基于CBL-CIPK信号通路的杜梨耐盐机制研究

Birch-leaf pear (Pyrus betulaefolia Bunge) is used as a pear rootsctock to improve salt tolerance of pear by reducing the accumulation of sodium ions in its roots and limiting the movement of sodium ions to aerial parts of the plant. Previous studies found that CBL-CIPK signal pathway plays an important role in regulating the distribution of sodium ions and the intracellular balance between sodium and potassium ions under salt stress in model plant. However, little is known about the mechanism of salt tolerance in pear. Some salt-tolerant individual plants of birch-leaf pear were screened in our lab which sodium ion absorption and transportation to shoot were significant reduction than ordinary seedlings. Furthermore, the CBL-CIPK signal pathway downstream SOS1 function gene expression levels in salt-tolerant birch-leaf pear seedlings were higher than ordinary seedlings. Four CBL genes (PbCBL1, PbCBL2, PbCBL4 and PbCBL10), responding salt stress obviously, have also been cloned from salt-tolerant birch-leaf pear seedlings. Moreover, Escherichia coli cells salt tolerance were enhanced by engineered expressing these CBL genes. Based on these results, RNA-Seq technology will be employed to sequence the transcriptomic libraries of the salt-tolerant individual plants and the control ones before and after salt stress. Then the differentially expression genes, which maybe including different members from CBL, CIPK, SOS1, NHX and AKT gene family, would be screened via bioinformatics analysis. At the same time, PbCBL1, PbCBL2, PbCBL4 and PbCBL10 will be transformed into birch-leaf pear and their functions will be analysis through evaluation salt tolerance abilities of transgenic plants. Then expression characteristics of the CBL-CIPK signal pathway members in transgenic plants, which differentially expressed in the transcriptomic libraries, will be comparative study. Finally, the spatiotemporal distribution of sodium and potassium ions in transgenic plants will be analysis. The salt tolerance mechanism based on CBL-CIPK signaling behind birch-leaf pear could be understood through a comprehensive analysis on the above results. We hope that this research would provide some theoretical basis for salinity and alkalinity resistance breeding of pear.

杜梨作为耐盐砧木，主要通过减少钠离子在根中积累和向地上部运输来提高梨的耐盐性。模式植物中的研究发现CBL-CIPK 信号通路起着调控盐胁迫下细胞内钠离子分布及钠、钾离子平衡的重要作用。梨耐盐机制相关报道很少。申请人前期筛选的杜梨耐盐单株对钠离子吸收和向地上部转运较普通单株显著减少，CBL-CIPK 信号通路下游SOS1功能基因表达量增加，并分离了4个响应盐胁迫提高大肠杆菌耐盐能力的CBL基因（PbCBL1、2、4和10）。在此基础上，拟构建耐盐及普通单株盐胁迫前后转录组文库，利用生物信息学筛选差异表达CBL、CIPK、SOS1、NHX、AKT 基因家族成员；将PbCBL1、2、4和10 基因转化杜梨，研究它们在耐盐胁迫中的作用、对转录组文库中差异表达CBL-CIPK 信号通路成员的调控及钠、钾离子时空分布的影响，揭示杜梨基于CBL-CIPK 信号通路的耐盐机制，为梨抗盐碱育种提供实验依据。

杜梨主要通过减少Na+在根中积累和向地上部运输来实现耐盐性。本项目借助杜梨耐盐单株盐胁迫转录组数据库，明确CBL-CIPK信号通路直接参与杜梨耐盐胁迫过程；分离鉴定通路中PbCBLs、PbCIPKs、PbNHXs基因，解析它们的基因结构及转录模式，明确耐盐相关的分子调控网络成员（PbCBL1/2/4/10、PbCIPK1/6/21/23/24、PbNHX1/2/7）及作用部位；通过耐盐单株/普通单株盐胁迫转录组的比较分析，明确耐盐能力不同株系中CBL-CIPK信号通路对盐胁迫的响应存在转录差异；利用CBL基因家族成员过量表达Meta分析，明确过量表达该类基因促进转基因植株耐盐的关键特性，转基因实验解析PbCBL1/2/4/10在耐盐胁迫中的作用：在盐胁迫下，转基因株系较野生型生长优势显著，生物量积累更多，耐盐能力更强，过表达PbCBL1/2/4/10能够改变受体植物体内的离子分布，使其维持较低的 Na+/K+比率以减少植株 Na+毒害，不同转基因株系中盐胁迫信号通路 Marker 基因（RD22/26/29B）表达增强，而CIPKs、NHXs、AKTs家族转录水平上调的成员有所区别，表明 PbCBL1/2/4/10是位于盐胁迫响应信号通路中的上游调控基因，各自参与的通路有所交叉、互为补充，但均在杜梨盐胁迫前后转录组特异DEGs内，与转录组结论相互验证。综上所述，PbCBL1 主要在叶中起作用，调控PbCIPK1/23/24、PbNHX1/2和PbAKT1；PbCBL2主要在根中起作用，调控PbCIPK1/21和PbNHX1/2；PbCBL4主要在叶中起作用，调控PbCIPK6/24、PbAKT2和PbNHX7；PbCBL10在全株起作用，调控PbCIPK23/24、PbAKT1和PbNHX1/2。以 PbCBL1/2/4/10为代表的CBL-CIPK 信号通路起着调控盐胁迫下杜梨胞内Na分布及Na+、K+平衡作用，使其更好的抵御高盐环境。本项目发表研究论文13篇（SCI 6篇），授权发明专利5项，培养硕士研究生2名，出站博士后3名；所建立的研究方法和技术方案可类推应用于蔷薇科其他砧木的相关研究，为解析杜梨抗逆胁迫相关性状的分子调控机制提供重要转录组信息基础，初步揭示杜梨基于CBL-CIPK 信号通路的耐盐机制，为梨抗盐碱育种提供候选基因及理论依据。