枸杞属野生种抗炭疽病相关基因的克隆和功能解析

Colletotrichum gloeosporioides Penz. is one of the most important diseases on wolfberry. It is very important to isolate and utilize resistance-related genes of wild wolfberry germplasm and to analyze their gene functions. In the present study, to select resistance-related genes, we conduct transcription profiling of Wild Wolfberry line NY-2 resistance to Colletotrichum gloeosporioides Penz. infected by the strain with high pathogenicity using cDNA-AFLP technique. By analyzing transcription profiling of the reaction across five sampling time points after inoculation, we want to isolate and sequence differentially expressed TDFs(transcript derived fragments).The expression patterns of TDFs related to resistance are identified in different treats by using real-time fluorescent quantitative reverse transcriptase PCR. After full-length cDNA sequences of TDFs are cloned via RACE technique, the sequence and structure characters of cDNA and corresponding protein are analyzed by using bioinfornatics. The functions of differential expressed genes are identified and characterized by using VIGS (virus induced gene silencing). Therefore, the current study aims at cloning and analyzing the functional gene related to resistance from wild wolfberry resistance to Colletotrichum gloeosporioides Penz. Furthermore, the study attempts to elucidate the gene expression patterns and functions of these differential expressed genes. This study will preliminarily reveal the resistant molecular mechanism of wolfberry to Colletotrichum gloeosporioides Penz. The result of study should be helpful to elucidate molecular mechanism of the interactions between wolfberry and Colletotrichum gloeosporioides Penz. And it will establish the theoretical basis and provide alternative resources for development of genetically improved disease resistance of the available wolfberry varieties, which will be also useful for rational use of resistant wild wolfberry germplasm for advanced wolfberry molecular breeding resistant to Colletotrichum gloeosporioides Penz.

枸杞炭疽病是严重危害枸杞生产的真菌性病害之一。对中国野生枸杞种质资源中抗病相关基因进行鉴别、挖掘以及利用是枸杞抗病研究的一项重要内容。本项目申请拟采用cDNA-AFLP技术，对枸杞抗病野生种质NY-2受炭疽病菌侵染前后的差异表达转录谱进行分析，分离筛选出抗病相关基因的转录衍生片段(Transcript Derived Fragments,TDFs)；进而利用cDNA末端快速扩增技术，克隆枸杞抗炭疽病相关基因cDNA全长序列，并通过生物信息学进行基因基本特征和功能的分析；采用VIGS(病毒诱导基因沉默)技术，对候选基因进行功能鉴定和验证分析。本项目研究目的和意义在于克隆枸杞抗炭疽病相关基因并阐明其表达特征和功能，初步解析枸杞抗炭疽病的分子机制，为研究枸杞与炭疽菌互作机制以及枸杞抗病基因的表达调控奠定基础，也为抗病枸杞野生种质的合理利用、现有品种抗病性遗传改良提供理论依据和新的基因资源。

枸杞炭疽病是严重危害枸杞生产的真菌性病害之一，对中国野生枸杞种质资源中抗病相关基因进行鉴别、挖掘以及利用是枸杞抗病研究的一项重要内容。本项目主要采用转录组学分析技术，对枸杞抗病野生种质受炭疽病菌侵染前后的差异表达转录谱进行分析，分离筛选抗病相关基因，分析和验证侵染后不同时间基因的表达模式，并鉴定其功能，从而初步解析枸杞抗炭疽病的分子机制。取得的重要结果包括：1、对抗性种质应答枸杞炭疽病菌的高通量转录组研究，共获得208290条Unigene，N50长度为1011bp，其中133076条Unigene得到SwissProt等数据库注释，检测到SSR共35386个，抗性种质接种病原菌后三个时间点与对照相比分别获得了10476、9677和899个差异基因，其中三个时间点特异表达差异基因分别为4106、2937和138个，共同表达的差异基因有331个。差异表达基因参与的抗病相关的Kegg Pathway有Plant-pathogen interaction(各时间点分别有45、51、8个基因参与)、Plant hormone signal transduction(三时间点分别有26、43、11个基因参与)、Peroxisome(各时间点分别有56、59、2个基因参与)、Phenylpropanoid biosynthesis(三时间点分别有3、52、17个基因参与)等。筛选出抗炭疽病相关候选基因有：病程相关蛋白1(PR1)；过氧化物酶；乙烯信号转导导途径中相关基因EIN3、乙烯受体ETR, ERS以及EBF1；参与苯丙烷等多种次生代谢产物途径的木质素合成关键酶咖啡酰辅酶A氧甲基转移酶；苯丙氨酸解氨酶；WRKY类型转录因子等。2、对差异表达基因的表达模式进行了实时荧光定量PCR分析，结果表明qRT-PCR表达趋势基本与RNA-Seq结果相符合，其中一个注释为PR1蛋白的基因在炭疽病菌接种后，表达量上调幅度较大。3、建立了枸杞基于烟草脆裂病毒载体的病毒诱导基因沉默体系，构建了PR1基因的TRV-VIGS载体。枸杞抗炭疽病的可能分子机制为：接种炭疽病菌后枸杞抗性种质产生的防御性植物激素类物质含量发生变化，诱导系统性抗性，合成积累病程相关蛋白，抵抗病原菌的侵染；另外，病原菌的侵染促使苯丙烷等代谢途径中的相关基因表达发生变化，产生更多抗性次生代谢产物(如木质素），参与抗病反应。

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