【目的】探明引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原及其遗传进化特征。【方法】对安徽省某鸭场的病死雏鸭中采集的出血肝脏开展鸭已知病原核酸检测、病原分离鉴定和动物回归试验,在明确其病原为鸭3型甲肝病毒(Duck hepatitis A v...【目的】探明引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原及其遗传进化特征。【方法】对安徽省某鸭场的病死雏鸭中采集的出血肝脏开展鸭已知病原核酸检测、病原分离鉴定和动物回归试验,在明确其病原为鸭3型甲肝病毒(Duck hepatitis A virus type 3,DHAV-3)的基础上分析其VP1基因序列分子特征。【结果】细菌分离结果显示,未分离到细菌;经病毒核酸(RT-)PCR检测结果显示,鸭3型甲肝病毒(DHAV-3)核酸阳性,未检测出其他已知引起鸭肝出血的病毒核酸。将该阳性样品经鸭胚进行病毒分离与传代,发现接种后鸭胚发生死亡,胚体全身出血,对第5代尿囊液经RT-PCR检测为DHAV-3,将其命名为AH230225。经测定,该分离株的鸭胚半数致死量(Effective lethal dose 50,ELD_(50))为10^(−4.17)/0.1 mL。动物回归试验表明,该毒株对樱桃谷雏鸭的致死率为80%,且攻毒死亡鸭肝脏和肾脏的剖检病变与临床典型病变相近。对该分离毒的VP1基因核苷酸序列进行同源性分析,显示AH230225株的VP1基因核苷酸序列与AH07株DHAV-3(安徽分离株)的同源性最高,为98.8%,与GenBank登录的10株DHAV-3分离株VP1基因核苷酸序列同源性为90.4%~98.8%,而与DHAV-1和DHAV-2的VP1基因核苷酸序列同源性分别为62.1%~63.0%、64.6%~64.9%;基于VP1蛋白氨基酸序列的遗传进化显示,该分离株与AH07株DHAV-3处于同一小进化分支上,亲缘关系最近;而与SD01株、G株和韩国株(AP-04009、AP-03337)等亲缘关系较远,即远离DHAV-1和DHAV-2进化分支。【结论】引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原为鸭3型甲肝病毒DHAV-3,同时明确了该毒株VP1基因的分子特征及遗传进化规律,为深入研究DHAV-3的致病机制和制定防控措施提供科学依据。展开更多
Hot pepper(Capsicum annuum var.conoides)is a significant vegetable that is widely cultivated around the world.Currently,global climate change has caused frequent severe weather events,and waterlogging stress harms the...Hot pepper(Capsicum annuum var.conoides)is a significant vegetable that is widely cultivated around the world.Currently,global climate change has caused frequent severe weather events,and waterlogging stress harms the pepper industry by affecting the planting period,growth conditions,and disease susceptibility.The gene CaABI3/VP1-1 could improve pepper waterlogging tolerance.In order to explore the upstream regulatory mechanism of CaABI3/VP1-1,a high-quality standardized yeast hybrid library was successfully constructed for yeast one-,two-,and threehybrid screening using pepper‘ZHC2’as the experimental material,with a library recombinant efficiency of up to 100%.The length of inserted fragments varied from 650 to 5000 bp,the library titer was 5.18×10^(6)colony-forming units(CFU)·mL-1,and the library capacity was 1.04×10^(7)CFU of cDNA inserts.The recombinant bait plasmid was used to successfully identify 78 different proteins through the yeast one-hybrid system,including one transcription factor within the ethylene-responsive factor family and the other within the growth-regulating factor family.The interaction happened between LOC124895848 and CaABI3/VP1-1 promoter by point-to-point yeast one-hybrid experiment.The expression level of the 12 selected protein-coding genes was then evaluated by quantitative real-time polymerase chain reaction.Results indicated the protein coding genes showed different responses to waterlogging stress and that the activity of the CaABI3/VP1-1 promoter could be inhibited or activated by up-regulating or down-regulating gene expression,respectively.The identification of these proteins interacting with the promoter provides a new perspective for understanding the gene regulatory network of hot pepper operating under waterlogging stress and provides theoretical support for further analysis of the complex regulatory relationship between transcription factors and promoters.展开更多
文摘【目的】探明引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原及其遗传进化特征。【方法】对安徽省某鸭场的病死雏鸭中采集的出血肝脏开展鸭已知病原核酸检测、病原分离鉴定和动物回归试验,在明确其病原为鸭3型甲肝病毒(Duck hepatitis A virus type 3,DHAV-3)的基础上分析其VP1基因序列分子特征。【结果】细菌分离结果显示,未分离到细菌;经病毒核酸(RT-)PCR检测结果显示,鸭3型甲肝病毒(DHAV-3)核酸阳性,未检测出其他已知引起鸭肝出血的病毒核酸。将该阳性样品经鸭胚进行病毒分离与传代,发现接种后鸭胚发生死亡,胚体全身出血,对第5代尿囊液经RT-PCR检测为DHAV-3,将其命名为AH230225。经测定,该分离株的鸭胚半数致死量(Effective lethal dose 50,ELD_(50))为10^(−4.17)/0.1 mL。动物回归试验表明,该毒株对樱桃谷雏鸭的致死率为80%,且攻毒死亡鸭肝脏和肾脏的剖检病变与临床典型病变相近。对该分离毒的VP1基因核苷酸序列进行同源性分析,显示AH230225株的VP1基因核苷酸序列与AH07株DHAV-3(安徽分离株)的同源性最高,为98.8%,与GenBank登录的10株DHAV-3分离株VP1基因核苷酸序列同源性为90.4%~98.8%,而与DHAV-1和DHAV-2的VP1基因核苷酸序列同源性分别为62.1%~63.0%、64.6%~64.9%;基于VP1蛋白氨基酸序列的遗传进化显示,该分离株与AH07株DHAV-3处于同一小进化分支上,亲缘关系最近;而与SD01株、G株和韩国株(AP-04009、AP-03337)等亲缘关系较远,即远离DHAV-1和DHAV-2进化分支。【结论】引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原为鸭3型甲肝病毒DHAV-3,同时明确了该毒株VP1基因的分子特征及遗传进化规律,为深入研究DHAV-3的致病机制和制定防控措施提供科学依据。
基金funded by the National Natural Science Foundation of China(grant no.32260760)the Science and Technology Program of Guizhou Province(grant no.20201Z002)the Platform Construction Project of Engineering Research Center for Protected Vegetable Crops in Higher Learning Institutions of Guizhou Province(Qianjiaoji[2022]No.040).
文摘Hot pepper(Capsicum annuum var.conoides)is a significant vegetable that is widely cultivated around the world.Currently,global climate change has caused frequent severe weather events,and waterlogging stress harms the pepper industry by affecting the planting period,growth conditions,and disease susceptibility.The gene CaABI3/VP1-1 could improve pepper waterlogging tolerance.In order to explore the upstream regulatory mechanism of CaABI3/VP1-1,a high-quality standardized yeast hybrid library was successfully constructed for yeast one-,two-,and threehybrid screening using pepper‘ZHC2’as the experimental material,with a library recombinant efficiency of up to 100%.The length of inserted fragments varied from 650 to 5000 bp,the library titer was 5.18×10^(6)colony-forming units(CFU)·mL-1,and the library capacity was 1.04×10^(7)CFU of cDNA inserts.The recombinant bait plasmid was used to successfully identify 78 different proteins through the yeast one-hybrid system,including one transcription factor within the ethylene-responsive factor family and the other within the growth-regulating factor family.The interaction happened between LOC124895848 and CaABI3/VP1-1 promoter by point-to-point yeast one-hybrid experiment.The expression level of the 12 selected protein-coding genes was then evaluated by quantitative real-time polymerase chain reaction.Results indicated the protein coding genes showed different responses to waterlogging stress and that the activity of the CaABI3/VP1-1 promoter could be inhibited or activated by up-regulating or down-regulating gene expression,respectively.The identification of these proteins interacting with the promoter provides a new perspective for understanding the gene regulatory network of hot pepper operating under waterlogging stress and provides theoretical support for further analysis of the complex regulatory relationship between transcription factors and promoters.
