MdSBP21调控下游关键基因参与苹果盐胁迫响应的分子机制

Soil salinization is one of the major limiting factors for apple planting in northern China, which seriously influences the growth and development of apple tree and greatly reduces the quality and yield of apple. In our preliminary study, we found that MdmiR156n degraded MdSBP21 to involve in salt stress response by analysis of their expression in apple stock and function of transgenic arabidopsis after salt stress. Based on this, we will identify the salt resistance function of MdmiR156n and MdSBP21 by over-expression and RNAi silencing expression in apple. Furthermore, the downstream genes regulated by MdSBP21 will be identified by ChIP-Seq and RNA-Seq. Then, we will go ahead and research the binding capacity and regulatory function of MdSBP21 with downstream genes by ChIP-PCR, EMSA and LUC/REN double fluorescence transient expression analyses. Finally, the downstream genes function will be identified by using over-expression and RNAi silencing in callus of apple. The results will not only help to declare the molecular mechanism of MdSBP21 involvement in salt stress response by regulating downstream genes in apple, but also help to provide theoretical basis and necessary gene resources for apple rootstocks salt-resistant breeding.

土壤盐渍化是我国北方产区苹果生产的主要限制因素之一，严重影响苹果树的生长发育，大大降低了果实的品质和产量。项目组前期通过分析盐胁迫后苹果砧木中的基因表达和异源转化拟南芥两项工作，证明MdmiR156n通过降解MdSBP21基因参与盐胁迫响应。在此基础上，本项目拟通过构建过表达和沉默载体转化苹果，获得MdmiR156n和MdSBP21参与苹果盐胁迫响应的直接证据。利用苹果转基因材料进行染色质免疫共沉淀测序和转录组测序关联分析获得MdSBP21直接调控的下游关键基因，用ChIP-PCR、EMSA和LUC/REN双荧光瞬时表达等技术深入研究MdSBP21与下游关键基因启动子的结合及调控功能。通过转化苹果愈伤组织进一步对获得的下游关键基因进行盐胁迫功能研究。由此阐明MdSBP21调控下游关键基因参与苹果盐胁迫响应的分子机制，为培育耐盐苹果砧木新材料提供基因储备和理论指导。

土壤干旱和盐渍化已成为我国北部苹果产区苹果生产的主要限制因素之一，严重影响苹果的生长发育，大大降低了果实的品质和产量。砧木抗性品种的选育和抗逆机制的研究一是苹果遗传育种领域重大的科学问题。本项目利用转基因技术获得MdmiRNA156n和MdSBP4参与苹果非生物胁迫响应的直接证据。胁迫处理后，WT和转基因株系的生长状态存在明显差异，35S:STTM156n苹果愈伤受到胁迫后颜色发白，几乎全死亡；WT野生型愈伤和35S:rMdSBP4愈伤大多数死亡，而35S:MdmiR156n受胁迫影响较小，仍呈生长状态，但积累了大量类黄酮。胁迫处理后，通过对不同株系的生理指标，酶活性，组织化学染色以及抗逆相关基因的表达量测定明确了MdmiR156n通过调控下游MdSBP4及相关胁迫基因提高了转基因株系的抗性。此外，利用诱饵载体pGBKT7-MdSBP4与苹果cDNA文库进行杂交，共获得9个调控生长发育及环境胁迫的互作蛋白，且通过点对点双杂交互作确定了MdSBP4与生长素相关基因SHOOT GRAVITROPISM5（MdSGR5）蛋白强烈互作。此外，进一步利用酵母单杂和转录组测序关联分析获得多个MdmiRNA156n的互作基因。其中，我们重点研究了MdNF-YB11基因功能。我们将pGADT7和重组质粒MdMdNF-YB11-pGADT7分别转化pmiR156n-pAbAi酵母感受态。然后在SD/-Leu/AbA600培养基上进行酵母点对点验证。试验结果表明，MdNF-YB11与miR156n启动子发生互作。构建原核表达载体，诱导并纯化重组蛋白MdMdNF-YB11-GST，EMSA试验结果显示MdNF-YB11可以直接结合miR156n启动子的CACGTC motif。通过构建 MdNF-YB11过量表达载体及proMdmiRNA156n::LUC 荧光表达载体（GreenII0800-LUC ）并瞬时共转化烟草叶片，证明MdNF-YB11可以正向激活miR156n的启动子。 最后，通过RT-PCR及苹果转基因技术进一步对MdNF-YB11进行非生物胁迫功能研究。明确了MdNF-YB11通过调控MdmiRNA156n启动子进而调控MdSBP4参与苹果非生物胁迫响应。本研究为创造优良的耐盐抗旱苹果砧木新材料提供基因储备和理论基础。