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为探索外源赤霉素(GA3)介导的无核葡萄果实膨大过程中的信号途径的调控基因,寻找验证主要调控元件,本研究以欧亚种无核葡萄品种‘无核白鸡心’(Vitis vinifera L.cv.Centennial Seedless)为材料,通过半定量RT-PCR技术分析属于GRAS基因家族的SCARECROW Like 14-Like(VvSCL14-Like)基因在葡萄不同组织器官和果实发育期的表达。通过启动子克隆、生物信息学分析、promoter∷GUS融合基因和GUS组织染色法对该基因的表达特征进行了研究。半定量RT-PCR结果表明,VvSCL14-Like基因主要在休眠芽中表达,在果实发育过程中无表达。利用GA3对葡萄幼果进行膨大处理,处理后1、3和7d的果实转录组结果表明VvSCL14-Like的转录水平无差异。对VvSCL14-Like启动子进行生物信息学分析,发现多个响应外源激素和逆境胁迫的作用元件。VvSCL14-Like启动子驱动的GUS基因,只在拟南芥萌发初期下胚轴处表达。以GA3和NaCl分别处理阳性拟南芥幼苗,48h后GUS基因均在叶柄和叶脉表达。干旱胁迫处理阳性拟南芥幼苗,15d后GUS基因只发现在根部表达。研究结果表明:VvSCL14-Like基因的表达具有组织特异性,参与外源赤霉素(GA3)和非生物胁迫响应过程,但不是外源赤霉素(GA3)介导的无核葡萄果实膨大过程的主要调控元件。
In order to explore the regulatory genes of GA3-mediated signal transduction in the seedless grapes, the main regulatory elements were identified. In this study, Eucommia seedless grapes, Vitis vinifera L.cv.Centennial Seedless) was used to analyze the expression of SCARECROW Like 14-Like (VvSCL14-Like) gene, which belongs to the GRAS gene family, in different organs and fruit developmental stages of grapevine by semi-quantitative RT-PCR. The gene expression characteristics were studied by promoter cloning, bioinformatics analysis, promoter :: GUS fusion gene and GUS tissue staining. The results of semi-quantitative RT-PCR showed that VvSCL14-Like gene was mainly expressed in dormant buds and no expression during fruit development. GA3 was used to enlarge the young fruits. The results of 1, 3 and 7 days after treatment showed that there was no difference in the transcriptional level of VvSCL14-Like. Bioinformatics analysis of the VvSCL14-Like promoter revealed a number of response elements that respond to exogenous hormones and stress. The GUS gene driven by the VvSCL14-Like promoter is only expressed in the hypocotyls of early Arabidopsis germination. The positive Arabidopsis thaliana seedlings were treated with GA3 and NaCl respectively. After 48h, the GUS gene was expressed in the petiole and veins. Drought stress treatment of positive Arabidopsis seedlings, GUS gene only found in roots 15d after expression. The results showed that the expression of VvSCL14-Like gene was tissue-specific and involved in the process of GA3 and abiotic stresses, but not the GA3-mediated enlargement of fruitless grapes The main regulatory components.