西瓜ClMYB46基因的耐低温功能鉴定及调控作用分析

发布时间：2018-09-17 07:23

【摘要】：西瓜(Citrullus lanatus)是重要的葫芦科作物,具有较高的经济价值。西瓜是典型的喜温作物,在早春和反季节设施栽培过程中,常受到低温胁迫而影响产量和经济效益。低温已成为西瓜生产中主要的非生物限制因子,严重制约西瓜的周年供应和种植效益提高。MYB转录因子在植物应答外界胁迫环境中起着重要作用,参与调控下游多个基因的表达。本课题前期研究中,通过挖掘低温胁迫下西瓜转录组数据发现多个基因参与低温逆境响应。进一步分析得出,Cla005622的表达量显著上调,且属于MYB-related型转录因子,但其耐低温功能及相关调控作用尚未明确。为回答上述问题,本研究拟采用如下方法解决:对MYB-related家族成员鉴定、分析Cla005622的结构特征、qRT-PCR鉴定Cla005622对西瓜低温胁迫的应答、亚细胞定位和转录活性鉴定Cla005622的特征、超量表达与烟草遗传转化后鉴定耐低温性功能验证、酵母双杂交探究蛋白互作及功能分析,旨在明确Cla005622对西瓜低温逆境的耐低温功能鉴定及调控作用,结果不仅可以解析西瓜的低温耐受性机制,同时还可为后续从分子育种角度改善西瓜低温抗性、提高西瓜产量与质量提供丰富的基因资源。本文的主要研究结果如下:1.Cla005622基因结构分析及西瓜MYB-related家族基因成员鉴定。使用NCBI预测Cla005622蛋白保守结构域得知,其属于典型的MYB-related基因。为进一步探索其在MYB-related家族基因中的特征,通过HMM算法在西瓜全基因组上共鉴定出50个MYB-related家族基因,编码序列长度240bp-4986bp,等电点4.8-9.97,分子量5890.67-181178.4。据基因在染色体上的分布顺序命名为ClMYB1-ClMYB50。Cla005622对应基因为ClMYB46,编码序列全长1040bp,编码334个氨基酸,等电点为6.70,分子量为35890.61,位于第10号染色体。2.ClMYB46对西瓜低温逆境的应答。以西瓜97103为实验材料,用qRT-PCR技术检测10℃低温处理西瓜叶片后6h、12h、24h、48h的基因表达量变化,变化趋势呈现先升高后降低再升高的过程。其中,6h点的表达量达到最高。因此,ClMYB46受低温胁迫诱导显著上调表达。3.ClMYB46的特征分析。亚细胞定位以本氏烟草为实验材料,构建载体GV3101[pH7LIC5.0-N-eGFP-ClMYB46]后瞬时表达,观测荧光信号位于细胞核。转录活性鉴定通过构建Y2HGold[pGBKT7-ClMYB46],激活HIS3、ADE2报告基因表达,具有转录活性。因此,ClMYB46表达部位在细胞核,且具有转录活性。4.ClMYB46的耐低温性功能鉴定。以野生栽培烟草为实验材料,构建载体LBA4404[pHellsgate8-ClMYB46]后进行烟草遗传转化,qRT-PCR检测ClMYB46表达量,筛选OE1、OE2株系进行后续实验。以野生型烟草WT、转基因株系OE1和OE2各75株为实验材料,4℃低温处理(光周期16 h/8 h,光强100μmol m-2s-1)0h、6h、12h、24h、48h时间点观察并取样。表型观察、冷害指数测定均得出不同时间点下野生型株系萎蔫程度均高于转基因株系,过表达ClMYB46能提高烟草对低温逆境的抗性。使用试剂盒测定不同时间点下电导率和丙二醛含量变化得知野生型细胞膜受损伤更严重,过表达ClMYB46能提高烟草对低温逆境的抗性。以低温处理不同时间点下野生型烟草WT、转基因株系OE1和OE2为材料,qRT-PCR检测ABA信号转导途径相关基因(rab18、ABI1、ABI2)与冷胁迫相关基因(DREB转录因子DREB2A、脯氨酸合成促进基因P5CS)的表达量变化。ClMYB46上调引起rab18上调表达、ABI1、ABI2下调表达、DREB2A、P5CS上调表达。5.ClMYB46互作蛋白筛选及点对点验证。重组构建无毒性的诱饵载体Y2HGold[pGBKT7-ClMYB46],AbA抑制其自激活后Mating法筛库鉴定并测序。初步筛选出10个有功能的互作蛋白,包含:维持细胞内环境离子平衡、盐胁迫响应、维持离子通道平衡、响应ABA信号转导途径、参与茉莉酸胁迫响应。其中,ClMYB46的互作蛋白Cla014285(AtCPK28)参与蛋白磷酸化途径、ABA信号转导途径、Ca2+信号转导途径从而引起植株对逆境胁迫的响应。
[Abstract]:Watermelon (Citrullus lanatus) is an important Cucurbitaceae crop with high economic value. Watermelon is a typical thermophilic crop. It is often affected by low temperature stress during early spring and off-season protected cultivation. Low temperature has become a major abiotic limiting factor in watermelon production, seriously restricting the annual supply of watermelon. MYB transcription factors play an important role in plant response to external stresses and participate in regulating the expression of many genes downstream. In the previous study, through mining the transcriptome data of Watermelon under low temperature stress, we found that many genes were involved in response to low temperature stress. Further analysis showed that Cla005622 expression was significant. To answer these questions, the following methods were proposed: identification of MYB-related family members, analysis of the structural characteristics of Cla005622, qRT-PCR identification of Cla005622 response to chilling stress in watermelon, subcellular localization and transduction. Characterization of Cla005622 by transcription activity assay, identification of low temperature tolerance function by overexpression and genetic transformation of tobacco, interaction and function analysis of proteins by yeast two-hybrid were studied in order to clarify the function identification and regulation of Cla005622 on low temperature tolerance of watermelon. The main results of this study are as follows: 1. Cla005622 gene structure analysis and identification of MYB-related family members in watermelon. In order to further explore its characteristics in MYB-related family genes, 50 MYB-related family genes were identified on watermelon genome by HMM algorithm. The coding sequence was 240 bp-4986 BP in length, 4.8-9.97 in isoelectric point and 5890.67-181178.4 in molecular weight. The gene was ClMYB46, with a coding sequence of 1 040 bp, encoding 334 amino acids. Its isoelectric point was 6.70, and its molecular weight was 35890.61. It was located on chromosome 10. 2. ClMYB46 responded to chilling stress in watermelon. The changes of gene expression in Watermelon Leaves at 6, 12, 24, and 48 h after chilling treatment were detected by qRT-PCR. The expression of ClMYB46 was up-regulated significantly by low temperature stress. 3. Characteristic analysis of ClMYB46. Subcellular localization of GV3101 [pH7LIC5.0-N-eGFP-ClMYB46] was carried out using Bennett tobacco as experimental material, and the fluorescence signal was observed to be fine. Nucleus. Identification of transcriptional activity by construction of Y2HGold [pGBKT7-ClMYB46], activation of HIS3, ADE2 reporter gene expression, with transcriptional activity. Therefore, ClMYB46 expression site in the nucleus, and has transcriptional activity. 4. ClMYB46 low temperature tolerance function identification. Wild tobacco as experimental materials, construction of vector LBA4404 [pHellgates 8-ClMYB46] Tobacco genetic transformation, qRT-PCR detection of ClMYB46 expression, screening OE1, OE2 strains for follow-up experiments. Wild-type tobacco WT, transgenic lines OE1 and OE2 75 strains as experimental materials, 4 C low temperature treatment (photoperiod 16 h/8 h, light intensity 100_ micromol m-2s-1) 0 h, 6 h, 12 h, 24 h, 48 h time point observation and sampling. At the same time, the wilting degree of wild-type strains was higher than that of transgenic strains. Overexpression of ClMYB46 could enhance the resistance of tobacco to low temperature stress. The expression of ABA signal transduction pathway related genes (rab18, ABI1, ABI2) and cold stress related genes (DREB transcription factor DREB2A, proline synthesis promoting gene P5CS) in wild type tobacco WT and transgenic lines OE1 and OE2 were detected by qRT-PCR. CLMYB46 interacting protein screening and point-to-point validation. Recombinant nontoxic bait vector Y2H Gold [pGBKT7-ClMYB46], AbA inhibited its self-activation after Mating method screening library identification and sequencing. ClMYB46 interacting protein Cla014285 (AtCPK28) is involved in protein phosphorylation pathway, ABA signal transduction pathway and Ca2+ signal transduction pathway, which induce plant response to stress.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S651

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