唐沙 陳靜 岳筠 李濤 李梅 文明 張雙翔 程振濤
摘要:【目的】分析不同品種羊Toll樣受體(TLRs)基因轉(zhuǎn)錄水平的差異性,為揭示TLRs基因轉(zhuǎn)錄水平與羊疫病間的關(guān)聯(lián)性提供參考依據(jù)。【方法】選取貴州省主要飼養(yǎng)的貴州黑山羊、貴州白山羊、黔北麻羊、波爾山羊和湖羊?yàn)檠芯繉?duì)象,根據(jù)GenBank已公布的TLRs基因序列設(shè)計(jì)熒光定量PCR特異性擴(kuò)增引物及TaqMan探針引物,利用TaqMan探針熒光定量PCR檢測(cè)不同品種羊血液和肺臟中TLR1~TLR10基因轉(zhuǎn)錄水平差異。【結(jié)果】TLR1~TLR10基因在不同品種羊血液和肺臟中均有轉(zhuǎn)錄表達(dá)。不同品種羊血液和肺臟中的TLRs基因轉(zhuǎn)錄水平均存在差異性,在波爾山羊血液中TLR2、TLR4和TLR5基因轉(zhuǎn)錄水平均極顯著低于其他4種羊(P<0.01,下同),TLR7和TLR8基因轉(zhuǎn)錄水平則極顯著低于除黑山羊外的其他3種羊;在白山羊血液中TLR4、TLR7、TLR8和TLR9基因轉(zhuǎn)錄水平極顯著高于其他4種羊;在湖羊肺臟中TLR2、TLR3、TLR4、TLR5和TLR8基因轉(zhuǎn)錄水平極顯著低于其他4種羊,而在黔北麻羊肺臟中TLR5和TLR8基因轉(zhuǎn)錄水平極顯著高于其他4種羊。此外,在5種羊血液中TLR2、TLR4、TLR7和TLR8基因轉(zhuǎn)錄水平均極顯著高于其他TLRs基因轉(zhuǎn)錄水平;羊肺臟中TLR2、TLR3、TLR4、TLR5和TLR8基因轉(zhuǎn)錄水平相對(duì)較高,在貴州白山羊、貴州黑山羊和波爾山羊均表現(xiàn)為極顯著高于其他TLRs基因轉(zhuǎn)錄水平?!窘Y(jié)論】不同品種羊血液和肺臟中TLRs基因轉(zhuǎn)錄水平具有差異性,以TLR2、TLR3、TLR4、TLR5和TLR8基因轉(zhuǎn)錄水平相對(duì)較高,提示TLRs在不同品種羊機(jī)體中發(fā)揮著清除病原體及維持機(jī)體穩(wěn)態(tài)的作用,而TLRs基因轉(zhuǎn)錄水平差異可能是造成不同品種羊?qū)σ卟∫赘胁町惖脑蛑弧?/p>
關(guān)鍵詞: 羊;Toll樣受體(TLRs);血液;肺臟;轉(zhuǎn)錄水平
中圖分類號(hào): S858.26? ? ? ? ? ? ? ? ? ? ? ? ? ? ?文獻(xiàn)標(biāo)志碼: A 文章編號(hào):2095-1191(2021)11-3130-09
Differential analysis of transcript levels of TLRs genes in the blood and lungs of different breeds of sheep
TANG Sha1, CHEN Jing1, YUE Jun2, LI Tao2, LI Mei1, WEN Ming1,3,
ZHANG Shuang-xiang2*, CHENG Zhen-tao1 ,3 *
(1College of Animal Science, Guizhou University, Guiyang? 550025, China; 2Animal Disease Prevention and Control Center in Guizhou Province, Guiyang? 550008, China; 3Key Laboratory of Animal Diseases and Veterinary
Public Health in Guizhou Province, Guiyang? 550025, China)
Abstract:【Objective】To analyze the variability of transcript levels of Toll-like receptors (TLRs) genes in different breeds of sheep, and to provide a reference basis for revealing the association between the transcript levels of TLRs genes and epidemic diseases in sheep. 【Method】In this study, Guizhou black goat, Guizhou white goat,Qianbei Ma goat,Boer goat and Hu sheep, which are the main breeding species in Guizhou Province, were selected for the study, and fluorescent quantitative PCR specific amplification primers and TaqMan probe primers were designed according to the published TLRs gene sequences in GenBank, and the differences in transcript levels of TLR1-TLR10 genes in blood and lung of different breeds of sheep were detected by using TaqMan probe fluorescent quantitative PCR.【Result】TLR1-TLR10 genes were transcriptionally expressed in the blood and lungs of different breeds of sheep. Differential transcript levels of TLRs genes were in both blood and lung of different breeds of sheep,the transcript levels of TLR2, TLR4 and TLR5 genes in the blood of Boer goats were extremely significantly lower than those of the other four species (P<0.01, the same below),the transcript levels of TLR7 and TLR8 genes were extremely significantly lower than those of the other three species of sheep except black goats. The transcript levels of TLR4, TLR7, TLR8 and TLR9 genes in the blood of white goats were extremely significantly higher than those of the other four species. The transcript levels of TLR2, TLR3, TLR4, TLR5 and TLR8 genes in the lungs of Hu sheep were extremely significantly lower than those of the other four sheep species, while the transcript levels of TLR5 and TLR8 genes were extremely highly significant higher in the lungs of Qianbei Ma sheep than in the other four species. In addition, the transcript levels of TLR2, TLR4, TLR7 and TLR8 genes in the blood of five sheep species were all extremely significantly higher than the transcript levels of other TLRs genes. Transcript levels of TLR2, TLR3, TLR4, TLR5 and TLR8 genes in sheep lungs were relatively high, in Guizhou white goat, Guizhou black goat and Boer goat all showed extremely significantly higher transcript levels than other TLRs genes. 【Conclusion】The transcript levels of TLRs genes in the blood and lungs of different breeds of sheep are different, with TLR2, TLR3, TLR4, TLR5 and TLR8 genes having relatively high transcript levels. It is suggested that TLRs play a role in the organism of different breeds of sheep in scavenging pathogens and maintaining the homeostasis of the organism, and the difference in the transcription level of TLRs genes may be one of the reasons for the difference in susceptibility of different breeds of sheep to epidemic diseases.
Key words: sheep; Toll-like receptors (TLRs); blood; lung; transcription level
Foundation item: National Natural Science Foundation of China (31660723, 32060786); Guizhou Provincial Scien-ce and Technology Project (Qiankehejichu〔2019〕1181, Qiankehezhicheng〔2018〕2271)
0 引言
【研究意義】貴州黑山羊、貴州白山羊和黔北麻羊具有生產(chǎn)性能優(yōu)、適應(yīng)范圍廣、抗病性強(qiáng)及耐粗飼等特點(diǎn),是貴州省養(yǎng)羊業(yè)重點(diǎn)發(fā)展的地方種質(zhì)資源(劉彬等,2014);波爾山羊和湖羊具有適應(yīng)性好、繁殖力強(qiáng)和耐粗飼等特點(diǎn),是貴州省引進(jìn)的外來(lái)品種(陳其新等,2012)。近年來(lái),貴州省羊疫病的頻發(fā)與蔓延極大阻礙其產(chǎn)業(yè)的健康發(fā)展。Toll樣受體(Toll-like receptors,TLRs)作為一類模式識(shí)別受體,能通過(guò)識(shí)別病原微生物保守的分子相關(guān)模式而實(shí)現(xiàn)對(duì)外來(lái)病原體的早期識(shí)別,具有激活機(jī)體天然免疫和調(diào)節(jié)機(jī)體獲得性免疫的能力(Vidya et al.,2018;趙飛等,2019)。疫病流行病學(xué)調(diào)查顯示,在應(yīng)對(duì)羊疫病發(fā)生時(shí)不同品種的易感性存在明顯差異,可能與不同品種羊的TLRs基因轉(zhuǎn)錄水平差異具有一定的相關(guān)性。因此,分析不同品種羊TLRs基因轉(zhuǎn)錄水平差異,對(duì)探究TLRs基因與羊疫病發(fā)生的關(guān)聯(lián)性具有重要意義。【前人研究進(jìn)展】至今,已知TLRs家族包括TLR1~TLR10,共10個(gè)成員,針對(duì)家畜TLRs的研究多見(jiàn)于馬、牛和豬等物種(Wassef et al.,2004;Alvarez et al.,2006;Tirumurugaan et al.,2010)。趙一萍等(2012)對(duì)蒙古馬不同組織器官中TLR4、TLR2、TLR1和TLR6的轉(zhuǎn)錄水平進(jìn)行檢測(cè),結(jié)果發(fā)現(xiàn)TLRs基因mRNA在蒙古馬各組織器官的轉(zhuǎn)錄水平差異明顯,可能與其識(shí)別和抵抗病原體的能力有關(guān)。李鵬和王雅春(2019)對(duì)豬TLRs的種類、進(jìn)化特點(diǎn)及免疫相關(guān)研究現(xiàn)狀進(jìn)行系統(tǒng)分析,發(fā)現(xiàn)豬TLRs廣泛分布在各種組織細(xì)胞內(nèi),且在免疫相關(guān)組織或細(xì)胞上的數(shù)量較多,在抵御病原入侵的過(guò)程中發(fā)揮重要作用。唐娜等(2020)研究表明,TLRs在水牛組織中也有廣泛分布,在脾臟、肺臟和肝臟等組織中均有表達(dá),并證實(shí)TLR1、TLR2及TLR4在??共∶庖叻矫姘l(fā)揮重要作用。近年來(lái),TRL2和TLR4作為抗病原體免疫應(yīng)答功能的家族成員得到廣泛關(guān)注及深入研究(Barton and Medzhitov,2003;O'Neill et al.,2013)。TLR2可調(diào)控細(xì)胞內(nèi)的信號(hào)傳導(dǎo),且在機(jī)體識(shí)別病原微生物方面發(fā)揮重要作用,在抗菌抗病毒過(guò)程中能直接或間接對(duì)炎癥因子的合成與釋放起促進(jìn)作用,與機(jī)體的免疫作用緊密相關(guān)(楊慶利和冷靜,2021)。TLR4能介導(dǎo)革蘭氏陰性菌及其脂多糖(LPS)的宿主反應(yīng),還能刺激機(jī)體產(chǎn)生相關(guān)細(xì)胞因子以抵抗病毒侵入,對(duì)機(jī)體識(shí)別病原體和增強(qiáng)抗病性能具有重要意義(趙明明等,2016)。劉潔等(2020)對(duì)支原體肺炎小鼠的TLR2/TLR4、MYD88和NF-κB20P65表達(dá)水平及其相關(guān)性進(jìn)行分析,結(jié)果發(fā)現(xiàn)TLR2和TLR4表達(dá)水平在支原體肺炎小鼠中升高,且參與支原體感染的免疫損傷修復(fù)?!颈狙芯壳腥朦c(diǎn)】目前,關(guān)于羊TLRs基因轉(zhuǎn)錄水平的研究尚無(wú)詳細(xì)報(bào)道,針對(duì)不同品種羊TLRs基因轉(zhuǎn)錄水平與其疫病易感性差異的研究更是鮮見(jiàn)報(bào)道。【擬解決的關(guān)鍵問(wèn)題】選取貴州省主要飼養(yǎng)的5種羊?yàn)檠芯繉?duì)象,基于TaqMan探針熒光定量PCR分析不同品種羊TLR1~TLR10基因轉(zhuǎn)錄水平的差異性,為揭示TLRs基因轉(zhuǎn)錄水平與羊疫病間的關(guān)聯(lián)性提供參考依據(jù)。
1 材料與方法
1. 1 試驗(yàn)材料
每個(gè)品種羊10份抗凝血液及10份肺臟組織,分別采自健康無(wú)病的貴州黑山羊、貴州白山羊、黔北麻羊、波爾山羊和湖羊;各品種羊樣本則由貴州省各品種羊規(guī)模養(yǎng)殖場(chǎng)提供,3月齡,未注射任何疫苗,且利用PCR和血清學(xué)診斷方法(趙萍等,2010;馮杰等,2016)對(duì)25只羊進(jìn)行山羊支原體山羊肺炎亞種(Mccp)、絲狀支原體山羊亞種(Mmc)、綿羊肺炎支原體(MO)、布氏桿菌(Brucella)、口蹄疫(FAM)、小反芻獸疫(PPR)、山羊痘(GP)的病原學(xué)和血清抗體檢測(cè),結(jié)果均呈陰性。RNAprep Pure血液總RNA提取試劑盒購(gòu)自天根生化科技(北京)有限公司;RNAiso Plus組織總RNA提取試劑和PrimeScript? RT Master Mix購(gòu)自TaKaRa公司。
1. 2 引物設(shè)計(jì)與合成
參考GenBank已公布的TLRs基因序列,選擇5種羊TLRs基因保守區(qū)域序列,使用Primer Premier 5.0分別設(shè)計(jì)TLR1~TLR10基因特異性擴(kuò)增引物和TaqMan探針引物(表1),并委托生工生物工程(上海)股份有限公司合成。
1. 3 RNA提取及cDNA合成
參照RNAprep Pure血液總RNA提取試劑盒說(shuō)明提取5種羊50份血液樣本總RNA,同時(shí)采用TRIzol法提取5種羊50份肺臟組織總RNA,以PrimeScriptTM RT Master Mix試劑盒制備cDNA。反轉(zhuǎn)錄過(guò)程:0.1~2.0 μg總RNA加RNase-free ddH2O至8.0 μL,再加入5×PrimeScriptTM RT Master Mix 2.0 μL,混勻。37 ℃反轉(zhuǎn)錄15 min,然后85 ℃滅活5 s,-20 ℃保存?zhèn)溆谩?/p>
1. 4 不同品種羊血液及肺臟組織中TLRs基因轉(zhuǎn)錄水平檢測(cè)
將已制作好的TLR1~TLR10標(biāo)準(zhǔn)質(zhì)粒稀釋成整數(shù)倍,再按10倍梯度進(jìn)行稀釋,選取其中5個(gè)梯度(107~103)繪制標(biāo)準(zhǔn)曲線,并擬合回歸方程。采用TaqMan探針熒光定量PCR對(duì)不同品種羊的10份血液和肺臟樣本cDNA進(jìn)行TLR1~TLR10基因檢測(cè),根據(jù)回歸方程與樣品檢測(cè)得到的Ct,計(jì)算出待測(cè)樣品中的基因拷貝數(shù),分別記錄5種羊血液及肺臟組織中TLR1~TLR10基因的拷貝數(shù)。
1. 5 TLRs基因轉(zhuǎn)錄水平差異顯著性分析
運(yùn)用SPSS 20.0對(duì)TLR1~TLR10基因在不同品種羊血液及肺臟組織中的轉(zhuǎn)錄水平進(jìn)行差異顯著性分析,并以Graphpad Prism 6.4進(jìn)行繪圖。
2 結(jié)果與分析
2. 1 TaqMan探針熒光定量PCR標(biāo)準(zhǔn)曲線
TLR1~TLR10基因TaqMan探針熒光定量PCR標(biāo)準(zhǔn)曲線見(jiàn)圖1,對(duì)應(yīng)的回歸方程分別為:yTLR1=-3.359x+41.996(R2=0.9990);yTLR2=-3.404x+44.028(R2=0.9982);yTLR3=-3.292x+42.556(R2=0.9998);yTLR4=-3.227x+46.483(R2=0.9988);yTLR5=-3.370x+46.124(R2=0.9995);yTLR6=-3.220x+41.190(R2=0.9930);yTLR7=-3.277x+43.399(R2=0.9985);yTLR8=-3.233x+43.393(R2=0.9908);yTLR9=-3.159x+43.389(R2=0.9996);yTLR10=-3.411x+43.059(R2=0.9992)。
2. 2 不同品種羊血液中TLRs基因轉(zhuǎn)錄水平差異
對(duì)5種羊血液中的TLR1~TLR10基因轉(zhuǎn)錄水平差異進(jìn)行分析,結(jié)果(圖2)顯示,不同品種羊血液中TLR1基因轉(zhuǎn)錄水平排序?yàn)橘F州白山羊<貴州黑山羊<波爾山羊<黔北麻羊<湖羊,TLR2基因轉(zhuǎn)錄水平排序?yàn)椴柹窖?lt;貴州黑山羊<湖羊<貴州白山羊<黔北麻羊,TLR3基因轉(zhuǎn)錄水平排序?yàn)橘F州黑山羊<波爾山羊<黔北麻羊<貴州白山羊<湖羊,TLR4基因轉(zhuǎn)錄水平排序?yàn)椴柹窖?lt;黔北麻羊<貴州黑山羊<湖羊<貴州白山羊,TLR5基因轉(zhuǎn)錄水平排序?yàn)椴柹窖?lt;湖羊<貴州黑山羊<黔北麻羊<貴州白山羊,TLR6基因轉(zhuǎn)錄水平排序?yàn)榍甭檠?lt;湖羊<貴州黑山羊<波爾山羊<貴州白山羊,TLR7基因轉(zhuǎn)錄水平排序?yàn)椴柹窖?lt;貴州黑山羊<黔北麻羊<湖羊<貴州白山羊,TLR8基因轉(zhuǎn)錄水平排序?yàn)椴柹窖?lt;貴州黑山羊<黔北麻羊<湖羊<貴州白山羊,TLR9基因轉(zhuǎn)錄水平排序?yàn)橘F州黑山羊<波爾山羊<黔北麻羊<湖羊<貴州白山羊,TLR10基因轉(zhuǎn)錄水平排序?yàn)榍甭檠?lt;貴州黑山羊<湖羊<貴州白山羊<波爾山羊。在波爾山羊血液中TLR2、TLR4和TLR5基因轉(zhuǎn)錄水平均極顯著低于其他4種羊(P<0.01,下同),TLR7和TLR8基因轉(zhuǎn)錄水平則極顯著低于除黑山羊外的其他3種羊;在白山羊血液中TLR4、TLR7、TLR8和TLR9基因轉(zhuǎn)錄水平極顯著高于其他4種羊。
2. 3 不同品種羊肺臟中TLRs基因轉(zhuǎn)錄水平差異
對(duì)5種羊肺臟中的TLR1~TLR10基因轉(zhuǎn)錄水平差異進(jìn)行分析,結(jié)果(圖3)顯示,不同品種羊肺臟中TLR1基因轉(zhuǎn)錄水平排序?yàn)橘F州黑山羊<湖羊<黔北麻羊<波爾山羊<貴州白山羊,TLR2基因轉(zhuǎn)錄水平排序?yàn)楹?lt;波爾山羊<貴州白山羊<黔北麻羊<貴州黑山羊,TLR3基因轉(zhuǎn)錄水平排序?yàn)楹?lt;黔北麻羊<波爾山羊<貴州白山羊<貴州黑山羊,TLR4基因轉(zhuǎn)錄水平排序?yàn)楹?lt;貴州白山羊<貴州黑山羊<波爾山羊<黔北麻羊,TLR5基因轉(zhuǎn)錄水平排序?yàn)楹?lt;貴州黑山羊<波爾山羊<貴州白山羊<黔北麻羊,TLR6基因轉(zhuǎn)錄水平排序?yàn)楹?lt;貴州白山羊<波爾山羊<貴州黑山羊<黔北麻羊,TLR7基因轉(zhuǎn)錄水平排序?yàn)榍甭檠?lt;貴州白山羊<湖羊<貴州黑山羊<波爾山羊,TLR8基因轉(zhuǎn)錄水平排序?yàn)楹?lt;貴州黑山羊<波爾山羊<貴州白山羊<黔北麻羊,TLR9基因轉(zhuǎn)錄水平排序?yàn)楹?lt;貴州白山羊<貴州黑山羊<波爾山羊<黔北麻羊,TLR10基因轉(zhuǎn)錄水平排序?yàn)榍甭檠?lt;貴州白山羊<湖羊<貴州黑山羊<波爾山羊。在湖羊肺臟中TLR2、TLR3、TLR4、TLR5、TLR6、TLR8和TLR9基因轉(zhuǎn)錄水平均低于其他4種羊,其中TLR2、TLR3、TLR4、TLR5和TLR8基因轉(zhuǎn)錄水平極顯著低于其他4種羊;而在黔北麻羊肺臟中TLR5和TLR8基因轉(zhuǎn)錄水平極顯著高于其他4種羊。
2. 4 同一品種羊血液中TLRs基因轉(zhuǎn)錄水平差異
對(duì)同一品種羊血液中的TLR1~TLR10基因轉(zhuǎn)錄水平差異進(jìn)行分析,結(jié)果(圖4)顯示,在波爾山羊血液中TLR2、TLR4、TLR7、TLR8和TLR10基因轉(zhuǎn)錄水平較高,均極顯著高于其他TLRs基因轉(zhuǎn)錄水平;在其他4種羊血液中TLR2、TLR4、TLR7和TLR8基因轉(zhuǎn)錄水平也極顯著高于其他TLRs基因轉(zhuǎn)錄水平。TLR2、TLR4、TLR7和TLR8基因轉(zhuǎn)錄水平在貴州白山羊、貴州黑山羊和湖羊血液中均表現(xiàn)為TLR2基因<TLR7基因<TLR8基因<TLR4基因,在黔北麻羊血液中表現(xiàn)為TLR7基因<TLR2基因<TLR4基因<TLR8基因,在波爾山羊血液中表現(xiàn)為TLR2基因<TLR8基因<TLR7基因<TLR4基因。可見(jiàn),在黔北麻羊血液中TLR8基因具有相對(duì)較高的轉(zhuǎn)錄水平,而其他4種羊均以TLR4基因具有相對(duì)較高的轉(zhuǎn)錄水平。
2. 5 同一品種羊肺臟中TLRs基因轉(zhuǎn)錄水平差異
對(duì)同一品種羊肺臟中的TLR1~TLR10基因轉(zhuǎn)錄水平差異進(jìn)行分析,結(jié)果(圖5)顯示,羊肺臟中TLR1基因轉(zhuǎn)錄水平相對(duì)較低,而TLR2、TLR3、TLR4、TLR5和TLR8基因轉(zhuǎn)錄水平相對(duì)較高,在貴州白山羊、貴州黑山羊和波爾山羊均極顯著高于其他TLRs基因。TLR2、TLR3、TLR4、TLR5和TLR8基因轉(zhuǎn)錄水平在貴州白山羊肺臟中表現(xiàn)為TLR2基因<TLR3基因<TLR8基因<TLR4基因<TLR5基因,在貴州黑山羊肺臟中表現(xiàn)為TLR8基因<TLR3基因<TLR2基因<TLR5基因<TLR4基因,在黔北麻羊肺臟中表現(xiàn)為TLR3基因<TLR2基因<TLR4基因<TLR8基因<TLR5基因,在波爾山羊肺臟中表現(xiàn)為TLR3基因<TLR2基因<TLR8基因<TLR4基因<TLR5基因,在湖羊肺臟中表現(xiàn)為TLR8基因<TLR2基因<TLR3基因<TLR4基因<TLR5基因??梢?jiàn),TLR4基因在貴州黑山羊肺臟中具有相對(duì)較高的轉(zhuǎn)錄水平,但在其他4種羊肺臟中均表現(xiàn)為TLR5基因具有相對(duì)較高的轉(zhuǎn)錄水平。
3 討論
TLRs可激活機(jī)體免疫應(yīng)答并對(duì)其免疫功能進(jìn)行調(diào)節(jié),在機(jī)體的炎癥反應(yīng)及相關(guān)信號(hào)傳導(dǎo)過(guò)程中發(fā)揮重要作用(趙飛等,2019)。TLRs在哺乳動(dòng)物各組織中廣泛表達(dá),已證實(shí)TLRs可減少和清除機(jī)體的細(xì)菌性病原感染(Albiger et al.,2010)。TLRs家族對(duì)不同組織病原體的識(shí)別作用直接受其在該組織中轉(zhuǎn)錄水平的影響,即TLRs轉(zhuǎn)錄水平?jīng)Q定了動(dòng)物個(gè)體或組織器官應(yīng)對(duì)外源微生物侵染的能力(Menzies and Ingham,2006;Tirumurugaan et al.,2010)。趙一萍等(2012)對(duì)蒙古馬不同組織器官的TLR3、TLR7、TLR8和TLR9基因轉(zhuǎn)錄水平進(jìn)行研究,結(jié)果發(fā)現(xiàn)TLRs基因轉(zhuǎn)錄水平在蒙古馬各組織器官中存在明顯差異,可能與其對(duì)病原體的識(shí)別和抵抗能力有關(guān);陳亞冰等(2014)對(duì)牦牛不同組織TLR3和TLR5基因轉(zhuǎn)錄水平進(jìn)行研究,發(fā)現(xiàn)TLRs基因在不同組織器官的轉(zhuǎn)錄水平差異明顯,推測(cè)其與病原體識(shí)別有關(guān)。本研究采用TaqMan探針熒光定量PCR檢測(cè)TLR1~TLR10基因在貴州黑山羊、貴州白山羊、黔北麻羊、波爾山羊及湖羊等5種羊血液和肺臟組織中的轉(zhuǎn)錄水平差,與程曉薇等(2017)檢測(cè)家禽β-連環(huán)蛋白基因在不同臟器表達(dá)水平應(yīng)用的絕對(duì)熒光定量PCR相同,但與陸珂靜等(2019)檢測(cè)小鼠TLRs mRNA表達(dá)量的相對(duì)熒光定量PCR不同。與相對(duì)熒光定量PCR相比,絕對(duì)熒光定量PCR更準(zhǔn)確,且特異性更好(朱振洪等,2011)。
病原致病性存在種間和種內(nèi)差異,即不同物種或同物種不同品種對(duì)同一病原的感染存在易感差異性,如Mmc在自然狀態(tài)下只感染山羊,一般不感染綿羊(樊慶德等,2011)。陶岳等(2006)對(duì)新疆石河子地區(qū)湖羊流行病學(xué)進(jìn)行調(diào)查,結(jié)果發(fā)現(xiàn)傳染性胸膜肺炎對(duì)不同品種羊的感染性存在明顯差異。本研究結(jié)果表明,不同品種羊血液和肺臟中的TLRs基因轉(zhuǎn)錄水平均存在差異,其中,波爾山羊血液中TLR2、TLR4和TLR5基因轉(zhuǎn)錄水平均極顯著低于其他4種羊,TLR7和TLR8基因轉(zhuǎn)錄水平則極顯著低于除黑山羊外的其他3種羊。這與本課題組前期對(duì)貴州省山羊支原體肺炎易感性調(diào)查發(fā)現(xiàn)波爾山羊較其他品種山羊具有更高發(fā)病率的結(jié)論相吻合,提示波爾山羊?qū)ρ蛑гw肺炎易感性可能受TLRs基因轉(zhuǎn)錄水平差異的影響。
越來(lái)越多研究證實(shí),同一TLRs基因在同科不同屬,甚至同種動(dòng)物間也存在微小的差異(曾爽等,2015)。TLRs基因在不同品種羊中的轉(zhuǎn)錄水平具有一定差異性,可能是造成不同品種羊?qū)σ卟∫赘胁町惖脑蛑弧1狙芯堪l(fā)現(xiàn),在貴州黑山羊、貴州白山羊、黔北麻羊、波爾山羊及湖羊等5種羊血液中TLR2、TLR4、TLR7和TLR8基因具有較高的轉(zhuǎn)錄水平,均極顯著高于其他TLRs基因轉(zhuǎn)錄水平;肺臟中TLR2、TLR3、TLR4、TLR5和TLR8基因轉(zhuǎn)錄水平相對(duì)較高,在貴州白山羊、貴州黑山羊和波爾山羊均極顯著高于其他TLRs基因轉(zhuǎn)錄水平。TLR3具有識(shí)別病原雙鏈RNA的能力,云云和黃升海(2007)從轉(zhuǎn)錄水平證實(shí)TLR3基因顯著上調(diào)表達(dá)受呼吸道合胞病毒感染的影響;TLR5是細(xì)菌鞭毛蛋白受體,家禽各組織中TLR5基因表達(dá)能促進(jìn)機(jī)體針對(duì)病原菌的免疫反應(yīng)(王琨等,2020);TLR7和TLR8能促進(jìn)樹突狀細(xì)胞的分化與遷移,進(jìn)而產(chǎn)生TNF-α、IFN-α和IL-2等炎性細(xì)胞因子(Crespo et al.,2013)。在黔北麻羊肺臟中TLR5和TLR8基因轉(zhuǎn)錄水平極顯著高于其他4種羊,是否與黔北麻羊在抵抗細(xì)菌及產(chǎn)生炎性細(xì)胞因子方面具有優(yōu)勢(shì)尚有待進(jìn)一步探究。
血液作為檢測(cè)TLRs基因轉(zhuǎn)錄水平的樣本具有易獲得且對(duì)動(dòng)物個(gè)體傷害小等優(yōu)點(diǎn),而肺臟是羊感染MO和Mmc后發(fā)生病變的主要器官。本研究以不同品種羊血液及肺臟為樣本進(jìn)行TLR1~TLR10基因轉(zhuǎn)錄水平差異檢測(cè),結(jié)果發(fā)現(xiàn)TLR2和TLR4基因在不同品種羊血液及肺臟中均有較高的轉(zhuǎn)錄水平,與Lanki等(2018)、石安惠和牟杰(2020)的研究結(jié)果一致,進(jìn)一步證實(shí)TLR2和TLR4是免疫應(yīng)答的重要調(diào)控因子,有助于機(jī)體抵抗病原菌侵染??梢?jiàn),TLRs在不同品種羊機(jī)體中發(fā)揮著清除病原體及維持機(jī)體穩(wěn)態(tài)的作用,為探究不同品種羊TLRs基因轉(zhuǎn)錄水平與羊疫病的相關(guān)性提供了重要的參考依據(jù)。
4 結(jié)論
不同品種羊血液和肺臟中TLRs基因轉(zhuǎn)錄水平具有差異性,以TLR2、TLR3、TLR4、TLR5和TLR8基因轉(zhuǎn)錄水平相對(duì)較高,提示TLRs在不同品種羊機(jī)體中發(fā)揮著清除病原體及維持機(jī)體穩(wěn)態(tài)的作用,而TLRs基因轉(zhuǎn)錄水平差異可能是造成不同品種羊?qū)σ卟∫赘胁町惖脑蛑弧?/p>
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收稿日期:2021-03-04
基金項(xiàng)目:國(guó)家自然科學(xué)基金項(xiàng)目(31660723,32060786);貴州省科技計(jì)劃項(xiàng)目(黔科合基礎(chǔ)〔2019〕1181號(hào),黔科合支撐〔2018〕2271號(hào))
通訊作者:張雙翔(1987-),https://orcid.org/0000-0002-9117-9960,高級(jí)獸醫(yī)師,主要從事動(dòng)物疫病防控研究工作,E-mail:51379577@ qq.com;程振濤(1979-),https://orcid.org/0000-0002-6723-5344,博士,教授,主要從事動(dòng)物病原學(xué)研究工作,E-mail:chengzhentao@sohu.com
第一作者:唐沙(1992-),https://orcid.org/0000-0001-7723-0574,研究方向?yàn)閯?dòng)物疫病防控,E-mail:670836771@qq.com
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