agric Res, 2014, 1:00024

[35] Zhang S P, Xiao Y N, Zhao J R, Wang F G, Zheng Y L. Digital gene expression analysis of early root infection resistance to Sporisorium reilianum f. sp. zeae in maize. Mol Genet Genomics, 2013, 288:21-37

[36] Liu B, Xue X D, Cui S, Zhang X Y, Han Q M, Zhu L, Liang X F, Wang X J, Huang L L, Chen X M, Kang Z S. Cloning and characterization of a wheat β-1,3-glucanase gene induced by the stripe rust pathogen Puccinia striiformis f. sp. tritici. Mol Biol Rep, 2010, 37:1045-1052

[37] Wang C J, Chan Y L, Shien C H, Yeh K W. Molecular characterization of fruit-specific class Ⅲ peroxidase genes in tomato (Solanum lycopersicum). J Plant Physiol, 2015, 177:83-92

[38] 姚璇. 植物逆境胁迫相关基因的研究-PROG1和OsHKT2基因的功能分析. 武汉大学博士学位论文, 湖北武汉, 2011 Yao X. The Characterization of Plant Stress-related Genes Functional Analyses on PROG1 and OsHKT2 Genes. PhD Dissertation of Wuhan University, Wuhan, China, 2011(in Chinese with English abstract)

[39] Anderberg R J, Walker-Simmons M K. Isolation of a wheat cDNA clone for an abscisic acid-inducible transcript with homology to protein kinases. Proc Natl Acad Sci USA, 1992, 89:10183-10187

[40] Novo-Uzal E, Gutiérrez J, Martínez-Cortés T, Pomar F. Molecular cloning of two novel peroxidases and their response to salt stress and salicylic acid in the living fossil Ginkgo biloba. Ann Bot, 2014, 114:923-936