| In this study, 6 gene fragments related to drought resistance were obtained from drought-resistant Chinese wild Vitis yeshanensis acc. Yanshan-1 by DDRT-PCR. Based on the obtained sequences, other members of the dehydrin gene family were cloned. The function divergence and sequence variation of dehydrin genes was studied. The main results are described as follows:1. Cloning of genes related to drought resistance in V. yeshanensis.The accession of Yanshan-1 of V. yeshanensis was used as material to isolate drought resistant-related genes by DDRT-PCR technique. 6 of differentially expressed cDNA fragments related to drought stress-inducible expression, T11VC/S1345-259 (GenBank no: JF900494), T11VG/S1052-312 (GenBank no: JF900496), T11VG/B0314-398 (GenBank no: JF900495), T11VA/S476-280 (GenBank no: JF900493), T11VG/S513-205 (GenBank no: JQ277737) and T11VG/S3-404 (GenBank no: JQ277736) were obtained.2. Cloning of grape dehydrin genes.The full-length of DHN1 gene (GenBank no: JF900497) was obtained from V. yeshanensis by using 5’RACE technique. Based on this sequence, the other 3 dehydrin genes were identified in GenBank database. The corresponding genes were cloned from V. yeshanensis acc. Yanshan-1, designated DHN2 (GenBank no: JF896556), DHN3 (GenBank no: JF896557), DHN4 (GenBank no: JF896558), respectively. According to genome information of V. vinifera cv. Pinot Noir, DHN1 is located in chromosome 4, DHN2 located in chromosome 18, DHN3 and DHN4 located in positive strand and negative strand of chromosome 3, respectively. The spacer between DHN3 and DHN4 is 616 kb.3. Structure and properties of grape dehydrin proteinsThere are significant differences in structure and isoelectric point among four Vitis dehydrin proteins. DHN1 is alkaline Y2SK2-type dehydrin, whereas DHN2 and DHN3 are both acidic SLysK2-type dehydrin, and DHN4 is acidic Y3SK2-type dehydrin. All four members possess phosphorylated CK2 recognition sites. DHN1 and DHN2 have the LXRXXS motifs, the phosphorylation site of SnRK2. Both DHN1 and DHN4 have nuclear location signal RRKK.4. Characterization of expression profiling of dehydrin family Grape dehydrin genes displayed different expression pattern. DHN1 can be induced by drought, cold, heat, powdery mildew and over-expressed at the late stage of the embryo development, which suggests that DHN1 mainly function as a stress-responsive gene. DHN2 is constitutively expressed in different tissues. Transcripts of DHN2 accumulate the leaves under cold, heat, and at the late stage of the embryo development, which suggests that DHN2 is not only involved in the metabolic process of plant growth, but also in response to stresses. DHN3 and DHN4 were only expressed during the late stage of embryo development, and there were no obvious responses to stress and signal molecues. DHN3 was expressed in a very low level. Grape dehydrin genes were regulated by diverse signal molecule. DHN1 was up-regulated by ABA, SA and MeJA. DHN2 was up-regulated by ABA only. DHN3 and DHN4 were not responsive to ABA, SA and MeJA. Only DHN1 played a role in drought tolerance among the four members of grape dehydrin family.5. Variation of DHN1 sequences in Vitis species72 DHN1 genes were cloned from 44 accessions of 13 species and 1 varietas native to China, 11 accessions of 7 species native to America, 9 cultivars of European species. Sequence analysis demonstrated that DHN1 sequences have low diversities in the regions of S-segments, K-segments, nuclear localization signal (NLS) and poly (A) signal, indicating that these functional areas is relatively conservative. There is a clear positive selection in N-terminal region of DHN1. The substitution of amino acid in the regions led to an increase of isoelectric point and hydrophilicity of DHN1, which could regulate and optimize the function of DHN1 gene. |
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