摘要
【目的】从大丽轮枝菌(Verticillium dahliae)中鉴定木糖苷酶基因,研究其与大丽轮枝菌致病力的关系,为解析大丽轮枝菌致病分子机制提供理论依据,同时为制定更好的棉花黄萎病防治策略提供科学依据。【方法】利用生物信息学方法从大丽轮枝菌基因组数据库中鉴定全部木糖苷酶基因,并对基因编码蛋白的结构域、基因的染色体定位及进化关系等进行分析。利用实时荧光定量PCR(qRT-PCR)技术检测木糖苷酶基因在不同抗/感棉花品种根系分泌物培养0、6、12、24和48 h大丽轮枝菌中的表达量。利用寄主诱导的基因沉默(host-induced gene silencing,HIGS)技术对木糖苷酶基因VdxyL3在大丽轮枝菌侵染过程中的功能进行初步分析。将VdxyL3的目标片段转化棉花,采用伤根法接种大丽轮枝菌Vd991,观察转化植株的表型,调查病情指数,同时利用qRT-PCR技术对植株中真菌生物量和VdxyL3的表达量进行检测。【结果】利用生物信息学方法从大丽轮枝菌中查找出13个木糖苷酶基因(VdxyL1—VdxyL13),其编码序列长度介于1461—2544 bp,蛋白质分子量介于38.78—90.97 kD,理论等电点介于4.67—5.89。结构域和进化树分析发现13个木糖苷酶基因中包括9个糖苷水解酶43家族成员、1个3家族成员和3个31家族成员。染色体定位分析发现13个基因分布在6条染色体上,未形成基因簇。qRT-PCR结果发现选取的6个基因均受到根系分泌物的诱导,在一种或多种根系分泌物中培养6 h或12 h后,表达量均明显升高,然后降低。其中VdxyL3受海岛棉根系分泌物诱导后表达量明显升高,表明该基因的表达明显受海岛棉根系分泌物的诱导。HIGS研究结果表明,接菌14 d和21 d后转化VdxyL3基因干扰片段的棉花发病明显较重,其病情指数(33.3和83.9)明显高于空载体对照(21.7和66.1)。qRT-PCR分析发现转化VdxyL3基因干扰片段的棉花植株茎中VdxyL3的表达量明显低于空载体对照,真菌生物量显著多于对照。【结论】利用HIGS技术将VdxyL3基因沉默后,棉株的抗病性明显降低,表明VdxyL3在大丽轮枝菌致病及宿主-病原体互作过程中可能发挥着重要的作用。
【Objective】The objective of this research is to identify xylosidase genes from Verticillium dahliae and study the relationship between xylosidase genes and pathogenicity of V.dahliae,which will provide a theoretical basis for exploring the molecular mechanism of pathogenicity of V.dahliae and a scientific basis for formulating better control strategies for verticillium wilt.【Method】All xylosidase genes were identified from the genome database of V.dahliae by bioinformatics,and their protein domain,chromosomal location and phylogenetic relationship were analyzed.Quantitative real-time PCR(qRT-PCR)was used to detect the expression pattern of xylosidase genes in V.dahliae cultured with different root exudates from resistant and susceptible cotton varieties for 0,6,12,24 and 48 h.The function of one of the xylosidase genes VdxyL3 in the infection of V.dahliae was analyzed by using host-induced gene silencing(HIGS)method.The target fragment of VdxyL3 was injected into cotton,and then V.dahliae Vd991 was inoculated to those plants injected with target fragment of VdxyL3 by using root-dip approach.The phenotype of transformed plants was observed and the disease index was counted,meanwhile,the biomass of fungi and the expression level of VdxyL3 in the plants were detected by qRT-PCR technique.【Result】Bioinformatics analysis showed that there were 13 xylosidase genes(VdxyL1-VdxyL13)in V.dahliae,whose coding sequences ranged from 1461 to 2544 bp,molecular weight of the encoded proteins ranged from 38.78 to 90.97 kD,and theoretical isoelectric point ranged from 4.67 to 5.89.Protein domain phylogenetic relationship analysis showed that there were 9 glycoside hydrolase 43 family members,1 glycoside hydrolase 3 family member and 3 glycoside hydrolase 31 family members included in xylosidase genes.The chromosomal location analysis showed that the 13 genes were distributed on 6 chromosomes and no gene clusters were formed.qRT-PCR analysis showed that the expression of 6 xylosidase genes was induced by cotton root exudates.After being cultured in one or more root exudates for 6 h or 12 h,expression levels of these genes were significantly increased and then decreased.Among 6 genes,the expression level of VdxyL3 increased significantly after sensing root exudates from sea island cotton.The results based on HIGS technology showed that after 14 and 21 days of inoculation,the disease symptom of cotton plants transformed with VdxyL3 interfering fragment was more serious,and the disease index(33.3 and 83.9)was significantly higher than that of empty vector control(21.7 and 66.1).qRT-PCR analysis showed that cotton plants transformed with VdxyL3 interfering fragment had higher fungal biomass but lower expression level of VdxyL3 compared to the empty vector control.【Conclusion】The VdxyL3 gene silencing by using HIGS technology lead to a significant decrease of cotton resistance to V.dahliae,indicating that VdxyL3 may play a certain role in the pathogenesis of V.dahliae and host-pathogen interaction.
作者
张小雪
孙天歌
张迎春
陈丽华
张新宇
李艳军
孙杰
ZHANG XiaoXue;SUN TianGe;ZHANG YingChun;CHEN LiHua;ZHANG XinYu;LI YanJun;SUN Jie(College of Agriculture,Shihezi University,Shihezi 832003,Xinjiang)
出处
《中国农业科学》
CAS
CSCD
北大核心
2021年第15期3219-3231,共13页
Scientia Agricultura Sinica
基金
国家自然科学基金(31460360)
石河子大学高层次人才科研启动项目(RCZK202019)。