根系内生真菌调控三叶青扩展蛋白基因表达促进根生长的机制研究

Tetrastigma hemsleyanum Diels & Gilg ex Diels is a rare medicinal plant in China. Its roots are full of medicinal values and have the anti-tumor activity. The previous report by us showed that an endophytic fungus strain TH15 was isolated from root of Tetrastigma hemsleyanum and significantly increased the expression of expansin gene in root to promote root growth of Tetrastigma hemsleyanum. However, its mechanism is unknown. In this project, we will carry out the following researches: (1) Determining and analyzing the whole genome sequences of endophytic fungus strain TH15, predicting the encoded protein and so on, combined with the analysis by HPLC-MS and NMR technique, we will identify the active ingredients in fermentation broth (metabolites) of endophytic fungus strain TH15. (2) Detecting and analyzing the difference of miRNAs in hairy roots of Tetrastigma hemsleyanum treatment with or without TH15’s fermentation broth, combined with related miRNAs overexpression and suppression expression, we will determinate the target genes of these different miRNAs. The results will reveal which ingredients are in fermentation broth of endophytic fungus strain TH15 and which miRNAs in Tetrastigma hemsleyanum respond to endophytic fungi TH15, and which of these ingredients and which of these different miRNAs are involved in regulating the expansin gene expression to promote root growth of Tetrastigma hemsleyanum. Finally, the project is expected to probe the molecular mechanism of endophytic fungus strain TH15 regulating the expression of expansin gene to promote root growth in Tetrastigma hemsleyanum.

三叶青是我国珍稀的药用植物，以根入药，有抗肿瘤等功效。本课题组前期开展的研究报道，从三叶青根中分离获得的内生真菌TH15显著提高扩展蛋白基因在三叶青根中的表达、促进根的生长，但其机制不明。本项目拟开展如下研究：（1）测定并分析内生真菌TH15全基因组序列，预测其编码的蛋白质等；再结合HPLC-MS和NMR分析，分类鉴别内生真菌TH15发酵液（代谢物）中的活性成分。（2）测定并分析内生真菌TH15发酵液处理前后三叶青毛状根中miRNA表达的差异；再结合相关的miRNA过表达和抑制表达，分析差异miRNA作用的靶基因。研究结果可望明确内生真菌TH15发酵液中有哪种成分，并参与调控三叶青扩展蛋白基因的表达和根的生长；明确三叶青中有哪些miRNA应答内生真菌TH15，并参与调控扩展蛋白基因的表达和根的生长，最终解析内生真菌TH15调控三叶青扩展蛋白基因表达、促进根生长发育的分子机制。

三叶青为葡萄科常绿藤本植物，既是珍稀的药用植物，以根入药，有抗肿瘤的功效；也可作为园林观赏植物。本项目以课题组前期从三叶青根中分离获得的内生真菌TH15为材料，多角度解析了内生真菌TH15促进根生长发育的机制。.（一）结合HPLC-MS和NMR及ELISA，发现内生真菌TH15代谢物中含5种植物激素（IAA、MeIAA、JA、JA-ILE和SA)）和玉米赤霉烯酮（zearalenone，ZEN）等活性成分；添加代谢物ZEN培养三叶青的实验表明，ZEN表现出低浓度促进三叶青根生长的效应。（二）以内生菌TH15代谢物ZEN处理和对照未处理三叶青根为材料，采用小RNA高通量测序、筛选出ZEN处理和对照组差异表达的miR156b和miR156f。通过转基因毛状根证实，ZEN提高三叶青根中miR156b和miR156f的表达、分别负向调控其靶基因SPL9和SPL10的表达、进而促进侧根的生长发育。（三）利用二代测序结合三代测序，测定内生菌TH15全基因组大小为46852391 bp，预测基因数为10442，其中包括植物激素合成代谢的同源基因；序列已提交GenBank (登录号JAHREO000000000)。（四）三叶青根的发育过程中有细根、棒状根和葫芦形根3种形态的根。生理生化和解剖学分析显示，葫芦形根中总黄酮含量最高、细胞质内含物最丰富；细根中维管束结构完整、而葫芦形根中维管束高度退化。对3种不同形态的根进行定量RNA-seq、并结合qRT-PCR验证揭示，在三叶青根的发育过程中，P450 711A1等10个上调基因、以及P450 83B1和LRR受体2个下调基因与葫芦形根的发育密切相关。.本项目首次发现内生真菌代谢物ZEN调控miR156、进而影响三叶青根的发育；在三叶青根的发育过程中，P450家族711A1和83B1起关键作用。研究结果丰富了内生真菌与植物互作、调控根发育的分子机理。