大豆转录因子GmWRI1基因的鉴定、表达及其调控油脂合成作用机制研究

Soybean (Glycine max (L.) Merrill.) is one of the most important crop for human and animal nutrition and industrial renewable energy in the world. However, the oil content of soybean in China is less than 2.69% of the foreign soybean and uncompetitive in the international market at present. Therefore developing high-quality, high lipid content soybean lines for the revitalization of Chinese soybean industry is imminent. During the seeds development, transcription factor WRINKLED1 (WRI1) can significantly improve lipid content by up-regulating a series of genes’ expression which involved in lipid synthesis. In this study, on the basis of identification about the transcription factor GmWRI1 by transcript profiling and the full-length cDNA of GmWRI1 was cloned, and the relationship between its expression characteristics and soybean lipid synthesis will be determined. Subcellular location and transcriptional activation property of GmWRI1 will also be researched. Biological function of the gene will be detected by Arabidopsis mutant, over-expression, and RNAi technology, and thus to clarify whether GmWRI1 plays an important role in soybean lipid synthesis process by regulating the structure gene (GmPK).On one hand, this project will lay a foundation for further research of soybean lipid synthesis mechanism. On the other hand, it will provide new ideas for cultivating new lines of high lipid content soybean by molecular breeding.

大豆（Glycine max(L.)Merrill.）是世界上最主要的油料作物之一，其种子油是人类与动物营养和工业可再生能源的重要来源。但目前我国大豆含油量低于国外大豆2.69%，在国际市场上缺乏竞争力，因此培育优质、高油大豆新品种对振兴我国大豆产业迫在眉睫。转录因子WRINKLED1(WRI1)在种子发育时，可从上游调控植物油脂代谢途径中的一系列基因的表达而使脂类含量大幅提高。本项目在通过表达谱鉴定出高油转录调控因子GmWRI1的基础上克隆该基因全长，明确其表达水平与大豆油脂合成间关系；确定GmWRI1的亚细胞定位和转录激活特性；在此基础上，通过超表达、RNAi及突变体回补探明大豆转录因子GmWRI1基因的生物学功能，阐明其是否通过调控结构基因GmPK进而在大豆油脂合成过程中起重要作用。一方面为深入研究大豆油脂合成调控机制奠定基础；另一方面为利用分子育种手段培育高油大豆新品系提供新思路。

大豆（Glycine max(L.)Merrill.）是世界上最主要的油料作物之一，其种子油是人类与动物营养和工业可再生能源的重要来源。但目前我国大豆含油量低于国外大豆2.69%，在国际市场上缺乏竞争力，因此培育优质、高油大豆新品种对振兴我国大豆产业迫在眉睫。转录因子WRINKLED1(WRI1)在种子发育时，可从上游调控植物油脂代谢途径中的一系列基因的表达而使脂类含量大幅提高。本项目在通过表达谱鉴定出高油转录调控因子GmWRI1的基础上克隆该基因全长；通过转录激活活性和亚细胞定位分析，确定其属于转录激活子，定位于细胞核和细胞膜中。在此基础上，通过超表达、RNAi及突变体回补探明大豆转录因子GmWRI1基因的生物学功能，将植物表达载体pBASTA-GmWRI1利用Floral Dip法转化拟南芥突变体获得转基因回补拟南芥植株，通过索氏抽提法对不同拟南芥株系进行种子油脂含量分析，结果显示高表达量的转Gm WRI1基因拟南芥种子油脂含量与野生型相比差异显著，缺失型拟南芥种子油脂含量与野生型比较油脂含量大幅度下降，将回补后的拟南芥种子油脂含量与野生型相比油脂含量差异不显著，初步说明Gm WRI1对转基因拟南芥种子油脂含量的提高有促进作用，并对基因缺失型的拟南芥种子有一定的回补作用。构建CRISPR-GmWRI1的植物表达载体，利用大豆子叶节转化法将超表达载体pBASTA-GmWRI1和基因敲除载体CRISPR分别转入大豆吉育72中，并获得超表达T3代转基因大豆5株，基因敲除T2代转基因大豆4株，结合油脂含量测定，证明该基因对大豆含量的提高具有促进作用。课题完成期间，发表研究性论文7篇，专利一项。一方面为深入研究大豆油脂合成调控机制奠定基础；另一方面为利用分子育种手段培育高油大豆新品系提供新思路。