‘红肉蜜柚’CmMYB330基因的分离与表达分析

发布时间：2019-03-30 15:58

【摘要】：【目的】分析‘红肉蜜柚’果实汁胞粒化相关的MYB转录因子基因特征与表达模式,探讨MYB转录因子在蜜柚汁胞木质素代谢过程中的作用机制,为研究蜜柚汁胞粒化发生机制提供借鉴。【方法】以‘红肉蜜柚’果实汁胞为试材,参考甜橙MYB全基因组分析,利用生物信息学分析和PCR技术克隆Cm MYB330的编码区序列及其5’端调控序列,并对序列进行生物信息学分析。利用实时荧光定量PCR技术对Cm MYB330在‘红肉蜜柚’果实汁胞不同发育时期和不同部位的表达情况进行分析。利用农杆菌介导法将Cm MYB330与GFP的融合表达载体注射转化到烟草叶片中进行亚细胞定位分析。利用酵母双杂交系统对Cm MYB330进行转录激活活性分析。【结果】Cm MYB330的编码区序列长度为942 bp,编码313个氨基酸,预测为核定位蛋白,为典型的R2R3 MYB转录因子。Cm MYB330与甜橙Cs1g19400.1、Am MYB330等亲缘关系近,都属于R2R3 MYB第4亚组。Cm MYB330的5’端调控序列为起始密码子上游-1 748 bp序列,预测该序列含有TATA-box、CAAT-box 2个启动子核心元件,3个MBS,1个AC-I,1个5UTR Py-rich stretch,还有大量激素(如赤霉素、乙烯、水杨酸等)响应元件。Cm MYB330表达量在‘红肉蜜柚’果实汁胞发育过程中有起伏,但总体呈现上升趋势,表达量在花后208 d达到最大,之后开始下降。在转化p-super1300-Cm MYB330-GFP的烟草叶片细胞核中检测到绿色荧光信号,与预测结果一致,说明Cm MYB330定位在细胞核中。在Y2H Gold酵母中,Cm MYB330表现为没有转录激活活性。【结论】克隆了‘红肉蜜柚’MYB转录因子Cm MYB330编码区序列及其5’端调控序列,Cm MYB330属于R2R3 MYB第4亚组,与汁胞粒化相关,为核定位蛋白,同时分析了其在‘红肉蜜柚’果实汁胞发育过程中的表达水平,总体呈上升趋势。为进一步研究Cm MYB330与蜜柚汁胞粒化的关系奠定了基础。
[Abstract]:[objective] to analyze the gene characteristics and expression patterns of MYB transcription factors related to the granulation of juice cells of 'Red Meat Honey Pomelo', and to explore the mechanism of MYB transcription factors in lignin metabolism of pomelo juice cells. [methods] the juice juices of 'Red Meat Pomelo' were used as test material, and the whole genome analysis of sweet orange MYB was used as reference. The coding region and its 5'- terminal regulatory sequence of Cm MYB330 were cloned by bioinformatics analysis and PCR technique, and the sequence was analyzed by bioinformatics. Real-time fluorescence quantitative PCR was used to analyze the expression of Cm MYB330 at different developmental stages and different parts of fruit juice cells of 'Red Meat Honey Pomelo'. The fusion expression vector of Cm MYB330 and GFP was injected into tobacco leaves by Agrobacterium tumefaciens-mediated transformation for subcellular localization analysis. The transcriptional activation activity of Cm MYB330 was analyzed by yeast two hybrid system. [results] the coding region length of Cm MYB330 was 942 bp, encoding 313 amino acids, which was predicted to be a nuclear localization protein. Cm MYB330 is a typical R2R3 MYB transcription factor. Cm MYB330 is closely related to Cs1g19400.1,Am MYB330 and belongs to R2R3 MYB subgroup 4. The 5 'terminal regulatory sequence of Cm MYB330 is upstream of the initiation codon-1748 bp, which is predicted to contain TATA-box,. CAAT-box has two promoter core elements, three MBS,1, AC-I,1, 5UTR Py-rich stretch, and a large number of hormones (such as gibberellin, ethylene, etc.) (salicylic acid, etc.) the expression of Cm MYB330 fluctuated during the fruit juice cell development of 'Red Meat Honey Pomelo', but showed an upward trend, and the expression level reached the maximum at 208th day after anthesis, and then began to decrease. Green fluorescence signals were detected in the nuclei of tobacco leaves transformed with p-super1300-Cm MYB330-GFP, which was consistent with the predicted results, indicating that Cm MYB330 was localized in the nuclei. In Y2H Gold yeast, Cm MYB330 showed no transcriptional activation activity. [conclusion] the Cm MYB330 coding region and its 5 'terminal regulatory sequence of MYB transcription factor were cloned. Cm MYB330 belongs to R2R3 MYB subgroup 4, which is related to juices granulation. [conclusion] the sequence of Cm MYB330 coding region and its 5' terminal regulatory sequence of 'red pomelo' MYB transcription factor have been cloned. As a nuclear localization protein, the expression level of the protein in the fruit juice cell development of 'Red Meat Honey Pomelo' was analyzed, and the expression level showed an increasing trend as a whole. It laid a foundation for further study on the relationship between Cm MYB330 and nectar Juice granulation.
【作者单位】：福建农林大学园艺学院·园艺产品贮运保鲜研究所;
【基金】：福建省科技重大专项(2013NZ0002) 国家科技支撑计划(2014BAD15B01) 福建省科技计划星火项目(2016S0022) 清远市科技计划(2015C002)
【分类号】：S666.3

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