DataSheet_2_Regulatory mechanism of MeGI on sexuality in Diospyros oleifera.doc

Dioecy system is an important strategy for maintaining genetic diversity. The transcription factor MeGI, contributes to dioecy by promoting gynoecium development in Diospyros lotus and D. kaki. However, the function of MeGI in D. oleifera has not been identified. In this study, we confirmed that MeGI, cloned from D. oleifera, repressed the androecium development in Arabidopsis thaliana. Subsequently, chromatin immunoprecipitation-sequencing (ChIP-seq), DNA affinity purification-sequencing (DAP-seq), and RNA-seq were used to uncover the gene expression response to MeGI. The results showed that the genes upregulated and downregulated in response to MeGI were mainly enriched in the circadian rhythm-related and flavonoid biosynthetic pathways, respectively. Additionally, the WRKY DNA-binding protein 28 (WRKY28) gene, which was detected by ChIP-seq, DAP-seq, and RNA-seq, was emphasized. WRKY28 has been reported to inhibit salicylic acid (SA) biosynthesis and was upregulated in MeGI-overexpressing A. thaliana flowers, suggesting that MeGI represses the SA level by increasing the expression level of WRKY28. This was confirmed that SA level was lower in D. oleifera female floral buds than male. Overall, our findings indicate that the MeGI mediates its sex control function in D. oleifera mainly by regulating genes in the circadian rhythm, SA biosynthetic, and flavonoid biosynthetic pathways.

雌雄异株（dioecy）系统是维持物种遗传多样性的重要策略。转录因子MeGI可通过促进君迁子（Diospyros lotus）与柿（D. kaki）的雌蕊发育，参与调控雌雄异株性状的形成。然而，MeGI在油柿（D. oleifera）中的功能尚未被阐明。本研究从油柿中克隆得到MeGI基因，并证实其能够抑制拟南芥（Arabidopsis thaliana）的雄蕊发育。随后，本研究采用染色质免疫共沉淀测序（chromatin immunoprecipitation-sequencing, ChIP-seq）、DNA亲和纯化测序（DNA affinity purification-sequencing, DAP-seq）与RNA测序（RNA-seq）技术，解析了MeGI介导的基因表达响应特征。结果显示，受MeGI诱导的上调表达基因与下调表达基因分别主要富集于生物钟节律相关通路与类黄酮生物合成通路。此外，本研究重点关注了经ChIP-seq、DAP-seq及RNA-seq共同筛选到的WRKY DNA结合蛋白28（WRKY28）基因。已有研究表明，WRKY28可抑制水杨酸（salicylic acid, SA）的生物合成，且在MeGI过表达的拟南芥花组织中呈上调表达，这提示MeGI可通过提升WRKY28的转录水平来降低植株体内水杨酸含量。后续验证实验证实，油柿的雌性花芽中水杨酸含量显著低于雄性花芽。综上，本研究结果表明，MeGI主要通过调控生物钟节律通路、水杨酸生物合成通路及类黄酮生物合成通路中的相关基因，实现对油柿性别决定过程的调控。