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钙信号是植物响应胁迫的重要系统之一,前期研究发现钙信号参与了连作地黄感知、传导和响应连作胁迫的进程。为了确证钙信号在地黄连作障碍形成过程中的作用方式,本研究在前期地黄大容量转录组基础上,利用生物信息学方法详细鉴定地黄钙信号转导体系相关蛋白的编码基因,并对其在连作和头茬地黄中的表达模式进行分析。同时,采用焦锑酸盐法和钙荧光染色法测定连作和头茬地黄根细胞中钙离子浓度差异。鉴定出5个CaMs、12个CBLs、21个CDPKs、21个CIPKs、16个CMLs和9个CRKs等84个钙信号体系相关基因。通过对连作和头茬地黄钙信号体系相关基因差异表达模式进行分析,发现连作效应增强了地黄钙信号体系中关键基因的表达。进一步利用qRT-PCR方法对12个关键的钙信号转导相关基因进行表达验证,确证了钙信号响应了连作胁迫进程。此外,2种钙离子浓度检测方法也均证实连作胁迫显著促进了地黄胞内钙离子的积累。本研究从分子和细胞水平上确证了钙信号在地黄连作障碍形成中扮演着重要角色,为进一步从分子水平上解读地黄连作障碍的形成机制奠定了基础。
Calcium signaling is one of the important systems in plant response to stress. Previous studies found that calcium signaling participates in the process of continuous cropping, transmitting and responding to continuous cropping stress. In order to confirm the role of calcium signaling in the process of Rehmannia glutinosa contingency, based on the large volume transcriptome of Rehmannia glutinosa, bioinformatics methods were used to identify the genes encoding digoxin signaling system in detail. The analysis of the expression pattern in continuous cropping and in stubble yellow. At the same time, the difference of Ca2 + concentration in roots and roots of Rehmannia glutinosa continuous cropping and stubble (Rehmannia glutinosa) was determined by pyroxidate method and calcium fluorescence staining. Eightty-four calcium signaling system-related genes were identified, including 5 CaMs, 12 CBLs, 21 CDPKs, 21 CIPKs, 16 CMLs and 9 CRKs. By analyzing the differential expression pattern of the genes related to Rehmannia glutinosa signal system, the continuous cropping effect enhanced the expression of key genes in Rehmannia glutinosa signal system. QRT-PCR method was further used to validate the expression of 12 key calcium signal transduction related genes, confirming that the calcium signal responded to the continuous cropping stress process. In addition, the two kinds of calcium concentration test methods also confirmed that cropping stress significantly promoted the accumulation of calcium ions in Rehmannia glutinosa. This study confirmed that calcium signaling played an important role in the formation of Rhizoma Coptidis at the molecular and cellular level, which laid the foundation for further understanding of the mechanism of Rhizoma Coptidis formation at the molecular level.