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| Cloning and Expression Analysis of VvMT2 Gene and Promoter in Grape (Vitis vinifera) |
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Abstract Metallothioneins (MTs) are rich in cysteine (Cys), playing an important role in detoxification, scavenging oxygen free radicals, fruit development and maturation and other processes. The MT gene VvMT2 (GenBank No. EU280163) was screened from cDNA library of Thompson Seedless (Vitis vinifera). And the full-length genomic DNA and promoter sequences were cloned by PCR. The experiment aimed at its coding structure and properties of VvMT2 gene and revealing the expression pattern of the gene in different stages of seed development and under different plant growth regulator treatments. The results indicated that the full-length of VvMT2 gene was 998 bp with 3 exons and 2 introns. The full-length cDNA was 527 bp and encoded 79 amino acids which contained 14 cysteines. VvMT2 gene had a Metallothio_2 structural sequence. The predicted molecular weight of VvMT2 was 7.71 kD and isoelectric point was 4.67, which was an acidic protein. There was a strong hydrophobic region in the middle. It had no signal peptide and was located outside the cell. The obtained VvMT2 promoter region sequence was 783 bp, which contained 12 upstream regulatory elements especially essential elements for transcription and stress response and hormone response elements. In the beginning of seed development, the expression level of VvMT2 gene in Thompsolln Seedless was higher than that in Pinot Noir significantly, and both were opposite in the later stage of seed development. The expression level of VvMT2 gene in Thompson Seedless was higher in 25 and 35 d after flowering. Soon after, it decreased rapidly and was below the full flowering level. The expression level of VvMT2 gene in Pinot Noir responded earlier (20 d after flowering), and significantly increased at the later period. On the other hand, the expression level of VvMT2 genes in grape leaves was rapidly reduced after spraying with plant growth regulators. And it returned to pre-spray level within 24 h. With the treatment of methyl jasmonate, the expression level of the gene in leaves did not increase. However it increased 12 h after ethylene treatment. And it was higher than that of the control significantly 24 h after ethylene treatment. With abscisic acid treatment, the gene expression recovered and increased fastest. The study provide theoretical basis for further function study of VvMT2 gene in hormone regulation and seed development process.
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Received: 18 April 2018
Published: 24 October 2018
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