LOB1在柑橘溃疡病中的功能及调控机制研究

Screening and exploitation of disease-resistant germplasm is the most effective way to control citrus canker disease caused by Xanthomonas citri subsp. Citri (Xcc). Kumquat and Citron-C05 have been shown to be resistant to citrus canker. Our preliminary research revealed that there is high level of nucleotide polymorphism in and around the binding sites of the TAL-type effectors PthAs from Xcc in the promoters of the host target gene LOB1 from these two resistant germplasm when compared to those from susceptible sweet orange. In addition, two novel LOB1 transcripts whose predicted LOB proteins lack a conserved domain from these two biotypes were induced by citrus canker infection. However, whether the sequence variation in the LOB1 promoters or the production of two novel transcripts in Kumquat and Citron-C05 are attributable to their resistance to the canker disease has not been determined. In this project, the two resistant germplasm and susceptible sweet orange will be used to identify key cis-acting elements that mediate or influence the interaction between the LOB1 promoter and PthAs using the yeast one-hybrid and dual-luciferase reporter assays. Next, targeted mutagenesis by the CRISPR/cas9 system will be employed to study the role of the LOB1 promoters in canker disease susceptibility or tolerance of different Citrus genotypes. Lastly, the expression patterns of different LOB1 transcripts in different citrus genotypes will be examined through comparative analyses, followed by functional characterization of such transcripts by CRISPR/cas9 system, and identification of potential downstream regulatory components by ChIP. Results from this study will help understand the function and regulatory mechanisms of the LOB1 gene during canker disease development and/or resistance reaction, thereby providing a theoretical basis for citrus canker disease resistance breeding.

通过抗性机制研究筛选或培育优良的抗病种质是根治柑橘溃疡病最有效的方法。金柑与枸橼-C05是独特的溃疡病抗性资源，前期研究表明这两种抗病种质的LOB1启动子上与致病效应子PthAs结合的位点及两侧序列具有极强多态性，且受溃疡病菌诱导后新增两个缺失LOB1保守结构域的转录本，但LOB1启动子及新增转录本的功能与调控机制尚不清楚。本项目拟以这两种抗病种质及感病种质冰糖橙为材料，通过酵母单杂交技术和双荧光素酶报告系统鉴定LOB1与PthAs互作的关键顺式作用元件并采用CRISPR技术明确其功能；同时对比研究受溃疡病菌诱导的LOB1不同转录本的表达模式，然后通过CRISPR技术验证其功能，进一步采用ChIP技术筛选下游调控基因。由此探明LOB1基因在溃疡病症形成与抗性反应中的作用与调控机制，从而为柑橘溃疡病抗性育种提供理论依据。

通过抗性机制研究筛选或培育优良的抗病种质是根治柑橘溃疡病最有效的方法。本项目以浏阳金柑和枸橼-C-05这两种独特的溃疡病抗病种质以及极易感病的冰糖橙为材料，解析了LOB1启动子与PthA4互作在溃疡病抗病和感病中的双重功能。本项目研究表明：在感病冰糖橙中，LOB1启动子的PthA3结合位点及两侧序列是决定LOB1启动子与PthA4有效结合启动感病的关键元件，当PthA3竞争性地结合该元件后，即可降低LOB1启动子与PthA4的结合能力；反之，在抗病浏阳金柑中，LOB1启动子的PthA3结合位点及两侧序列是抑制LOB1启动子与PthA4有效结合启动抗病的关键元件，当PthA3竞争性地结合该元件后，即可提高LOB1启动子与PthA4的结合能力；枸橼-C-05的LOB1启动子PthA3结合位点及两侧序列不是其抗病的关键元件，其抗溃疡病的机理与浏阳金柑不同，有待进一步研究。本研究结果深入地解析了感病冰糖橙和抗病浏阳金柑LOB1启动子与PthA4互作产生差异的原因，是对目前LOB1的国际研究热点集中在感病机制中的补充，具有很好的科学意义。同时本研究鉴定到的关键顺式作用元件中的多态性位点可开发成抗病和感病性鉴定的基因标记，辅助溃疡病抗病育种，具有良好的应用前景。此外，本研究针对LOB1基因的功能开展了横向研究，从进化角度研究了整个柑橘LOB基因家族，结果表明柑橘LOB基因家族在柑橘组织分化和溃疡病响应中起着重要作用，并且大片段复制和串联复制促进了柑橘LOB基因家族成员的功能分化，研究结果为柑橘溃疡病的发生机理研究提供了更多候选基因，同时也为柑橘LOB基因家族的功能研究提供有效线索，具有较强的科学意义。