PEG介导的玉米原生质体瞬时转化方法优化及其在ZmMYC2功能研究中的应用

	PEG介导的玉米原生质体瞬时转化方法优化及其在ZmMYC2功能研究中的应用; Optimization of PEG Mediated Transient Transformation of Maize Protoplast and Its Application in ZmMYC2 Function Study
	孙艳
导师	王蕾
摘要	Protoplast is the inclusion of plant or microorganism cell after removing cell wall, which keeps all the activity. Protoplast transient transformation system can be used to analyze gene overexpression, silencing, protein localization, promoter research and protein-protein interaction in a short time, which is a powerful tool for gene function research. Before, our group established a maize protoplast transient transformation system, which can be used for gene overexpression, silencing, secondary metabolite content analysis, gene expression level analysis and so on. However, the protoplast isolation and transformation steps are time-consuming, the transformation efficiency is low and the results are unstable. To optimized the method of maize protoplast transient transformation, we reduced the stimulation of light on protoplasts in the processes of seedling growing, protoplast isolation, and transformation by strictly avoiding light expose; reduced the times of centrifugation to maintains the integrity of protoplasts; deleted the vacuum steps. In general, the experimental procedure was simplified and the experimental stability was increased. The transformation efficiency was increased to 80%, which is sufficient for function study of candidate genes. This improved protoplast transient transformation system was applied to study the function of five ZmMYC2-regulated transcription factors, which are ZmMYB140 (Zm00001d020569), Zmhb42 (Zm00001d014628), ZmNAC87 (Zm00001d052390), ZmbHLH115 (Zm00001d031167), ZmbHLH119 (Zm000016257). In this study, we overexpressed these five transcription factors in maize protoplast, and detected the transcriptional changes of nine benzoxazinoids (BXs) biosynthesis genes, BX6, BX7, BX8, BX9, BX10/11, BX12, BX13, and BX14, and two terpene synthase genes, TPS7 and TPS10, as well as the metabolite content of BXs. The results show that ZmMYB140, Zmhb42, ZmNAC87, regulates the synthesis of benzoxazinoids as well as terpene volatiles, the insect resistant secondary metabolites, through transcriptional regulation of BXs and TPS. ZmbHLH115 regulates the synthesis of benzoxazinoids through transcriptional regulation of BXs. And ZmbHLH119 regulates the terpene synthesis through transcriptional regulation of TPS10. In addition, the improved maize protoplast transformation method was applied to study the interaction between ZmMYC2 and its downstream five transcription factors. The result showed that ZmMYC2 did not interact with them. Furthermore, the system was also used to detect the subcellular localization of ZmMYC2. It was found that ZmMYC2 mainly exists in the nucleus.
	2021-05
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/74497
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	孙艳. PEG介导的玉米原生质体瞬时转化方法优化及其在ZmMYC2功能研究中的应用, Optimization of PEG Mediated Transient Transformation of Maize Protoplast and Its Application in ZmMYC2 Function Study[D],2021.

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