Cloning and Expression Analysis of Sulfur Dioxygenase Gene VvETHE1 in Grapevine (Vitis vinifera)
YAN Jing1,2,*, SONG Jia-Ning1,2,*, WANG Ling1,2, WANG Yue-Jin1,2, ZHANG Chao-Hong1,2,**
1 State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling 712100, China; 2 Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northwest Region), Ministry of Agriculture, Yangling 712100, China
Abstract：Sulfur dioxygenase 1 (ethylmalonic encephalopathy protein 1, ETHE1), the key enzyme in the oxidation of sulfides into sulfates, is mainly involved in plant stress tolerance, hormone response, seed development and survival. In this study, two VvETHE1s were cloned from Vitis vinifera. 'Pinot Noir' through reverse transcription PCR (RT-PCR), and named VvETHE1A (GenBank No. LOC100248855) and VvETHE1B (GenBank No. LOC100262655). The ORF of VvETHE1A was 867 bp, encoding 288 amino acids; while VvETHE1B was 861 bp encoding 286 amino acids. Multiple sequence alignment indicated that VvETHE1s were highly conserved in the domain of metallo-β-lactamase family. Evolutionary analysis showed that the genetic distance between VvETHE1A and jujube (Ziziphus jujuba) ETHE1 was closer than VvETHE1B. By semi-quantitative reverse transcription and PCR (sqRT-PCR), tissue-specific expression analysis showed both VvETHE1A and VvETHE1B were expressed in all tissues with different levels, among which VvETHE1B was highly expressed in root. qRT-PCR analysis showed that the expression levels of VvETHE1A and VvETHE1B were significantly different in the ovules of Vitis vinifera 'Pinot noir' and 'Thompson Seedless'. And both of them decreased in the ovules of 35~45 d after full-bloom of Thompson Seedless. VvETHE1A responded strongly to exogenous abscisic acid (ABA), followed by ethylene (ETH), while VvETHE1B strongly responded to methyljasmonate (MeJA), weakly to ABA and ETH. Moreover, the prokaryotic expression vectors of VvETHE1s were constructed and 2 fusions of about 35 kD were induced in the form of inclusion body. Decrease of induction temperature and increase of time resulted in high yield of recombinant VvETHE1s. The optimal induction condition was 28 ℃ for 6 h, and more soluble proteins were induced at 16 ℃. This study provide a basis for further studies on the regulatory mechanism of VvETHE1s in seed abortion and hormone response, and on the physiological and biochemical characteristics of VvETHE1s.
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