BnMYB93s转录因子基因调控甘蓝型油菜侧根发育的分子机制研究

Lateral root is a key organ for plant anchorage, acquisition of water and nutrient uptake etc, thereby is important for crop productivity. Therefore, robust lateral roots are crucial for Brassica napus root-lodging resistant and acquisition of water and mineral nutrients distributed in soil, thereby contribute to increase the yield and defense responses. MYB transcription factor is one of the largest transcription factor gene families in land plants, and plays a key role in regulating lateral root development. Thus, two MYB candidate genes, BnMYB93-1 and BnMYB93-2 that were involved in the regulation of lateral root development were identified in our previous study, based on the available genome data of Brassica napus. Based on our results, the present project will focus on seeking the underlying regulating mechanism of the candidate genes, exploring their genomic variation profiles in 600 Brassica napus gerplasm resources that were re-sequenced as well as the correlation between the genomic variation profiles of candidates and the efficiency of plant nutrient uptake, yield traits and root-lodging resistant. Our results will provide key gene resources for molecular breeding to improve the yield and root-lodging resistant of Brassica napus. This project is expected to get 1 to 2 relative inventions patented, publish 3 to 4 SCI papers, including 2-3 papers published in journals with impact factors around 6, and train 3 to 4 graduate students.

侧根(Lateral root)是根系锚定植株、吸收水分和矿质养分等的重要器官，与作物生产密切相关。发育良好的侧根是油菜抗根倒的必要保障，可以促进植株获取土壤养分和水分，进而提高其产量和抗逆胁迫能力。MYB类转录因子是植物中最大的转录因子基因家族之一，对侧根发育具有重要的调控作用。鉴于此，我们前期基于甘蓝型油菜基因组测序数据，鉴定了2个调控油菜侧根发育的MYB基因(BnMYB93-1和BnMYB93-2)。本项目拟在此基础上，深入解析候选基因调控油菜侧根发育的分子机制；结合600份重测序材料分析候选基因在群体中的遗传变异规律及其与油菜营养高效利用、产量性状、根倒等的相关性。本项目的研究成果将为油菜高产和抗根倒分子育种提供新的策略和关键的基因资源。本项目拟申请发明专利1-2项，发表SCI期刊学术论3-4篇，其中影响因子大于6的文章2-3篇，培养研究生3-4名。

侧根对锚定植株、吸收土壤中的水分和矿质养分非常重要，其生长发育与油菜营养高效利用、抗逆（如抗根倒、抗旱等）、产量等紧密相关。因此，挖掘油菜中调控侧根发育的关键基因，阐明其分子机制，兼具理论研究价值和应用前景。鉴于此，本研究利用生物信息学的方法挖掘油菜基因组中调控侧根发育的候选基因，并联合分子生物学等多学科的技术深入研究候选基因的分子作用机制。本研究的主要结果包括：（1）我们在油菜基因组中筛选并克隆到2个可能调控侧根发育的R2R3-MYB类候选基因，BnMYB93-1和BnMYB93-2。（2）RNA-Seq分析表明他们在油菜苗期根中优势表达；qRT-PCR分析表明其表达受外源生长素（IAA）、脱落酸（ABA）、细胞分裂素（6-BA）和赤霉素（GA3）的显著正诱导。（3）我们克隆了BnMYB93-1基因上游1600bp启动子序列并驱动GUS报告基因在拟南芥中表达，结果显示GUS基因在侧根发育起始阶段的细胞中高表达，随着侧根原基形成至突破皮层GUS基因的表达量逐渐降低，表明BnMYB93-1基因可能调控侧根起始过程。（4）过表达BnMYB93-1基因后拟南芥和油菜的侧根密度均显著降低，而沉默该基因（CRISPR-Cas9）后油菜的侧根密度显著增加，证明该基因负调控侧根密度。（5）ChIP-Seq实验证明BnMYB93-1的靶基因编码4个高度同源的细胞周期蛋白BnCYCD3;1s，其靶位点为靶基因启动子上游500bp处的“T[A/G]GTTA”基序；EMSA和双荧光素酶分析证明侧翼序列影响BnMYB93-1与靶基序的结合能力；该调控模式在拟南芥、白菜、甘蓝和油菜中是保守的；atcycd3;1突变体的侧根密度显著增加，证明BnMYB93-1对BnCYCD3;1s基因起正调控作用。（6）Y2H、BILC和GST-Pull down实验共同证明BnMYB93-1与BnSY1-like蛋白的C端互作；双荧光素酶分析发现BnSY1-like能降低BnMYB93-1与靶基序的结合能力；双过表达BnSY1-like和BnMYB93-1基因后拟南芥的侧根密度高于过表达BnMYB93-1并回复到WT水平，说明BnSY1-like部分沉默BnMYB93-1的功能。