低温响应转录因子CdCAMTA1在野生狗牙根抗寒中的功能及其抗寒分子机理研究

Low temperature is considered as the major factor limiting bermudagrass utilization. Discovering key genes and then improving bermudagrass cold resistance by modern breeding technology, is one of the most important methods to obtain bermudagrass varieties resistant to cold stress. In previous study，we identified a CdCAMTA1 gene, encoding calmodulin binding transcription activator. The gene was induced by low temperature and its expression level was higher in cold resistant genotype than that of in cold sensitive accession. On this basis, our present research will analyze the basic characteristic of CdCAMTA1 as transcription factor; To identify the function of CdCAMTA1 gene, over-expression and RNAi vectors will be constructed and transformed into bermudagrass; The differential expression of CBF genes will be investigated on CdCAMTA1 transgenic and control plants under normal and low temperature condition. The target CBF gene regulated by CdCAMAT1 will be further identified by yeast one-hybrid methods; Moreover, our previous yeast two- hybrid experiments revealed that CdCAMTA1 interacts with JAZ1, the repressor of jasmonate signaling. The experiment that JAZ1 proteins repress the transcriptional function of CAMTA1 will be determined. The study will reveal the function of CdCAMTA1 gene in response to cold stress, hence provide elite gene resource for bermudagrass breeding for cold resistance. Moreover, the study will provide important evidence for elucidating the signaling pathway that the interaction between CdCAMTA1 and JAZ1 is involved in response to JA-regulated cold stress. Therefore, the study has important theoretical significance and potential application value.

低温是限制狗牙根推广应用的主要因素，从狗牙根中发掘抗寒关键基因，进而通过现代育种技术改良狗牙根的抗寒性，是获得狗牙根抗寒品种的重要手段之一。前期研究中，我们从野生狗牙根中鉴定到一个编码转录因子的CdCAMTA1基因，发现其受低温诱导且在抗寒材料中表达水平高于不抗寒材料。在此基础之上，本项目将研究该转录因子的基本特征，并通过超表达和RNAi转基因技术鉴定CdCAMTA1在抗寒中的功能；通过差异表达分析和酵母单杂交等技术，鉴定CdCAMTA1调控的CBF基因；此外，前期研究发现CdCAMTA1能与JA信号通路中转录抑制子JAZ1相互作用。基于此，我们将开展JAZ1抑制CdCAMTA1的转录功能研究。项目实施将阐明CdCAMTA1抗寒功能，为狗牙根抗寒育种提供重要的基因资源，并为解析CdCAMTA1与JAZ1互作参与JA调控植株耐寒性的信号网络提供重要科学依据，具有重要的理论意义和潜在应用价值。

野生狗牙根中蕴藏着丰富的抗逆优异基因，是狗牙根品种改良的遗传资源宝库。本项目主要研究野生狗牙根抗寒基因发掘、功能鉴定及调控机制解析。我们以耐寒和低温敏感狗牙根种质为材料，利用RNA-Seq结合生理生化技术，在低温胁迫和正常温度条件下，分析了全基因组水平上基因的差异表达情况，发掘了一批参与狗牙根应答低温胁迫的基因。研究还揭示了狗牙根耐寒种质相比低温敏感种质，能够更快地调节基因表达来应答低温胁迫；经过4 °C低温驯化后，狗牙根耐寒种质能够更加有效地激活基因表达来应答冷冻胁迫（零下低温）；差异基因富集的代谢通路分析揭示狗牙根可能通过激活光和代谢、氮代谢、碳固定、脂类代谢、糖代谢以及激素信号转导等通路来应答低温胁迫。我们对在耐寒种质中优势表达的关键基因CdCAMTA1和CdERF1进行了耐寒功能鉴定。利用病毒介导的基因沉默技术（VIGS）成功下调了狗牙根内源CdCAMTA1和CdERF1基因的表达，CdCAMTA1-VIGS和CdERF1-VIGS狗牙根植株的耐寒能力明显低于对照。进一步利用RNA-Seq技术，分析了WT、CdERF1-VIGS与CdCAMTA1-VIGS狗牙根植株在低温胁迫下的差异基因表达模式，揭示了CdCAMTA1和CdERF1调控耐寒性的分子网络。我们进一步构建了CdCAMTA1和CdERF1的超表达载体并转化了模式植物拟南芥，对转基因和野生型WT植株进行耐寒能力比较，发现低温胁迫下，转基因植株的耐寒能力明显增强。研究还表明CdCAMTA1 基因可能参与到JA正向调控植株耐寒性的信号网络，有关CdJAZ1 与CdCAMTA1 互作并抑制CdCAMTA1 的转录功能研究，正在深入研究当中。我们研究还揭示了激素和小分子物质，包括乙烯和褪黑素，通过调节光合系统、抗氧化系统、代谢物质合成和关键基因表达来调节狗牙根耐寒性。基于关联分析策略，我们发掘到15个SSR位点与狗牙根耐寒性紧密相关联，并且有10个位点与多个功能性状相关联，为狗牙根抗寒分子育种提供了重要科学依据。本项目总结发表论文10余篇，取得重要研究进展，达到预期研究目标，为狗牙根抗寒分子育种提供了有价值的科学资料。