辣椒CaCP1基因在疫病和盐胁迫诱导叶片衰老中的功能和调控机理研究

Pepper (Capsicum annuum L.), widely cultivated in the world, is an important vegetable crop. Recently, Recently, with the large-scale protective culture of pepper, phytophthora diseases and secondary salinization caused by high cropping index and excessive use of chemical fertilizer have severely affected the regular growth of pepper plants, resulting in the premature of the plant, and finally affect the yield and quality of fruits. Previous studies have found a senescence-related gene CaCP1 was cloned from the premature aging variety of B12. In this study, in order to explore the function of CaCP1 during leaf senescence induced by Phytophthora bligh and salt stress, a series of technologies will be applied, including over-expression and RNAi of CaCP1 by transformation of tobaccoand pepper, silencing of CaCP1 induced by VIGS technology, detection of physiological indexes and expression levels of senescence-related genes and histocytological observations. Secondly, Yeast one-hybrid and ChIP-Seq assays were used to find the upstream regulation genes and downstream target gene of CaCP1. Moreover, the promoter activity of CaCP1 will be preformed by phytophthora capsici and salt and hormone treatment. This research is aimed to provide genetic resources for anti-senescence molecular breeding in pepper, and also to provide theory basis for pepper variety improvement.

辣椒（Capsicum annuum L.）是一类重要的蔬菜作物，在全球范围内广泛种植。近年来由于大规模进行辣椒保护地栽培，复种指数高，化肥过量使用，引起的疫病危害和次生盐渍化严重影响辣椒植株的正常生长，导致植株早衰，影响辣椒果实的产量和品质。项目组前期从早衰材料B12中克隆得到衰老相关基因CaCP1。本项目拟通过超表达和RNAi对烟草“百日红”品种和辣椒B12进行遗传转化，运用VIGS技术对辣椒B12进行诱导沉默。结合生理生化指标测定、防御相关基因表达以及组织细胞学观察，探讨CaCP1在辣椒疫病和盐胁迫诱导叶片衰老中的功能；其次，通过酵母单杂和染色质免疫共沉淀筛选其上游调控基因和下游靶基因，明确其调控机制；再者，通过启动子活性分析，探讨CaCP1基因启动子在辣椒疫霉菌、激素和盐处理下的响应情况。本项目的开展，旨在为辣椒抗衰老分子育种提供基因资源，为辣椒品种改良提供理论基础。

本项目在辣椒的基因组中鉴定了35个PLCPs，根据其进化系统发育，将其分为9个亚家族，其中包括有4个RD21基因、2个CEP基因、2个XCP基因、3个XBCP3基因、1个THI基因、10个SAG12基因、3个RD19基因，1个ALP基因和8个CTB基因。通过分析PLCPs的保守基序、结构域和基因结构，揭示了PLCPs的特性。项目中的CaCP1基因属于一类SAG12基因，编号为CaCP11。定量分析发现叶片衰老、激素处理和逆境胁迫均可诱导CaCP1基因不同程度的上调表达。盐和渗透胁迫处理可诱导CaCP1基因过表达的烟草T2代纯系植株的早衰，最终确定CaCP1基因正向调控在盐和渗透胁迫诱导的早衰。CaCP1基因启动子的功能分析发现，其主要转录位点在0-1000bp之间。另外，对35个候选CaPLCPs基因进行表达分析发现，辣椒CaPLCPs家族成员在不同组织和叶片衰老过程中的模式不同，且以不同的表达模式响应不同的激素处理和逆境胁迫。利用VIGS技术初步研究CaCP13、CaCP15、CaCP20、CaCP21、CaCP25、CaCP28和CaCP34的功能发现，CaCP13、CaCP15和CaCP28沉默可增强辣椒叶片盐和渗透胁迫的耐受性，CaCP20和CaCP34基因则相反。CaCP21基因沉默降低辣椒叶片盐胁迫的耐受性，增强渗透胁迫的耐受性，CaCP25基因则相反。通过本项目的研究有助于全面揭示 CaCP1 在盐和渗透胁迫诱导叶片早衰中的功能以及调控机制，也为其他相关PLCPs的功能研究提供良好基础，同时为辣椒抗衰老育种提供理论依据。