摘要
【目的】菌核病是由核盘菌(Sclerotinia sclerotiorum)引起的一类真菌病害,核盘菌寄主范围广泛,严重危害多种作物的品质。本研究利用寄主诱导基因沉默(HIGS)的方法在寄主中诱导核盘菌致病相关基因的沉默从而增强寄主的菌核病抗性,为菌核病抗病育种提供新的思路。【方法】铜锌超氧化物歧化酶是一种重要的抗氧化剂,以核盘菌铜锌超氧化物歧化酶铜伴侣基因(copper chaperone for copper/zinc superoxide dismutase,SsCCS)为靶基因,通过生物信息学分析该基因的结构特点,并利用MEGA6.0软件构建系统发育树;通过分别比对拟南芥及核盘菌基因组,选择特异的干扰片段进行扩增;采用农杆菌介导的浸花序法,将HIGS载体转入拟南芥Col-0,通过DNA鉴定以及标记筛选出稳定的HIGS-CCS转基因拟南芥;选取4—5周龄的HIGS-CCS转基因拟南芥植株叶片接种核盘菌野生菌株1980,于接种24 h后统计病斑面积,分析转基因株系的菌核病抗性;通过qRT-PCR分析核盘菌侵染转基因植株过程中SsCCS的表达情况;同时在接种6、12、24 h后利用DAB染色的方法检测转基因植株与核盘菌互作过程中H2O2的积累。【结果】生物信息学分析结果表明,SsCCS(SS1G_00102)全长为1010 bp,编码序列长759 bp,共编码253个氨基酸,该蛋白分子质量为27176.96 Da,等电点(PI)为5.04,与灰霉病菌BcCCS(EDN25358)亲缘关系最近,氨基酸同源性达到87%,与拟南芥AtCCS(AT1G12520.1)亲缘关系较远;通过与核盘菌以及拟南芥基因组比对,选择314 bp特异干扰片段,成功构建SsCCS的HIGS表达载体,转化拟南芥。T1及T2代转基因拟南芥接种核盘菌24 h后的病斑面积均小于野生型拟南芥。从T2代中获得3个稳定表达的T3代HIGS-CCS转基因拟南芥株系:HIGS-CCS-5、HIGS-CCS-8、HIGS-CCS-13;与野生型拟南芥相比,转基因拟南芥在接种核盘菌24 h后,病斑面积显著减小46%—61%;qRT-PCR结果显示核盘菌在侵染转基因植株过�
【Objective】Sclerotinia stem rot is a kind of fungal disease caused by Sclerotinia sclerotiorum.The host range of S.sclerotiorum is wide,which seriously endangers the quality of many crops.The objective of this study is to enhance the resistance to stem rot by silencing the virulence genes of S.sclerotiorum in host via the host-induced gene silencing(HIGS)technology,and to provide new ideas for breeding of sclerotinia stem rot resistance.【Method】The gene encoding a copper chaperone for copper/zinc superoxide dismutase of S.sclerotiorum(SsCCS)was selected as the target gene,the sequences were analyzed by bioinformatics tools,and the phylogenetic tree was constructed using MEGA6.0 software.The specific interference fragment was selected for amplification after comparing the genome of Arabidopsis thaliana and S.sclerotiorum,respectively.HIGS vector containing the RNAi structure of SsCCS was transferred into wild type A.thaliana Col-0 mediated by Agrobacterium,and the stable HIGS-CCS transgenic A.thaliana lines were screened by DNA identification and labeling.The leaves of HIGS-CCS transgenic plants grown for 4-5 weeks were selected to analyze the resistance to sclerotinia stem rot according to the lesion area at 24 h after inoculated with S.sclerotiorum.The relative expression level of SsCCS during infecting was analyzed by qRT-PCR.The accumulation of H2O2 during the period of interaction between transgenic plants and S.sclerotiorum was detected by DAB staining at 6,12 and 24 hpi.【Result】Bioinformatics analysis showed that the length of genome sequence of SsCCS(SS1G_00102)is 1010 bp,while the length of its coding sequence(CDS)is 759 bp,encoding a protein with 253 amino acids,the molecular weight is 27176.96 Da,the isoelectric point(PI)is 5.04.SsCCS has 87%amino acid homology to BcCCS(EDN25358)while far to AtCCS(AT1G12520.1).By aligning with the genome of S.sclerotiorum and A.thaliana,a 314 bp specific interference fragment was selected and constructed the HIGS vector successfully and transformed into A.th
作者
柴亚茹
丁一娟
周思钰
杨文静
闫宝琴
远俊虎
钱伟
CHAI YaRu;DING YiJuan;ZHOU SiYu;YANG WenJing;YAN BaoQin;YUAN JunHu;QIAN Wei(College of Agronomy and Biotechnology,Southwest University,Chongqing 400715)
出处
《中国农业科学》
CAS
CSCD
北大核心
2020年第4期761-770,共10页
Scientia Agricultura Sinica
基金
国家自然科学基金（31801395,31971979）
中国博士后科学基金（2018M633305）
重庆市基础与前沿面上项目（cstc2019jcyj-msxm2511,cstc2019jcyj-zdxmX0012）
关键词
拟南芥
寄主诱导的基因沉默
核盘菌
SsCCS
抗病性
菌核病
Arabidopsis thaliana
host-induced gene silencing(HIGS)
Sclerotinia sclerotiorum
SsCCS
disease resistance
sclerotinia stem rot
