棉属D基因组3个野生棉种Bet v1基因鉴定及功能分析  

Identification and Functional Analysis of Bet v 1 in Three D Genome-containing Wild Cotton Varieties

作　　者：董琪 Magwanga Richard Odongo 路普 蔡小彦 周忠丽[2] 王省芬[1] 王星星 许艳超 侯宇清 王艳情 王坤波[2] 刘方 马峙英[1] Dong Qi;Magwanga Richard Odongo;Lu Pu;Cai Xiaoyan;Zhou Zhongli;Wang Xingfen;Wang Xingxing;Xu Yanchao;Hou Yuqing;Wang Yanqing;Wang Kunbo;Liu Fang;Ma Zhiying(College of Agronomy,Hebei Agricultural University/North China Key Laboratory for Crop Germplasm Resources of Ministry of Education,Baoding,Hebei 071001,China;Institute of Cotton Research,Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology,Anyang,Henan 455000,China)

机构地区：[1]河北农业大学农学院/教育部华北作物种质资源研究与利用重点实验室,河北保定071001 [2]中国农业科学院棉花研究所/棉花生物学国家重点实验室,河南安阳455000

出　　处：《棉花学报》2019年第5期361-380,共20页Cotton Science

基　　金：国家科技支撑计划(2013BAD01B03);国家重点研发计划(2016YFD0100203-18)

摘　　要：【目的】对棉花D基因组中Bet v 1基因家族进行分析,比较其在不同抗性棉种间的表达模式差异,为深入研究Bet v 1基因在棉花抗黄萎病中的作用提供理论依据。【方法】通过生物信息学方法对D基因组中Bet v 1基因进行鉴定。通过雷蒙德氏棉(Gossypium raimondii,D5)、三裂棉(G.trilobum,D8)和瑟伯氏棉(G.thurberi,D1)转录组和实时荧光定量聚合酶链式反应(qRT-PCR)分析Bet v 1基因在黄萎病菌处理下的表达模式。利用病毒诱导的基因沉默技术(VIGS)对Bet v 1基因进行功能鉴定。【结果】[棉花D基因组中包含59个成员,其中57个基因带有内含子,分布于8条染色体上,多数为亲水性蛋白并定位于细胞质。]黄萎病菌胁迫条件下3个野生棉种的Bet v 1基因表达量与其抗病水平一致。将不同表达水平的基因分为3组,其中第3组基因响应黄萎病菌侵染并在抗病棉种瑟伯氏棉中高表达,表明该组基因可能与黄萎病胁迫应答反应有关。从中筛选出高水平表达的Bet v 1基因,在陆地棉中沉默相应的同源基因,棉株感病加重,揭示该基因在棉花抵御黄萎病菌侵染过程中起正调控作用。【结论】响应黄萎病菌胁迫的Bet v 1基因在棉花抗黄萎病复杂的生物过程中至关重要。本研究结果为棉花Bet v 1家族基因的深入研究提供依据,为进一步解析棉花Bet v 1基因的功能奠定基础。[Objective]By analyzing the major birch pollen allergen Bet v 1 gene family in D genomes of cotton and comparing the expression patterns of three diploid D-genome cotton varieties with different Verticillium wilt resistance levels,we aimed to provide a theoretical basis for further studies on the role of Bet v 1 genes in cotton resistant to Verticillium wilt.[Method]The Bet v 1 genes were identified,and a bioinformatics analysis of the physicochemical properties of their encoded sequences in Gossypium raimondii(D5)was performed.The transcriptome sequencing and quantitative real time-PCR of G.raimondii(D5),Gossypium trilobum(D8)and Gossypium thurberi(D1)were used to verify the expression patterns of Bet v 1 genes under Verticillium dahlia infection stress.Bet v 1 genes were silenced by virus-induced gene silencing in G.hirsutum to identify their functions.[Result]The D genome of cotton contains 59 members,58 of which have introns and are distributed on eight chromosomes,and most encode hydrophilic proteins that localize to the cytoplasm.The expression levels of Bet v 1 genes in three wild cotton species having D genomes after being inoculated with V.dahliae were consistent with their disease resistance levels.The genes were separated into three groups based on their expression levels.Genes of Group 3 responded to V.dahliae infection and were highly expressed in disease-resistant cotton species G. thurberi. This indicated that Group 3 genes may be involved in the immune response of Verticillium wilt. A gene with a high expression level was screened out of Group 3. A corresponding homologous gene was silenced in G. hirsutum by virus-induced gene silencing, and gene-silenced plants were more susceptible to V. dahliae, indicating that the gene plays a positive regulatory role in the progress of Verticillium wilt resistance in cotton. [Conclusion] The Bet v 1 genes act in response to V. dahliae infection and are critical in cotton resistance to Verticillium wilt. The information obtained provides a basis for further st

关 键 词：野生棉 黄萎病 BET v 1基因 转录组 实时荧光定量聚合酶链式反应 病毒诱导的基因沉默 

分 类 号：S43[农业科学—农业昆虫与害虫防治]