【摘要】：西瓜(Citrullus lanatus)是重要的葫蘆科作物,具有較高的經(jīng)濟(jì)價(jià)值。西瓜是典型的喜溫作物,在早春和反季節(jié)設(shè)施栽培過程中,常受到低溫脅迫而影響產(chǎn)量和經(jīng)濟(jì)效益。低溫已成為西瓜生產(chǎn)中主要的非生物限制因子,嚴(yán)重制約西瓜的周年供應(yīng)和種植效益提高。MYB轉(zhuǎn)錄因子在植物應(yīng)答外界脅迫環(huán)境中起著重要作用,參與調(diào)控下游多個(gè)基因的表達(dá)。本課題前期研究中,通過挖掘低溫脅迫下西瓜轉(zhuǎn)錄組數(shù)據(jù)發(fā)現(xiàn)多個(gè)基因參與低溫逆境響應(yīng)。進(jìn)一步分析得出,Cla005622的表達(dá)量顯著上調(diào),且屬于MYB-related型轉(zhuǎn)錄因子,但其耐低溫功能及相關(guān)調(diào)控作用尚未明確。為回答上述問題,本研究擬采用如下方法解決:對MYB-related家族成員鑒定、分析Cla005622的結(jié)構(gòu)特征、qRT-PCR鑒定Cla005622對西瓜低溫脅迫的應(yīng)答、亞細(xì)胞定位和轉(zhuǎn)錄活性鑒定Cla005622的特征、超量表達(dá)與煙草遺傳轉(zhuǎn)化后鑒定耐低溫性功能驗(yàn)證、酵母雙雜交探究蛋白互作及功能分析,旨在明確Cla005622對西瓜低溫逆境的耐低溫功能鑒定及調(diào)控作用,結(jié)果不僅可以解析西瓜的低溫耐受性機(jī)制,同時(shí)還可為后續(xù)從分子育種角度改善西瓜低溫抗性、提高西瓜產(chǎn)量與質(zhì)量提供豐富的基因資源。本文的主要研究結(jié)果如下:1.Cla005622基因結(jié)構(gòu)分析及西瓜MYB-related家族基因成員鑒定。使用NCBI預(yù)測Cla005622蛋白保守結(jié)構(gòu)域得知,其屬于典型的MYB-related基因。為進(jìn)一步探索其在MYB-related家族基因中的特征,通過HMM算法在西瓜全基因組上共鑒定出50個(gè)MYB-related家族基因,編碼序列長度240bp-4986bp,等電點(diǎn)4.8-9.97,分子量5890.67-181178.4。據(jù)基因在染色體上的分布順序命名為ClMYB1-ClMYB50。Cla005622對應(yīng)基因?yàn)镃lMYB46,編碼序列全長1040bp,編碼334個(gè)氨基酸,等電點(diǎn)為6.70,分子量為35890.61,位于第10號染色體。2.ClMYB46對西瓜低溫逆境的應(yīng)答。以西瓜97103為實(shí)驗(yàn)材料,用qRT-PCR技術(shù)檢測10℃低溫處理西瓜葉片后6h、12h、24h、48h的基因表達(dá)量變化,變化趨勢呈現(xiàn)先升高后降低再升高的過程。其中,6h點(diǎn)的表達(dá)量達(dá)到最高。因此,ClMYB46受低溫脅迫誘導(dǎo)顯著上調(diào)表達(dá)。3.ClMYB46的特征分析。亞細(xì)胞定位以本氏煙草為實(shí)驗(yàn)材料,構(gòu)建載體GV3101[pH7LIC5.0-N-eGFP-ClMYB46]后瞬時(shí)表達(dá),觀測熒光信號位于細(xì)胞核。轉(zhuǎn)錄活性鑒定通過構(gòu)建Y2HGold[pGBKT7-ClMYB46],激活HIS3、ADE2報(bào)告基因表達(dá),具有轉(zhuǎn)錄活性。因此,ClMYB46表達(dá)部位在細(xì)胞核,且具有轉(zhuǎn)錄活性。4.ClMYB46的耐低溫性功能鑒定。以野生栽培煙草為實(shí)驗(yàn)材料,構(gòu)建載體LBA4404[pHellsgate8-ClMYB46]后進(jìn)行煙草遺傳轉(zhuǎn)化,qRT-PCR檢測ClMYB46表達(dá)量,篩選OE1、OE2株系進(jìn)行后續(xù)實(shí)驗(yàn)。以野生型煙草WT、轉(zhuǎn)基因株系OE1和OE2各75株為實(shí)驗(yàn)材料,4℃低溫處理(光周期16 h/8 h,光強(qiáng)100μmol m-2s-1)0h、6h、12h、24h、48h時(shí)間點(diǎn)觀察并取樣。表型觀察、冷害指數(shù)測定均得出不同時(shí)間點(diǎn)下野生型株系萎蔫程度均高于轉(zhuǎn)基因株系,過表達(dá)ClMYB46能提高煙草對低溫逆境的抗性。使用試劑盒測定不同時(shí)間點(diǎn)下電導(dǎo)率和丙二醛含量變化得知野生型細(xì)胞膜受損傷更嚴(yán)重,過表達(dá)ClMYB46能提高煙草對低溫逆境的抗性。以低溫處理不同時(shí)間點(diǎn)下野生型煙草WT、轉(zhuǎn)基因株系OE1和OE2為材料,qRT-PCR檢測ABA信號轉(zhuǎn)導(dǎo)途徑相關(guān)基因(rab18、ABI1、ABI2)與冷脅迫相關(guān)基因(DREB轉(zhuǎn)錄因子DREB2A、脯氨酸合成促進(jìn)基因P5CS)的表達(dá)量變化。ClMYB46上調(diào)引起rab18上調(diào)表達(dá)、ABI1、ABI2下調(diào)表達(dá)、DREB2A、P5CS上調(diào)表達(dá)。5.ClMYB46互作蛋白篩選及點(diǎn)對點(diǎn)驗(yàn)證。重組構(gòu)建無毒性的誘餌載體Y2HGold[pGBKT7-ClMYB46],AbA抑制其自激活后Mating法篩庫鑒定并測序。初步篩選出10個(gè)有功能的互作蛋白,包含:維持細(xì)胞內(nèi)環(huán)境離子平衡、鹽脅迫響應(yīng)、維持離子通道平衡、響應(yīng)ABA信號轉(zhuǎn)導(dǎo)途徑、參與茉莉酸脅迫響應(yīng)。其中,ClMYB46的互作蛋白Cla014285(AtCPK28)參與蛋白磷酸化途徑、ABA信號轉(zhuǎn)導(dǎo)途徑、Ca2+信號轉(zhuǎn)導(dǎo)途徑從而引起植株對逆境脅迫的響應(yīng)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S651

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本文編號：2245151