【作者】 李洋；

【导师】 巩振辉；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2023， 博士

【摘要】 辣椒（Capsicum annuum L.）是世界上重要的经济作物之一,具有较高的营养价值。尽管现代育种和栽培技术已经大大提高,但病原菌危害仍然是辣椒产业发展中的关键限制因素。其中,由炭疽菌（Colletotrichum spp.）引起的炭疽病是辣椒采收前后破坏力很强的真菌病害之一,严重影响了辣椒的产量和品质,而Colletotrichum scovillei被认为是一种常见且毁坏性很强的炭疽菌。为了研究炭疽病的抗病机理,我们通过正向遗传学的方式挖掘抗病相关基因,同时也通过反向遗传学的方式去验证WRKY转录因子在抗病中的重要调控功能。数量性状位点（quantitative trait locus,QTL）和图位克隆技术为挖掘抗病基因提供了快速有效的方法。此外,WRKY转录因子调控植物响应各种应激反应,在植物抗病和抗逆反应中发挥着至关重要的作用,但在辣椒抗炭疽病中的功能尚不清楚。在本研究中通过分析课题组前期的转录组数据以及利用病毒诱导的基因沉默（virus-induced gene silencing,VIGS）技术,鉴定出2个响应辣椒炭疽病的CaWRKY20和CaWRKY50基因,并探究它们在辣椒响应C.scovillei侵染中的调控机理。同时,对控制辣椒炭疽病抗性相关的QTLs进行定位。主要研究结果如下:1.通过对课题组前期的转录组数据分析,筛选出了7个差异表达的CaWRKYs基因。利用VIGS技术在离体的辣椒果实上沉默这些CaWRKYs基因,并接种C.scovillei进行抗病性鉴定。结果表明CaWRKY20和CaWRKY50沉默果实的病斑直径明显小于对照果实,其余基因沉默的果实病斑直径与对照果实相比没有显著性差异。因此,推测CaWRKY20和CaWRKY50可能参与了辣椒对炭疽病侵染的防御反应。2.CaWRKY20负向调控辣椒对炭疽病的抗性。用水杨酸（salicylic acid,SA）处理离体的辣椒果实,结果表明CaWRKY20能够被SA诱导表达。CaWRKY20蛋白定位于细胞核且具有转录自激活活性。在离体的辣椒果实中瞬时过表达CaWRKY20或在番茄中稳定过表达CaWRKY20都增加了丙二醛和H₂O₂的含量,降低了活性氧（reactive oxygen species,ROS）清除能力,通过抑制SA防御相关基因（CaPR1、CaPR10和CaSAR8.2）和ROS清除酶基因（CaCAT、CaPOD和CaSOD）的表达降低了对C.scovillei的抗性。而在离体的辣椒果实上沉默CaWRKY20增强了辣椒对C.scovillei的抗性。此外,通过酵母双杂交（Yeast two-hybrid,Y2H）技术筛选到与CaWRKY20互作的两个E3泛素连接酶蛋白CaMIEL1和CaCHYR1,并通过荧光素酶互补成像（Luciferase complementation imaging,LCI）和双分子荧光互补（Bimolecular fluorescence complementation,Bi FC）技术证明了CaWRKY20在细胞核内与CaMIEL1和CaCHYR1相互作用。同时,CaWRKY20直接结合SA防御基因CaACD6和CaSARD1启动子中的W-box（TTGACT/C）元件,并抑制其表达。以上结果表明,CaWRKY20在辣椒响应炭疽病的防御反应中发挥负向调控作用,该过程是通过SA信号通路及抗氧化防御系统进行调节。3.CaWRKY50负向调控辣椒对炭疽病的抗性。CaWRKY50能在SA处理下明显诱导表达。CaWRKY50蛋白定位于细胞核但不具有转录自激活活性。在离体的辣椒果实上沉默CaWRKY50增强了辣椒对C.scovillei的抗性。同时,在离体的辣椒果实中瞬时过表达CaWRKY50或在番茄中稳定过表达CaWRKY50增加了丙二醛和H₂O₂的含量,降低了ROS清除能力,通过抑制SA防御相关基因（CaPR1、CaPR10和CaSAR8.2）和ROS清除酶基因（CaCAT、CaPOD和CaSOD）的表达降低了对C.scovillei的抗性。通过Y2H、LCI和Bi FC技术表明CaWRKY50可以与CaWRKY42和CaMIEL1在细胞核内相互作用,可能参与辣椒对C.scovillei的防御反应。此外,CaWRKY50直接与SA防御基因CaEDS1和CaSAMT1启动子中的W-box（TTGACT/C）结合,并抑制其转录活性。以上结果表明,CaWRKY50通过SA和ROS信号途径在辣椒对炭疽病的抗性中发挥负向调控作用。4.利用抗病亲本R24和感病亲本R25构建的F₂、F_2:3和F_3:4分离群体,通过QTL定位鉴定了与炭疽病抗性相关的两个脂氧合酶基因CaLOX6和CaLOX7。遗传分析表明辣椒对C.scovillei的抗性是多基因控制的显性遗传。利用186株F₂群体构建了遗传连锁图谱,该图谱覆盖长度约为1614.02 c M。通过F₂群体和F_2:3家系在Chr01染色体的01g088-01g085标记之间都检测到了辣椒炭疽病抗性相关QTL位点q AR1.1。随后利用F_3:4家系将q AR1.1位点缩小至标记01g282-01g256之间,物理距离为1.56 Mb,区间内有18个基因。通过辣椒基因功能注释信息及q RT-PCR分析,推测两个脂氧合酶基因CaLOX6（Capana01g001574）和CaLOX7（Capana01g001578）可能通过茉莉酸（jasmonic acid,JA）信号途径参与辣椒对炭疽菌的防御反应。综上所述,本研究揭示了CaWRKY20和CaWRKY50通过调控SA和ROS信号途径,在辣椒响应C.scovillei的防御反应中发挥负向调控作用。并对辣椒炭疽病抗性相关QTL进行定位,预测CaLOX6和CaLOX7可能参与了辣椒对C.scovillei的防御反应。研究结果丰富了辣椒WRKYs及LOXs参与抗病的调控网络,为辣椒抗炭疽病育种奠定了基础。更多还原

【Abstract】 Pepper（Capsicum annuum L.）is one of the most important economic crops around the world,with high nutritional value.Although modern breeding and cultivation techniques have greatly improved,the harm of pathogens remains a key limiting factor in the development of the pepper industry.Among them,anthracnose caused by Colletotrichum spp.has greatly affected the yield and the high quality of pepper,which is one of the most destructive fungal diseases in the pre-and post-harvest stages of pepper.Colletotrichum scovillei is considered a relatively common and destructive of anthracnose.In order to study the disease resistance mechanism to anthracnose,we screened for disease resistance related genes through forward genetics,and verified the important regulatory function of WRKY transcription factor（TF）in disease resistance based on reverse genetics.Quantitative trait locus（QTL）and map-based cloning technology provide a rapid and effective ways to screen for the resistance genes.In addition,WRKY TF regulates the response of plants to various stress and plays a crucial role in disease resistance and stress of plants.However,the function of WRKY TF in pepper resistance to anthracnose is still unclear.In this study,two CaWRKY genes responding to pepper anthracnose,CaWRKY20 and CaWRKY50,were identified by analyzing the transcriptome data in the previous study of our research group and using virus-induced gene silencing（VIGS）technology.We also studied the regulatory mechanism of these two genes under C.scovillei infection.At the same time,the QTLs related anthracnose resistance was mapped in pepper.The main research results are as follows:1.By analyzing the transcriptome data based on the previous studies in our group,seven differentially expressed CaWRKYs genes were screened.These CaWRKYs genes were silenced by VIGS technology on detached pepper fruits and then inoculated them with C.scovillei to identify the disease resistance of pepper fruits.The results showed that the lesion diameter of CaWRKY20 and CaWRKY50-silenced fruits were significantly smaller than the control fruits.While the lesion diameter of other genes silenced fruits were not significantly different from the control.Therefore,it is speculated that CaWRKY20 and CaWRKY50 may be involved in the defense response of pepper against C.scovillei infection.2.CaWRKY20 negatively regulated the resistance of pepper to C.scovillei.We treated detached pepper fruits with salicylic acid（SA）.The results showed that CaWRKY20 can be induced by SA.The CaWRKY20 protein is localized in the nucleus and has transcriptional activation activity.CaWRKY20 transiently expressed on detached pepper fruits or CaWRKY20stable overexpression in tomato increased the content of malondialdehyde（MDA）and H₂O₂,reduced the scavenging ability of reactive oxygen species（ROS）,and inhibited the expression of SA defense-related genes（CaPR1,CaPR10 and CaSAR8.2）and ROS scavenging genes（CaCAT,CaPOD and CaSOD）resulted in decreasing the resistance to C.scovillei.While silencing of CaWRKY20 on detached pepper fruits enhanced the resistance to C.scovillei.Additionally,it was found that CaWRKY20 interacts with two E3 ubiquitin ligase proteins CaMIEL1 and CaCHYR1 by using yeast two-hybrid（Y2H）technique.Through luciferase complementation imaging（LCI）and bimolecular fluorescence complementation（Bi FC）technique,it was demonstrated that CaWRKY20 interacts with CaMIEL1 and CaCHYR1 in the nucleus.Meanwhile,CaWRKY20 inhibits the expression of the SA defense genes CaACD6 and CaSARD1 by directly binds to the W-box（TTGACT/C）element in the promoters of them.These results indicated that CaWRKY20 plays a negative regulatory role in the defense response to C.scovillei in pepper,which is regulated by SA signaling pathway and antioxidant defense system.3.CaWRKY50 negatively regulated the resistance of pepper to C.scovillei.CaWRKY50can be induced by SA treatment.The CaWRKY50 protein is localized in the nucleus but has no transcriptional activation activity.Silencing of CaWRKY50 on detached pepper fruits enhanced the resistance to C.scovillei.Meanwhile,CaWRKY50 transiently expressed on detached pepper fruits or CaWRKY50 stable overexpression in tomato increased the content of MDA and H₂O₂,reduced the scavenging ability of ROS,and inhibited the expression of SA defense-related genes（CaPR1,CaPR10 and CaSAR8.2）and ROS scavenging genes（CaCAT,CaPOD and CaSOD）resulted in decreasing the resistance to C.scovillei.Through the Y2H,LCI and Bi FC technique,it was demonstrated that CaWRKY50 could interact with CaWRKY42 and CaMIEL1 in the nucleus,which may be involved in pepper defense response against C.scovillei.Besides,CaWRKY50 could directly binds to the W-box（TTGACT/C）in the promoters of SA defense genes CaEDS1 and CaSAMT1 to inhibits their activity.These results indicated that CaWRKY50 plays a negative regulatory role in the defense response to C.scovillei in pepper through SA and ROS signaling pathways.4.Two lipoxygenase genes related to anthracnose,CaLOX6 and CaLOX7,were identified by QTL mapping using segregation populations F₂,F_2:3 and F_3:4 constructed from crossing resistant parent R24 with susceptible parent R25.The genetic analysis showed that the pepper resistance to C.scovillei was a dominant inheritance controlled by multiple genes.The genetic linkage map was constructed using 186 F₂ populations.This linkage map covers a length of about 1614.02 c M.The QTL locus q AR1.1 for anthracnose resistance in pepper was detected between 01g088-01g085 markers on chromosome 01 using F₂ population and F_2:3 families.Then F_3:4 families was used to narrow the q AR1.1 locus to markers 01g282-01g256,with a physical distance of 1.56 Mb and 18 genes within the interval.Based on functional annotation information of pepper gene and q RT-PCR analysis,it is speculated that two lipoxygenase genes CaLOX6（Capana01g001574）and CaLOX7（Capana01g001578）may be involved in the defense response to C.scovillei in pepper through jasmonic acid（JA）signaling pathway.In summary,this study revealed that CaWRKY20 and CaWRKY50 played a negative regulatory role in the defense response to C.scovillei in pepper by regulating SA and ROS signaling pathways.It was predicted that CaLOX6 and CaLOX7 might be involved in the defense response to C.scovillei in pepper by QTLs mapping associated with anthracnose resistance.These results enriched the regulatory network of pepper WRKYs and LOXs participating in disease resistance to C.scovillei,laying the foundation for breeding pepper resistance to anthracnose.更多还原