【作者】 杨亚州；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 本研究利用DDRT-PCR技术从中国原产的抗旱种质燕山葡萄株系燕山-1中获得了6个抗旱相关基因片段,在此基础上克隆葡萄脱水素基因家族的其他成员,并分析了脱水素基因家族成员间的功能差异和脱水素基因DHN1的序列变异,为鉴定脱水素基因的抗旱性提供依据和参考。主要研究结果如下:1.中国野生葡萄抗旱相关基因的克隆以燕山葡萄株系燕山-1为材料,通过DDRT-PCR技术获得6个干旱胁迫诱导表达的差异cDNA片段T11VC/S1345-259 (GenBank登录号:JF900494),T11VG/S1052-312 (GenBank登录号:JF900496),T11VG/B0314-398 (GenBank登录号:JF900495),T11VA/S476-280 (GenBank登录号:JF900493),T11VG/S513-205 (GenBank登录号:JQ277737)和T11VG/S3-404 (GenBank登录号:JQ277736)。2.葡萄属植物脱水素基因家族成员的克隆通过5’RACE方法获得了燕山-1脱水素基因DHN1全序列(GenBank登录号:JF900497)。以此为基础,从GenBank数据库鉴定出其他3个葡萄脱水素基因,并从燕山-1中克隆到对应的脱水素基因序列,命名为DHN2 (GenBank登录号:JF896556),DHN3 (GenBank登录号:JF896557)和DHN4 (GenBank登录号:JF896558)。根据已知黑比诺葡萄基因组信息分析,DHN1和DHN2分别位于染色体4和染色体18的正链上;DHN3和DHN4分别位于染色体3的正链和负链上,二者相距约616 kb。3.葡萄属植物脱水素基因家族成员的结构和性质葡萄脱水素基因家族成员的结构和性质不同。DHN1为Y2SK2型,呈碱性;DHN2和DHN3为SLysK2型,呈酸性;DHN4为Y3SK2型,呈酸性。四个脱水素基因都具有CK2磷酸化识别位点,DHN1和DHN2具有SnRK2磷酸化识别位点LXRXXS,DHN1和DHN4具有核定位序列RRKK。4.脱水素基因家族成员的胁迫诱导表达葡萄脱水素基因家族成员的功能分化非常明确,4个成员的胁迫诱导表达模式不同。DHN1在干旱、低温、高温和白粉病等逆境胁迫条件下以及胚发育后期大量表达。DHN2在不同组织中组成型表达,低温和高温胁迫以及胚发育后期表达量增加、并且受ABA诱导表达,但不受干旱和病原菌诱导。DHN3和DHN4仅在胚发育后期中表达,而DHN3的表达量极低。说明DHN1和DHN2都参与植株对逆境胁迫的响应,DHN3和DHN4仅参与葡萄的胚发育。DHN1受ABA、SA和MeJA等信号分子诱导表达;DHN2受ABA诱导表达,不受SA和MeJA诱导;DHN3和DHN4不受ABA、SA和MeJA等信号分子诱导。脱水素基因家族的四个成员中仅脱水素基因DHN1具有抗旱的功能。5.葡萄属植物DHN1基因的序列变异从14个中国野生葡萄种或变种的44个株系,7个美洲野生葡萄种的11个品种或株系,以及欧洲葡萄的9个品种中共克隆到72条DHN1基因序列。脱水素DHN1在S-片段、K-片段、核定位信号以及poly(A)信号附近核苷酸多样性较低,说明这些功能区域相对保守。DHN1基因N-端的非同义置换率较高,存在明显的正向选择。这些区域的氨基酸替代导致DHN1亲水性增加,等电点升高,能够起到调节和优化脱水素DHN1功能的作用。更多还原

【Abstract】 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.更多还原