doi:

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


Abstract:
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



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