关键词: 甘蔗, NADP异柠檬酸脱氢酶, 基因克隆, 表达分析

Abstract: 【Objective】The aim of this study was to clone the full-length cDNA of sugarcane NADP⁺- dependent lsocitrate dehydrogenase, a key enzyme gene relating to tricarboxylic acid cycle (TCA) in sugarcane, to provide a candidate gene for breeding of sugarcane disease resistance and other stress tolerance.【Method】Two-dimensional electrophoresis (2-DE) was used to study the variation expression of proteins, and the differential proteins were identified by MALDI TOF/TOF. The SoNADP-IDH gene cDNA sequence was cloned from sugarcane variety GT11 using RT-PCR techniques. Online software was used to analyze the putative amino acid sequence, and qRT-PCR method was used to study the expression of SoNADP-IDH gene in different tissues and under different stresses. 【Result】 The full-length cDNA of SoNADP-IDH (GenBank accession number KF808326) in sugarcane was cloned. The full-length cDNA was 1 497 bp with an intact open reading frame of 1 239 bp, encoding a polypeptide of 412 amino acids. The bioinformatics analysis showed that the protein was a hydrophilic protein, did not contain a signal peptide, but contained four transmembrane regions. This protein was a soluble and stable protein. Secondary structure analysis showed that the protein contains alpha helix, bend region, extended strand, random coil, and alpha helix and random curl occupied most of the protein secondary structure. Online software analysis showed that the gene contains 19 phosphorylation sites, 4 N-glycosylation sites, 4 casein kinase phosphorylation sites Ⅱ, 4 N-myristoylation site of action, 6 protein kinase C phosphorylation sites and 1 tyrosine kinase phosphorylation site. Homology analysis showed that the deduced SoNADP-IDH protein was highly homologous to other IDH proteins from different species. The qRT-PCR analyses showed that the SoNADP-IDH expressed in root, stalk and leaf. Furthermore, SoNADP-IDHtranscription level was impacted under the treatment of the pathogen of ratoon stunting disease, low temperature, PEG, NaCl and ABA stresses, but the expression patterns were different. In PEG simulated drought treatment, the transcript of SoNADP-IDH showed first increase and then decrease, reached the peak at 6 h, then continued to decline, and reached the minimum at 48 h. In the 4℃ cold stress treatment, the expression of SoNADP-IDH reached the minimum at 3 h, with the extension after treatment, the expression of SoNADP-IDH increased, reached the peak at 24 h, then slightly decline at 48 h. After ABA application, the transcript of SoNADP-IDH showed a “suppression-increase-suppression-increase” pattern, the transcript of SoNADP-IDH levels decreased at 3 h, reached the peak at 6 h, to a minimum at 24 h, and rose again at 48 h. In salt-treatment, the expression of SoNADP-IDH showed an “increase-suppression-increase” pattern, reached the peak at 6 h, then began to decline sharply, down to the lowest at 24 h, then began to rise, and basically flatted with the control at 48 h 【Conclusion】 The gene SoNADP-IDH was firstly isolated and characterized from sugarcane, and the pathogen of ratoon stuning disease, low temperature, PEG, NaCl and ABA stresses impacted the expression of the gene, indicating that the SoNADP-IDH gene may play an important role in resistance to oxidative stress of sugarcane.

Key words: sugarcane, NADP-IDH, gene cloning, expression analysis