枣树抗病品种对枣疯病植原体增殖、遗传变异和相关分子调控的作用

The jujube witches'-broom disease caused by phytoplasma is the most serious problem of jujube cultivation and fruit production in China. Significant achievements on pathogen molecular biology and screening of resistant cultivars provide the disease control with new expectation and challenge. However, due to the difficulty of in vitro culturing phytoplasmas and the knowledge and information shortage about pathogenic growth, propagation, metabolism, regulation and strain variation as well as resistance mechanism of the cultivars, the disease control is greatly retarded. By means of techniques for gene or operon cloning, sequence and function analyses, this project will investigate in detail the molecular variation of phytoplasma growth and propagation-related genes as well as their promoters of thymidylate kinase (TMK), elongation factor (TUF), tRNA-isopentenyl pyrophosphate transferase (tRNA-IPT) and superoxide dismutase (SOD) and further identify the genotypes based on graft inoculation reaction and sequence data. The structure and function of corresponding promoters will be identified and compared through determining the prokaryotic and host plant expression of constructed plasmid vectors using the phytoplasma promoters and their target genes or promoter-less report genes. The possible phytoplasma-inhibiting substances will be extracted and analyzed from resistance cultivars and their effects on related gene expression and regulation will be evaluated. These researches should be helpful for revealing the jujube resistance mechanism and pathogen variation rules, understanding the key gene expression and regulation modes, searching new types of bactericides, preventing from the resistance loss of cultivars and increasing the level of the disease management eventually.

由植原体引起的枣疯病是我国枣树的严重问题。在病菌分子生物学和枣树抗病品种选育方面取得的重要进展给这一难防治病害治理带来希望和契机。但病原不能人工培养，生长繁殖、代谢调控、株系变异及品种抗病机制不清，严重地影响了病害防治效果。本项目用基因和操纵子克隆、序列功能分析手段，系统调查感染不同抗病枣树品种的植原体菌株生长繁殖密切相关的胸苷酸激酶（TMK）、蛋白延伸因子（TUF）、转移RNA异戊烯基转移酶（tRNA-IPT）和超氧岐化物酶（SOD）等基因及启动子的分子变异，结合嫁接接种鉴定植原体基因型；构建目的基因启动子报告表达载体测定其在细菌和植物体内的表达，鉴定不同启动子的结构与功能；抽提和鉴定枣树抗菌活性物质，测定其对植原体基因表达和调控的影响。研究结果将有助于揭示枣树抗病机制和植原体变异规律、阐明植原体一些关键基因表达调控方式、开发新型治疗剂、防止品种抗病性丧失和最终提高枣疯病综合治理水平。

由植原体引起的枣疯病是我国枣树的严重问题。我国抗枣疯病品种资源丰富，在病菌分子生物学和枣树抗病品种选育方面取得的重要进展给这一难防治病害治理带来希望和契机。但病原不能人工培养、生长繁殖、代谢调控、株系变异及品种抗病机制不清等问题严重地制约了病害防治效果。 因而本研究重点开展了枣树抗病品种对枣疯病植原体侵染、增殖、编码基因和非编码序列（启动子）适应性变异以及分离鉴定枣树叶片抗病活性物质与抗病机理的研究。我们选择收集和用嫁接接种鉴定了10余个抗枣疯病（JWB）品系，采用新建立的LAMP和实时荧光定量PCR等技术准确测定了抗病株对植原体侵染的不同的症状反应和病原繁殖的抑制作用。 发现部分抗病品系枣叶片甲醇抽提物对芽孢杆菌有特异性抑菌作用，用薄层层析、HPLC、组织化学定位等方法鉴定出水杨酸、茉莉酸、活性氧（H2O2）及荧光物质等与抗病性密切相关的物质。活性抽提物处理染病枣树和泡桐组培苗能减轻丛枝症状和促进生根。利用TAIL-PCR扩增和测定JWB的蛋白延伸因子基因tuf上游未知序列，用启动子探针载体pSUPV4成功构建了JWB 和泡桐丛枝(PaWB)植原体tuf基因上游序列的大肠杆菌异源表达体系，发现PaWB-sdyz等株系tuf基因上游非编码区129bp片段具有启动子活性，而JWB等16SrV组株系fusA和tuf基因间区长53-54bp，无完整启动子结构与活性。根据对所采集JWB菌株的tuf上游多个基因编码序列和非编码序列， 以及多拷贝tmk基因的直接测序和克隆测序分析，比较鉴定了JWB等4种16SrV组和PaWB等5种等16SrI组植原体的tuf基因上游序列的明显差异，确定了与不同地区和品种相关的JWB植原体株系序列变异与类型，其中tmk存在三类序列型，tmkX, tmkY 和tmkXY。以JWBtuf基因和上游非编码序列相邻区域为靶标设计特异性探针定制的管芯片，用杂交验证实验证实该探针能特异性地鉴定16SrV组的枣疯病、槐树丛枝、丝棉木丛枝、重阳木丛枝等植原体，而健康对照和其他组植原体在该探针位置均未出现阳性杂交信号。本研究初步揭示了枣树对植原体的抗病机制、并为进一步深入研究植原体基因表达与分子调控、开发病害快速诊断试剂盒、新型治疗药剂和合理利用抗病品种防控枣疯病奠定了更坚实基础。