基于转录组测序的金银花提取物抗鸽源新城疫病毒感染的作用机制研究  

Transcriptome Sequencing Analysis Reveals the Potential Molecular Mechanisms of Honeysuckle Extract in Protecting Newcastle Disease Virus Infection in Pigeon

作　　者：莫红芳 石宗承 杨燕燕 俸云 虞霖田 石德顺[1] 熊晓妍 MO Hongfang;SHI Zongcheng;YANG Yanyan;FENG Yun;YU Lintian;SHI Deshun;XIONG Xiaoyan(College of Animal Science and Technology,Guangxi University,Nanning 530004,China;College of Animal Science and Technology,Guangxi Vocational University of Agriculture,Nanning 530007,China;Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]广西农业职业技术大学动物科技学院,广西南宁530007 [3]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150069

出　　处：《畜牧与饲料科学》2024年第4期10-19,共10页Animal Husbandry and Feed Science

基　　金：国家转基因生物新品种培育科技重大专项(2011 ZX08007-003);2021年广西农业职业技术学院院级课题资助项目(YKJ2117)。

摘　　要：[目的]探究金银花提取物抗鸽源新城疫病毒(Newcastle disease virus,NDV)感染的作用机制。[方法]选取未接种NDV疫苗的健康28日龄美国王鸽40只,适应性饲养7 d,于35日龄随机分为金银花预防(KO)组和攻毒对照(OE)组,每组20只。KO组试验鸽于35日龄开始在饮水中添加1.2%的金银花提取物,持续饮用7 d,42日龄时肌肉注射HA效价为5log2的鸽源NDV Pi/NJ/CH/4416/2016株0.5 mL/只;OE组试验鸽于35日龄进入正式试验,正常饮水,42日龄时肌肉注射HA效价为5log2的鸽源NDV Pi/NJ/CH/4416/2016株0.5 mL/只。攻毒7 d后,每组选取3只试验鸽采集脾脏组织样本制备组织切片,同时提取总RNA构建转录组文库,利用转录组测序(RNA-sequencing,RNA-Seq)技术进行差异表达基因(differentially expressed genes,DEGs)鉴定、GO功能富集分析和KEGG通路富集分析。随机选取4个显著差异表达基因,利用qRT-PCR验证测序结果。[结果]OE组鸽子脾细胞核异常增大且出现白色空泡,并伴有大量粒细胞浸润,而KO组脾脏组织仅见较多白色空泡和少量粒细胞浸润,未见脾细胞核异常增大。利用RNA-Seq技术从KO组鸽脾脏中共鉴定出374个DEGs,其中,112个基因表达显著上调,262个基因表达显著下调。GO功能富集分析显示,共有875个DEGs富集到312个GO条目,其中,生物学过程富集到149个条目,细胞组成过程富集到33个条目,分子功能过程富集到130个条目。KEGG通路富集分析显示,共有226个DEGs主要被富集在信息传递通路、代谢通路、天然免疫相关通路、细胞凋亡通路等93条KEGG信号通路。qRT-PCR验证结果显示,从DEGs中随机选取的4个显著差异表达基因(LOC110360702、LOC110360688、SFRP5、CHAD)的相对mRNA丰度变化趋势与RNA-Seq结果显示的4个基因变化趋势一致。[结论]金银花提取物通过激活宿主的信息传递、代谢调节及免疫应答等重要生理过程发挥抗鸽源NDV感染作用。研究结果为进一步探究鸽源NDV�[Objective]This study was conducted to assess the mechanisms underlying the protective role of honeysuckle extract against Newcastle disease virus(NDV)infection in pigeon.[Method]A total of 40 healthy 28-day-old American king pigeons without NDV vaccination were selected and subjected to a 7-day adaptive rearing.At the age of 35 days,the experimental pigeons were randomly assigned into a honeysuckle extract protective(KO)group(n=10)or a virus challenge control(OE)group(n=10).The pigeons in KO group drank the water containing 1.2%honeysuckle extract for 7 consecutive days,and at the age of 42 days,they were intramuscularly injected with 0.5 mL NDV Pi/NJ/CH/4416/2016 strain of pigeon origin with HA potency of 5log2.The pigeons in OE group drank the normal water and were challenged with the same NDV strain at the same dosage at the age of 42 days.Seven days after challenge,3 pigeons were chosen from both KO and OE groups.The spleen samples were collected for paraffin-embedded tissue section preparation and for total RNA extraction to construct the transcriptome libraries.RNA-Seq was used to identify differentially expressed genes(DEGs)and to perform GO functional enrichment analysis as well as KEGG pathway enrichment analysis.Four randomly selected significant DEGs identified by RNA-Seq were verified by using qRT-PCR.[Result]The splenocytes of the pigeons in OE group had abnormally enlarged nuclei and white vacuoles,accompanied by a large number of granulocyte infiltration in splenic tissues,while those of the pigeons in KO group had many white vacuoles and minor granulocyte infiltration alone,and no abnormally enlarged splenocyte nuclei in splenic tissues were observed.A total of 374 DEGs were identified in the spleens of the pigeons in KO group by RNA-Seq,of which 112 were significantly up-regulated and 262 were significantly down-regulated.GO functional enrichment analysis demonstrated that 875 DEGs were enriched in 312 GO entries,of which 149 belonged to biological process,33 belonged to cellular component,and 1

关 键 词：鸽 新城疫病毒 金银花 转录组测序 差异表达基因 天然免疫 

分 类 号：S853.74[农业科学—临床兽医学] S858.395.3[农业科学—兽医学]