miRNA-ARF调控网络在根域限制栽培葡萄根系发育中的作用机制研究

As a widely used cultivation technique, the root-zone restriction cultivation can improve the development and fruit quality of grapevine, significantly. The source of the plant aboveground regulation was comes from the root development differentiation, such as the increased total number of root, fine root ratio, larger root cortical area and so on. However, the mechanism of root development in the root-zone restriction system is still unknown. MiRNA is an important transcription regulator and plays an important role in plant root development. Our previous work showed that there were many miRNAs, such as Vvi-miR160, who target the ARF gene family numbers showed difference expression patterns between the root-zone restriction cultivation and traditional cultivation grapes. Those results indicate that the miRNA-ARF regulatory system function in root development in grape. Therefore, the RNA-Seq will be used to detect the differentially expressed miRNAs between the root-zone restriction cultivation and traditional cultivation grape roots. The miRNAs who targeted the ARF family gene will be analyzed by bioinformatics analysis. And the miRNA-ARF regulatory network will be established by using the miRNA family analysis, miRNA-mRNA targeting analysis and transgenic phenotypic analysis. This study provides a new understanding for the gene regulation pathway and regulatory mechanism of root development. It also offers a new direction for improving the root development regulation method in grape cultivation.

根域限制作为一项广泛应用的栽培技术，能显著改善葡萄树体生长发育，提高果实品质。而其对地上部的调控源自于根系发育状态的差异，如根系总量增加、细根比例增多、皮层面积增大等。但根域限制栽培根系发育变化的机理还不清楚。MiRNA是重要的基因表达调控因子，在植物根系发育中具有重要作用。申请人研究表明Vvi-miR160等多个靶向ARF家族基因的miRNA的表达量在根域限制和地栽葡萄根系中存在显著差异，推测miRNA-ARF调控体系可能参与葡萄根系发育调控。因此本课题将利用RNA-Seq测定限根与地栽葡萄根系发育的差异表达miRNA，利用生物信息学分析聚焦靶向ARF家族基因的miRNAs，通过miRNA家族分析、miRNA-mRNA靶向调控分析、转基因表型分析等手段建立根系发育的miRNA-ARF调控网络，为解析植物根系发育的调控途径、调控机制提供新认知，也为开发调控葡萄根系发育栽培技术提供新的方向。

作为一项广泛应用的栽培技术，根域限制能显著改善葡萄树体生长发育，提高果实品质。MiRNA是重要的基因表达调控因子，在植物根系发育中具有重要作用。本项目通过对比限根与地栽葡萄根系构型，明确了根域限制栽培葡萄根系构型变化特点及形成过程。栽培70天后两种栽培方式根系构型出现显著变化，具体表现为限根后根尖退化早、侧根和不定根发生量增加，且根系出现螺旋卷曲和更新速度加快的特点。以差异变化节点的根系为材料进行小RNA测序，筛选并验证到多个影响葡萄根系发育miRNA。小RNA测序共得到153条已知miRNA和119条预测的新miRNA。预测的新miRNA与miRBase数据库进行进一步默认参数比对分析后得到96个同源于其他物种的葡萄miRNA以及23个葡萄特有的新 miRNA。在不同的比较组中分别有26、33、26和32个差异表达的miRNA（RR7 vs nR7；RR12 vs nR12；nR12 vs nR7；RR12 vs RR7）。不同样本间差异表达miRNA的靶基因预测分析得到24个miRNA的靶基因可能参与根系发育过程。RLM-RACE验证得到6个VvARF能被miRNA直接靶切。其中vvi-miR160c能靶切VvARF10/10-like/16基因并作用于根尖发育，转基因拟南芥后vvi-miR160c株系的根尖退化并分化出大量侧根；vvi-miR167b能靶切VvARF6/6-like/8基因并作用于初生根和侧根发育。综上所述，本研究揭示了葡萄根系周年发生规律，并明确了根域限制栽培后根系构型变化方式及时间点；通过小RNA测序鉴定到4个影响葡萄初生根和侧根发生的miRNA，并证明miRNA-ARF调控网络能影响葡萄初生根、侧根和根尖的发生。该研究为解析植物根系发育的调控途径、调控机制提供新认知，也为开发调控葡萄根系发育栽培技术提供新的方向。