李 潔，魏振宇，彭 坤，張少麗，黨瑞華，雷初朝，陳 宏，藍(lán)賢勇

(1.西北農(nóng)林科技大學(xué)動物科技學(xué)院，陜西 楊凌 712100；2. 西北農(nóng)林科技大學(xué)創(chuàng)新實(shí)驗(yàn)學(xué)院，陜西 楊凌 712100)

朊蛋白基因(PRNP)不僅與動物傳染性海綿樣腦病(TSEs)密切相關(guān)，還與反芻家畜的表型性狀密切相關(guān)。已有研究表明：牛、水牛、牦牛、綿羊、山羊等反芻家畜PRNP基因具有豐富的多態(tài)性，同時，這些遺傳變異位點(diǎn)在這些物種中又具有顯著差異性；其中，牛、綿羊和山羊PRNP基因多態(tài)性與BSE、瘙癢病顯著相關(guān)；牛、綿羊和山羊PRNP基因多態(tài)性與生產(chǎn)性能有密切相關(guān)性。為此，本文從反芻家畜PRNP基因結(jié)構(gòu)比較、反芻家畜PRNP基因多態(tài)性研究、反芻家畜PRNP多態(tài)性與疾病的關(guān)系、反芻家畜PRNP多態(tài)性與生產(chǎn)性能的關(guān)系等四方面進(jìn)行綜述，以期為反芻家畜優(yōu)良個體及品種的高效選育提供理論參考。

反芻家畜;朊蛋白基因(PRNP);多態(tài)性;遺傳效應(yīng);性狀

作為人獸共患疾病的典型代表，傳染性海綿樣腦病(transmissible spongiform encephalopathies，TSEs)因其高傳染性與致死性對畜牧產(chǎn)業(yè)乃至整個社會造成了極大的危害[1]。作為調(diào)控哺乳動物TSEs的主要基因，朊蛋白基因(prion protein gene，PRNP)突變或其編碼蛋白構(gòu)象改變，會引起朊病毒疾病的發(fā)生。目前，已有研究證實(shí)反芻家畜PRNP基因存在豐富的遺傳多態(tài)性，同時這些遺傳變異位點(diǎn)在這些物種中又具有顯著差異性；其中牛[2]、山羊[3]和綿羊[4]的PRNP基因多個多態(tài)性位點(diǎn)與TSEs的易感性及潛伏期有顯著的相關(guān)關(guān)系。典型的朊病毒病包括人的克雅氏病、吉斯特曼斯特勞斯綜合征、致死性家族性失眠癥、散發(fā)型致死性失眠病、庫魯病、變異型克雅氏病、牛傳染性海綿狀腦病或稱瘋牛病和羊瘙癢病等[5]。疾病的發(fā)生會影響家畜的生長速度，產(chǎn)子數(shù)以及其他生產(chǎn)性能等，從而造成了嚴(yán)重的經(jīng)濟(jì)生產(chǎn)損失[6]。隨著研究的逐漸深入，也有研究發(fā)現(xiàn)PRNP基因多態(tài)性可以影響未感染TSEs的健康動物的表型性狀[7,8]，因此，研究PRNP基因多態(tài)性對家畜生產(chǎn)性能的提高具有重要意義。

為此，本文對反芻家畜PRNP基因結(jié)構(gòu)比較、反芻家畜PRNP基因多態(tài)性研究、反芻家畜PRNP多態(tài)性與疾病的關(guān)系、反芻家畜PRNP多態(tài)性與生產(chǎn)性能的關(guān)系等方面進(jìn)行綜述，以期為分子標(biāo)記輔助優(yōu)良個體選擇及家畜育種研究提供了重要思路與理論支持。

1 反芻家畜PRNP基因結(jié)構(gòu)的比較

由于PRNP基因廣泛存在于動物體內(nèi)，目前研究人員已對多種動物的PRNP基因進(jìn)行了研究。在常見反芻家畜中，例如山羊、綿羊、家牛、瘤牛、水牛、牦牛等，PRNP基因大多被定位于家畜13號染色體(牦牛未定位)，大多含有三個或五個外顯子，PRNP 基因的開放閱讀框(ORF)都編碼一個約由250個氨基酸組成的PrP蛋白，基因結(jié)構(gòu)較簡單(表1)。此外，在已研究的哺乳動物中，PRNP基因ORF中的DNA序列和PrP氨基酸序列的相似性分別達(dá)到90%和95%以上[9]。由此可見，PRNP基因具有高度保守性，這也為朊蛋白跨物種傳播提供了分子基礎(chǔ)。

表1 常見反芻家畜PRNP基因結(jié)構(gòu)統(tǒng)計表

2 反芻家畜PRNP基因多態(tài)性研究進(jìn)展

雖然PRNP基因在反芻家畜之間具有高度同源性，但該基因在不同物種之間多態(tài)性卻具有很大差異性，這也是PRNP基因多態(tài)性一直是研究熱點(diǎn)的原因之一。目前，反芻家畜PRNP基因已被報道的多態(tài)性位點(diǎn)包括單核苷酸多態(tài)性(SNPs)和插入/缺失(indel)突變，拷貝數(shù)變異(CNV)卻未見報道。

2.1 反芻家畜PRNP基因SNP多態(tài)性研究進(jìn)展

反芻家畜PRNP基因中存在大量的SNP位點(diǎn)，且不同品種間的SNP多態(tài)性差異較大。早在2010年，Shimogiri等通過對mythun，日本黑牛以及蒙古牛群體進(jìn)行檢測，發(fā)現(xiàn)K3T和S154N兩個無義突變[10]。Xi等通過對125頭大額牛的PRNP基因編碼區(qū)序列進(jìn)行檢測，共發(fā)現(xiàn)10個SNP位點(diǎn)，包括6個同義突變(C60T，G75A，A108T，G126A，C357T和C678T)和4個非同義突變(C8A，G145A，G461A和C756G)，相對應(yīng)的氨基酸上的改變(T3K，G49S9，N154S和I252M)也得到了驗(yàn)證[11]。與此同時，Choi等首次對300頭Hanwoo牛(韓國)，澤西牛，日本黑牛等群體的PRNP基因編碼區(qū)序列進(jìn)行檢測，發(fā)現(xiàn)4個同義突變(G234A，C555T，C576T，和C630T) 和2個非同義突變[12]。在中國延邊牛和中國草原紅牛群體中，PRNP基因也存在3個非同義突變造成了氨基酸的改變(K119N，S154N和 M177V)和一個沉默突變(A234G)[13]。

在安托利亞水牛及摩拉水牛PRNP基因編碼區(qū)檢測到3個同義突變(126，234及285位)與一個非同義突變(322位，G108S)。其中，126位存在G/A/T三種堿基[14]；水牛PRNP基因也具有較高多態(tài)性，但牦牛PRNP基因多態(tài)性的相關(guān)研究還未見報道。

綿羊PRNP基因中有大量SNP位點(diǎn)，例如，密碼子85 (G/R)，112 (M/T/I)，116 (A/P)，127(G/A/V/S)，136 (A/V/T)，137 (M/T)，138 (S/N)，141(L/F)，143 (H/R)，151 (R/C)，152 (Y/F)，154 (R/H)，167 (R/S)，168 (P/L)，171 (Q/R/H/K)，175 (Q/E)，176(K/N)，180 (H/Y)，189 (G/L/R)，195 (T/S)，196 (T/S)，211 (R/Q)和161 (P/S)[15-19]。Lan等對來自中國16個地方品種的486只綿羊進(jìn)行了檢測，共檢測到154位密碼子(R或H)和171 位密碼子(具有4種突變，分別編碼Q，R，H或K)具有多態(tài)性。在所有檢測品種中，136位密碼子均為純合AA，未檢測到A/V突變。此外，在所有檢測品種中，密碼子21，101，112，127，138，141，143，146，153和189 均具有多態(tài)性。密碼子171位的優(yōu)勢等位基因?yàn)镼(頻率高達(dá)88.68%)[20]。

在山羊群體中，有大量SNP位點(diǎn)，分別位于密碼子：V21A，G22C，L23P，G37V，G42A，G49S，P63L，W102G，T110N，T110P，G127S，L133Q，M137I，I142M，I142T，H143R，N146S，N146D，R151H，R154H，P168Q，T194P，F(xiàn)201L，R211Q，R211G，I218L，T219I，Q220H，Q222K和P240S[21-31]。Zhou等對來自中國11個地方品種的337只山羊羊進(jìn)行了檢測，共在山羊PRNP基因上檢測到10種氨基酸的多態(tài)性(分別位于密碼子102，127，143，146，154，211，218，219，222和240)[32]。

2.2 反芻家畜PRNP基因indel多態(tài)性研究進(jìn)展

反芻家畜PRNP基因中除了存在大量的SNPs位點(diǎn)，還存在一些indel位點(diǎn)，如表2所示。

目前，關(guān)于人類PRNP基因CNV研究有極少報道，例如：Ana等[33]通過對1147名克雅氏病人進(jìn)行PRNP CNV掃描，經(jīng)多重檢驗(yàn)后發(fā)現(xiàn)CNVs >100，>500，或者 >1000 kb，與人克雅氏疾病的發(fā)生并無顯著關(guān)聯(lián)性。但關(guān)于反芻家畜PRNP基因拷貝數(shù)變異(CNV)卻未見報道，反芻動物PRNP 是否存在CNV變異及其遺傳效應(yīng)仍有待挖掘。

表2 常見反芻家畜PRNP基因indel位點(diǎn)統(tǒng)計表

3 反芻家畜PRNP多態(tài)性與疾病的關(guān)系

朊蛋白基因(prion protein gene，PRNP)突變或其編碼蛋白構(gòu)象發(fā)生改變，是引發(fā)哺乳動物TSEs的主要原因。Sander等[34,35]分析了43頭已感染BSE的德國肉牛與48頭健康的德國肉牛，也是第一次比較了23-bp indel和12-bp indel這兩個indel多態(tài)性位點(diǎn)與BSE抗病性的相關(guān)關(guān)系，結(jié)果發(fā)現(xiàn)PRNP基因多態(tài)性與BSE有顯著相關(guān)，并且也可以調(diào)節(jié)BSE的潛伏期，與瘋牛病的易感性相關(guān)聯(lián)。并且，他們發(fā)現(xiàn)這兩個indel位點(diǎn)可以影響轉(zhuǎn)錄因子RP58和Sp1的結(jié)合位點(diǎn)。Xue等通過對日本黑牛的研究，發(fā)現(xiàn)轉(zhuǎn)錄因子Sp1和RP58可能對PRNP基因的表達(dá)有負(fù)反饋調(diào)節(jié)作用[36]。同時，Kashkevich等(2007)研究PRNP基因啟動區(qū)域的多態(tài)性，發(fā)現(xiàn)通過降低PRNP基因的表達(dá)可以增強(qiáng)瘋牛病的抗性[37]。而Neibergs等[38]報道了開放閱讀框并不影響瘋牛病的易感性。Hreko等研究發(fā)現(xiàn)，PRNP基因啟動子區(qū)的23-bp缺失的純合子插入基因型似乎已經(jīng)對瘋牛病起保護(hù)作用[39]。此外，由于水牛與家牛PrP的表達(dá)量不同，因此牛與水牛對瘋牛病的易感性也明顯不同[40-41]。至今還未在水牛群體中見到爆發(fā)瘋牛病。大量研究表明牛PRNP啟動子區(qū)23-bp及第一內(nèi)含子12-bp的多態(tài)性與瘋牛病感染性及抗性密切相關(guān)，但在安納托利亞水牛及摩拉水牛中只發(fā)現(xiàn)了插入/插入(II)型，未檢測到突變型，推測II型為抗性基因型[42]。

綿羊PRNP基因多態(tài)性研究多集中于外顯子區(qū)域，尤其是第三外顯子且突變多以單核苷酸突變?yōu)橹鳌?36[43]，141[44]，146[45]，154[46]和171[47]位密碼子的變異被證實(shí)與綿羊瘙癢病的發(fā)生或病癥密切相關(guān)。研究表明，在患有TSE的病羊中，其136 位點(diǎn)(A-V)154 位點(diǎn)(R-H)和171 位點(diǎn)(Q-R/H)均會發(fā)生突變。其中，Q171R多態(tài)性對羊瘙癢病的感染具有很強(qiáng)的抗性而A136V多態(tài)性則極易感染。并且，基因型ARR/ARR 或ARR/ARQ 是抗性基因型，而ARQ/ARQ、VRQ/ARQ、AHQ/VRH 等其它基因型羊則為敏感型[48-51]。同時，一些非編碼區(qū)域的突變也被報道影響綿羊瘙癢病的易感性[52]。

類似地，山羊PRNP基因的多態(tài)性，如I142M，H143R，N146S/D，R154H，R211Q和Q222K也對羊瘙癢病的感染表現(xiàn)出明顯的抗性[5]。自2001年開始，歐盟各國開始培育有抗性基因的羊，其中ARR/ARR純合子具有最高的抗羊瘙癢病的能力，并且這些純合子綿羊的產(chǎn)量、生長、繁殖和健康都和其他羊沒有明顯的差異[53]。隨著家畜抗TSEs分子育種工作在歐盟各國成功開展，這些著名的致病性位點(diǎn)經(jīng)過人工選擇逐漸被淘汰，綿羊瘙癢病和瘋牛病等神經(jīng)退行性疾病的爆發(fā)也基本已得到控制。

4 PRNP多態(tài)性與生產(chǎn)性能的關(guān)系

生產(chǎn)性能是決定畜牧養(yǎng)殖業(yè)效益的主要指標(biāo)，包括家畜生長性狀及繁殖性能。除了參與調(diào)控TSEs疾病，PRNP基因多態(tài)性也被證實(shí)與動物的一些表型性狀顯著相關(guān)。例如，綿羊PRNP基因的VRQ和ARQ等位基因變異體(在密碼子136、154和171的多態(tài)性位點(diǎn))與綿羊瘙癢病的高易感性呈顯著相關(guān)，導(dǎo)致綿羊的生產(chǎn)性能顯著降低從而造成了嚴(yán)重的經(jīng)濟(jì)生產(chǎn)損失[6]。與此同時，也有研究表明PRNP基因多態(tài)性可以影響未感染TSEs的健康動物的表型性狀。

在健康牛群體中，PRNP八肽重復(fù)序列多態(tài)性被報道與荷斯坦奶牛的產(chǎn)奶性狀密切相關(guān)[54]。在我國地方黃牛群體中，PRNP基因多樣性與生產(chǎn)性能的關(guān)系也被揭示。Yang[7-8]等首次在中國良種黃牛(秦川牛，南陽牛，郟縣紅牛，吉安牛，夏南牛和皮南牛)群體中，驗(yàn)證了PRNP基因3′ UTR區(qū)14-bp indel，啟動子區(qū)23-bp indel和第一內(nèi)含子12-bp indel突變與牛的生長性狀顯著相關(guān)。例如，3′ UTR區(qū)14-bp indel與秦川牛的體長，夏南牛的體重和腰圍顯著相關(guān)(P<0.05)。南陽牛在23-bp indel位點(diǎn)上基因型為缺失/缺失(DD)基因型的個體以及12-bp indel位點(diǎn)基因型為插入/插入(II)基因型的個體表現(xiàn)出了更優(yōu)良的表型性狀，在六個研究的牛品種中南陽牛具有較低的雜合子(ID)單倍型頻率[8]。此外，PRNP啟動子區(qū)23-bp indel顯著影響南陽牛的體長和胸圍，第一內(nèi)含子12-bp indel突變也與吉安牛的管圍密切相關(guān)[7]。

在健康綿羊群體中，PRNP基因多態(tài)性被報道影響羔羊生長性狀[55-57]，母綿羊的繁殖力[58-60]和產(chǎn)奶量[61-62]等生產(chǎn)性能。Sawalha等研究發(fā)現(xiàn)，攜帶ARQ等位基因的蘇格蘭黑面母綿羊往往具有較強(qiáng)的季節(jié)性脂肪組織動員能力[63]。同時，他們也發(fā)現(xiàn)171位密碼子的多態(tài)性與薩?？司d羊的產(chǎn)羔性狀顯著相關(guān)[25]。Allais-Bonnet[64]等人也發(fā)現(xiàn)PRNP基因型的差異(例如，ARQ/ARQ和 ARR/ARQ)及八肽重復(fù)序列多態(tài)性可以導(dǎo)致Latxa綿羊產(chǎn)奶性能(產(chǎn)奶量，奶蛋白，奶脂含量)的差異。還有研究表明PRNP基因型與Texel羊的產(chǎn)仔數(shù)和135日齡的體重有顯著相關(guān)[65]。先前我們課題組在來自中國和蒙古5個品種的768名健康綿羊個體PRNP基因中共發(fā)現(xiàn)了四種新的indel多態(tài)性：第一內(nèi)含子7-bp(I1-7bp)，第二內(nèi)含子15-bp(I2-15 bp)，第二內(nèi)含子19-bp(I2-19 bp)和3 'UTR-7 bp(3'UTR-7 bp)。并且，這4種indel與13個不同的生長性狀(例如，毛長，背高，胸圍等)呈顯著相關(guān)(P<0.05)[66]。尤其是I2-15 bp對小尾寒羊(母羊)的胸寬(P= 0.001，DD為優(yōu)勢基因型)，3'UTR-7 bp對湖羊的胸圍(P= 0.003，DD為優(yōu)勢基因型)，12-19 bp 對小尾寒羊(公羊)的胸圍指數(shù)(P=1.122E-4，DD為優(yōu)勢基因型)以及同羊的尾長(P= 0.001，II為優(yōu)勢基因型)，呈現(xiàn)極顯著關(guān)聯(lián)性(P<0.01)[66]。

在健康山羊群體中，山羊PRNP基因啟動子區(qū)28-bp的indel突變與內(nèi)蒙古白絨山羊的平均體重，1歲羊平均體重以及3歲羊毛厚度顯著相關(guān)，并且II型均為這三種性狀的優(yōu)勢基因型[67]。此外，該 indel 突變也與西農(nóng)薩能奶山羊的平均產(chǎn)奶量密切相關(guān)，II型個體產(chǎn)奶量顯著高于雜合型(ID)個體，但I(xiàn)D型個體夜間總固體量要顯著高于II型個體[67]。此外，Lan等通過對來自4個綿羊品種(西農(nóng)薩能奶山羊，內(nèi)蒙古白絨山羊，新疆絨山羊，陜北白絨山羊)2002只綿羊的PRNP基因進(jìn)行多態(tài)性位點(diǎn)掃描，發(fā)現(xiàn)第42位密碼子的SNP位點(diǎn)(42CCG>42CCA)與7歲內(nèi)蒙古白絨山羊的體重顯著相關(guān)，并且GG型個體體重要顯著優(yōu)于AA基因型個體[68]。與此同時，該位點(diǎn)還與內(nèi)蒙古白絨山羊的產(chǎn)絨量和毛長顯著相關(guān)。此外，在西農(nóng)薩能奶山羊中，GG型個體的早、晚奶密度以及奶中非脂質(zhì)固體含量要顯著優(yōu)于其他基因型。總體而言，GG型薩能奶山羊所產(chǎn)奶質(zhì)量往往要優(yōu)于其他基因型[68]。

5 展望

對于PRNP基因多態(tài)性與TSEs發(fā)病機(jī)理的關(guān)系，Telling等(1996年)學(xué)者認(rèn)為PRNP基因點(diǎn)突變的發(fā)生增加了不穩(wěn)定的中間分子的數(shù)量，同時加快了PrPc向PrPsc的轉(zhuǎn)換速率，從而形成穩(wěn)定性更好的PrPsc構(gòu)象[69]。此外，有研究證明Pr P在形成二級結(jié)構(gòu)過程中,該區(qū)域很容易發(fā)生α螺旋轉(zhuǎn)變?yōu)棣?折疊，增加了發(fā)病機(jī)率[70]解釋了PRNP 多態(tài)性與TSEs發(fā)病機(jī)理的關(guān)系。

在醫(yī)學(xué)領(lǐng)域中已有報道稱PrPc參與胚胎自我修復(fù)，組織分化和癌干細(xì)胞的生成等過程[71-72]，這為朊蛋白基因調(diào)控動物的生產(chǎn)性狀提供了理論基礎(chǔ)。先前研究報道DNA序列上堿基的突變可能會改變mRNA的穩(wěn)定性，以及mRNA加工和成熟過程，進(jìn)一步影響等位基因的表達(dá)和翻譯后肽鏈的折疊[73]。因此，我們預(yù)測PRNP這些indel突變序列中可能存在其他生長主效基因轉(zhuǎn)錄因子的結(jié)合位點(diǎn)，使得這些生長基因的表達(dá)受到影響[74]。另外，有報道稱PRNP基因3′ UTR區(qū)的indel多態(tài)性可以通過微小RNA介導(dǎo)的轉(zhuǎn)錄后機(jī)制來調(diào)控性狀或疾病易感性[75]。此外，基因間的相互作用也可能是PRNP基因調(diào)控生長性狀的原因之一。已有研究證明在綿羊群體中，PRNP多態(tài)性位點(diǎn)往往與其臨近同源基因-疊朊蛋白基因(PRND)的多態(tài)性位點(diǎn)相互關(guān)聯(lián)[76]，而我們先前的研究結(jié)果表明，綿羊PRND基因編碼區(qū)上游的20-bp indel突變與湖羊的管圍指數(shù)顯著相關(guān)[77]。此外，綿羊PRNP和GnRH基因也被報道可能存在連鎖關(guān)系，影響綿羊性成熟時的體重，對乳腺分化、母性行為、免疫特征及細(xì)胞凋亡都存在影響[78]。然而，這其中確切的分子調(diào)控機(jī)制還需要繼續(xù)探究。

迄今為止，我國還未見有關(guān)瘋牛病和瘙癢病大規(guī)模爆發(fā)的報道，且相關(guān)研究表明我國部分地方品種感染TSEs的可能性較低[79]，因此如何在健康群體中高效準(zhǔn)確的篩選優(yōu)良個體及培育優(yōu)良畜種成為養(yǎng)殖業(yè)的關(guān)鍵。因DNA標(biāo)記具有標(biāo)記范圍廣，標(biāo)記數(shù)量不受限制，遺傳相對穩(wěn)定等優(yōu)點(diǎn)，分子標(biāo)記輔助選擇(MAS)在畜牧業(yè)被廣泛的應(yīng)用，發(fā)展也十分迅速。目前已有應(yīng)用的DNA分子標(biāo)記包括：RFLP標(biāo)記、SSR標(biāo)記、indel 標(biāo)記以及SNP標(biāo)記等多種標(biāo)記方法，這些方法能夠加快育種改良進(jìn)程、減少生產(chǎn)成本。與此同時，上述大量研究已表明PRNP基因在反芻家畜中具有較高的多態(tài)性，可作為MAS選擇靶向基因。伴隨著畜牧產(chǎn)業(yè)的快速發(fā)展，相信未來會有更多的研究關(guān)注PRNP的遺傳多樣性與動物經(jīng)濟(jì)性狀的關(guān)系，從而為分子標(biāo)記輔助選擇(MAS)在畜牧業(yè)中的廣泛應(yīng)用提供新思路與理論依據(jù)，加快家畜優(yōu)良經(jīng)濟(jì)品種的選育進(jìn)程。

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