江西铅山红芽芋超低温疗法脱毒抑制细胞凋亡抗早衰的机理研究

江西铅山红芽芋是国家地理标志农产品，长期无性繁殖易导致其病毒积累、种性退化、产量和品质下降。脱毒能恢复种性是学界共识。超低温疗法是一种新型高效脱毒技术。我们前期研究表明，与常规茎尖脱毒相比，超低温疗法脱毒可抑制细胞凋亡抗早衰，促进增产增收。目前超低温疗法脱毒在后期大田生产抑制细胞凋亡抗早衰的研究尚未见报道。本课题以铅山红芽芋为研究对象，获得超低温疗法脱毒苗，模拟蚜虫的病毒田间传播进行病样汁液接种，模拟田间气候进行人工气候室无菌培养，采用RNA-Seq技术进行超低温疗法脱毒抑制细胞凋亡抗早衰的转录组分析，筛选相关候选基因，克隆其全长cDNA序列，并通过遗传转化过量表达研究其功能及其亚细胞定位，分析其作用机制，再结合光合生理、生理生化、线粒体特性、Cyt C基因、抗氧化酶基因和激素相关基因表达等分析，诠释超低温疗法脱毒抑制细胞凋亡抗早衰的机理，为铅山红芽芋种质保存及其优质高效栽培提供理论依据。

Yanshan County of Jiangxi Province is the hometown of red bud taro in China. Red bud taro is a national geographical indication agricultural product. Long term asexual propagation is easy to result in its virus accumulation, variety degeneration, yields and quality decline. It is a common understanding in the academic field that plant virus-free can restore its variety. Cryotherapy is a new and efficient virus-free technique. Our previous studies showed that, compared with conventional shoot-tip virus-free, virus-free by cryotherapy could inhibit cell apoptosis, resist premature aging, and promote production and income in the later field planting process. At present, the research of virus-free by cryotherapy is mostly focused on apoptosis in cryopreservation process, but there is no report about the study of virus-free by cryotherapy inhibiting apoptosis and resisting premature aging in the later field planting process. In this project, red bud taro in Yanshan County of Jiangxi Province will be used as the research object and its virus-free plantlets will be obtained through cryotherapy treatment and be inoculated with virus-infected sample juice to simulate virus transmission in the field by aphids. Then the aseptic culture in the artificial climate chamber will be carried out by simulating the field climate and RNA-Seq technique will be used to analyze the transcriptome of virus-free by cryotherapy inhibiting apoptosis and resisting premature aging. The candidate genes will be screened, and the full-length cDNA sequence will be cloned. The exact function and accurate subcellular determination will be studied through the over expression of genetic transformation, and the mode of action mechanism will be analyzed. At last combined with the analysis of photosynthetic physiology, physiology and biochemistry, mitochondrial characteristics, expression of Cyt C gene, antioxidant enzyme gene and hormone related gene, the mechanism of virus-free by cryotherapy inhibiting apoptosis and resisting premature aging will be explained, which will provide theoretical basis for the germplasm conservation of red bud taro in Yanshan County of Jiangxi Province and its high yield, stable yield, high quality and high efficiency cultivation in the field.