李法君付春鵬李明爽李群峰傅洪拓

(1. 濰坊科技學(xué)院，壽光 262700; 2. 中國(guó)水產(chǎn)科學(xué)研究院淡水漁業(yè)研究中心，農(nóng)業(yè)部淡水漁業(yè)和種質(zhì)資源利用重點(diǎn)實(shí)驗(yàn)室，無(wú)錫 214081; 3. 全國(guó)水產(chǎn)技術(shù)推廣總站，北京 100125)

RNAi在甲殼動(dòng)物中的研究進(jìn)展

李法君1,2付春鵬1李明爽3李群峰1傅洪拓2

(1. 濰坊科技學(xué)院，壽光 262700; 2. 中國(guó)水產(chǎn)科學(xué)研究院淡水漁業(yè)研究中心，農(nóng)業(yè)部淡水漁業(yè)和種質(zhì)資源利用重點(diǎn)實(shí)驗(yàn)室，無(wú)錫 214081; 3. 全國(guó)水產(chǎn)技術(shù)推廣總站，北京 100125)

RNA干擾(RNA interference, RNAi)是一類在真核生物中廣泛存在的, 由雙鏈RNA介導(dǎo)的轉(zhuǎn)錄后基因沉默機(jī)制。作為一項(xiàng)研究基因功能的有力工具, RNAi技術(shù)已經(jīng)被廣泛應(yīng)用在線蟲(chóng)、果蠅、斑馬魚(yú)和小鼠等生物的基因組學(xué)研究中。近來(lái)在甲殼動(dòng)物中, 通過(guò)RNAi技術(shù)取得了眾多的科研成果。文章從免疫、生長(zhǎng)發(fā)育、蛻皮、生殖、性別調(diào)控、滲透壓調(diào)節(jié)和代謝等幾個(gè)方面進(jìn)行了綜述。進(jìn)而對(duì)RNAi技術(shù)在甲殼動(dòng)物中的研究前景進(jìn)行了展望, 旨在為以后更好地研究甲殼動(dòng)物的基因功能和調(diào)控網(wǎng)絡(luò)提供參考。

RNA干擾; 基因沉默; 甲殼動(dòng)物; 基因功能; 調(diào)控機(jī)制

甲殼動(dòng)物種類繁多, 其中的許多物種, 特別是十足目蝦蟹類是我國(guó)乃至世界范圍內(nèi)重要的養(yǎng)殖對(duì)象。近年來(lái)在甲殼動(dòng)物的養(yǎng)殖過(guò)程中出現(xiàn)了疾病頻發(fā)的問(wèn)題; 而且多數(shù)甲殼動(dòng)物雌雄之間存在明顯的生長(zhǎng)差異現(xiàn)象, 因此單性化養(yǎng)殖一直是甲殼動(dòng)物養(yǎng)殖領(lǐng)域極具吸引力的研究方向。解決上述問(wèn)題的關(guān)鍵在于闡明其內(nèi)在基因的功能及調(diào)控機(jī)制。隨著測(cè)序技術(shù)的發(fā)展, 不但甲殼動(dòng)物不同組織、不同發(fā)育時(shí)期的mRNA序列得以明確, 而且像中華絨螯蟹(Eriocheir sinensis)這樣重要經(jīng)濟(jì)種類的全基因組DNA序列也已經(jīng)測(cè)序完成[1]。如何有效利用這些基因序列, 闡明甲殼動(dòng)物諸如免疫、生長(zhǎng)、蛻皮和性別分化等生理過(guò)程的分子機(jī)制, 從而更好地解決甲殼動(dòng)物養(yǎng)殖過(guò)程中出現(xiàn)諸多問(wèn)題, 就成為當(dāng)前亟待解決的課題。

雙鏈RNA (Double-stranded RNA, dsRNA)介導(dǎo)的RNA干擾(RNA interference, RNAi)現(xiàn)象是普遍存在線蟲(chóng)、果蠅、斑馬魚(yú)和小鼠等真核生物中的轉(zhuǎn)錄后調(diào)控機(jī)制, 在生物體的生長(zhǎng)發(fā)育、穩(wěn)定轉(zhuǎn)座子和抵御外來(lái)病毒的入侵等方面發(fā)揮重要作用。RNAi技術(shù)于2001年被《Science》雜志評(píng)為十大科學(xué)進(jìn)展之一, 2002年更是位居十大科學(xué)進(jìn)展之首。作為研究基因功能的重要工具, 近年來(lái)RNAi技術(shù)也在甲殼動(dòng)物中得以廣泛應(yīng)用, 并取得了眾多研究成果。鑒于此, 本文對(duì)RNAi在甲殼動(dòng)物中的研究進(jìn)展進(jìn)行了綜述, 旨在為應(yīng)用此技術(shù)解析甲殼動(dòng)物重要基因的功能及調(diào)控機(jī)制提供理論參考。

1 RNAi簡(jiǎn)介

RNAi是指在進(jìn)化過(guò)程中高度保守的、由dsRNA誘發(fā)的同源mRNA高效特異性降解的現(xiàn)象。因此, RNAi技術(shù)又被形象地稱為基因敲除(Knock-down)或基因沉默(Gene silencing)。1995年, Guo和Kemphues[2]利用反義RNA阻斷線蟲(chóng) (Caenorhabditis ele-gans) 的part-1基因表達(dá)時(shí), 意外發(fā)現(xiàn)作為對(duì)照組的正義RNA也可以抑制part-1基因的表達(dá); 1998年, Fire等[3]分別將part-1基因的正義、反義和dsRNA導(dǎo)入線蟲(chóng), 發(fā)現(xiàn)dsRNA的沉默效果明顯高于單鏈RNA。于是將這種由dsRNA抑制特定基因表達(dá)的現(xiàn)象稱為RNAi。隨后, RNAi以其獨(dú)有的高效性、特異性、穩(wěn)定性和可傳播性迅速成為研究基因功能的重要工具。

研究表明, 由dsRNA引發(fā)的RNAi作用機(jī)制分兩步進(jìn)行: 起始階段和效應(yīng)階段。起始階段: 外源或內(nèi)源的dsRNA進(jìn)入細(xì)胞后, 被Dicer酶劈成21—23個(gè)堿基的小片段, 稱為小分子干擾RNA (Small interfering RNA, siRNA); 效應(yīng)階段: siRNA和RNA誘導(dǎo)沉默復(fù)合物(RNA Induced Silencing Complex, RISC)相結(jié)合, 結(jié)合后的復(fù)合物具有核酸酶的作用,能識(shí)別并降解目標(biāo)RNA。

2 RNAi技術(shù)在甲殼動(dòng)物中的研究

甲殼動(dòng)物隸屬節(jié)肢動(dòng)物門, 處在由無(wú)脊椎動(dòng)物向脊椎動(dòng)物進(jìn)化的特殊階段, 獨(dú)特的進(jìn)化地位也決定了發(fā)育過(guò)程和組織構(gòu)造的特殊性。RNAi作為真核生物普遍存在的機(jī)制, 也在甲殼動(dòng)物中調(diào)控多個(gè)生物學(xué)過(guò)程。

2.1 免疫

病毒病已經(jīng)給對(duì)蝦養(yǎng)殖業(yè)造成重大損失, 對(duì)蝦白斑綜合癥病毒(White spot syndrome virus, WSSV)、黃頭病毒(Yellow head virus, YHV)和桃拉病毒(Taura syndrome virus, TSV)是危害我國(guó)對(duì)蝦養(yǎng)殖業(yè)的三種主要病毒。其中以WSSV的危害最為嚴(yán)重, 已經(jīng)給對(duì)蝦養(yǎng)殖業(yè)造成巨大的經(jīng)濟(jì)損失。RNAi被認(rèn)為是一種古老的抗病毒機(jī)制, 是生物體中先天性存在的免疫因子, 甲殼動(dòng)物也不例外。研究表明,免疫系統(tǒng)是后生動(dòng)物普遍存在的抵抗外來(lái)病原入侵的防御體系, 分為先天性免疫和獲得性免疫。甲殼動(dòng)物體內(nèi)不能產(chǎn)生免疫球蛋白, 缺乏獲得性免疫,因此僅具有先天性免疫[4,5]。RNAi技術(shù)最早應(yīng)用在甲殼動(dòng)物也是在此方面。Kim等[6]首次在感染W(wǎng)SSV和TSV的凡納濱對(duì)蝦(Litopenaeus vannamei)中分別注射綠頭鴨(Anas platyrhynchos)、鯰(Ictalurus punctatus)和野豬(Sus scrofa)的免疫球蛋白基因dsRNA。結(jié)果顯示, 注射異源物種dsRNA的實(shí)驗(yàn)組死亡率下降到對(duì)照組的50%—70%, 由于凡納濱對(duì)蝦體內(nèi)不存在上述脊椎動(dòng)物的免疫球蛋白基因, 所以脊椎動(dòng)物的dsRNA分子不可能參與到凡納濱對(duì)蝦的抗病毒干擾體系中去。引起染病凡納濱對(duì)蝦死亡率降低的原因在于, 脊椎動(dòng)物免疫球蛋白基因dsRNA作為外源性物質(zhì)注射到凡納濱對(duì)蝦體內(nèi), 從而激活了凡納濱對(duì)蝦先天性的免疫反應(yīng)。

WSSV是具有雙層囊膜的桿狀型病毒, 目前已確定VP19、VP24、VP26、VP28等10余種膜蛋白[7,8], 這些膜蛋白通常參與WSSV病毒粒子的吸附、入侵、包裝和釋放等過(guò)程, 對(duì)病毒的感染起著至關(guān)重要的作用[9,10]。由于WSSV屬于雙鏈DNA病毒, 因此科研人員通常利用RNAi技術(shù)定向沉默編碼上述蛋白的mRNA來(lái)抑制WSSV的繁殖。Kim等[11]將VP28和VP281的長(zhǎng)鏈dsRNA注射到感染W(wǎng)SSV的中國(guó)對(duì)蝦(Fenneropenaeus chinensis)體內(nèi), 有效提高存活率。Xu等[12]將短鏈VP28的dsRNA注射到日本囊對(duì)蝦(Penaeus japonicus)體內(nèi), 也發(fā)現(xiàn)有類似的結(jié)果。近來(lái)Thammasorn等[13]在凡納濱對(duì)蝦中利用基因重組技術(shù), 將VP28和WSSV051 (WSSV的極早期基因之一)兩個(gè)基因干擾位點(diǎn)構(gòu)建到同一條dsRNA中, 并將其飼喂凡納濱對(duì)蝦。7d后此實(shí)驗(yàn)組的死亡率為40%, 顯著低于VP28和WSSV051的單獨(dú)干擾組,表現(xiàn)出良好的治療效果。RNAi在甲殼動(dòng)物不同WSSV結(jié)構(gòu)基因方面的具體研究在表 1中列出。

與WSSV不同, TSV和YHV都是單鏈RNA病毒[14,15], RNAi對(duì)其結(jié)構(gòu)基因的研究?jī)H限于YHV的gp116和gp64兩個(gè)功能基因, 而且是在體外細(xì)胞中進(jìn)行的[16]。有關(guān)RNAi在YHV和TSV中的研究多體現(xiàn)在阻斷其傳輸途徑中的相關(guān)基因方面。Rab是GTP結(jié)合蛋白家族中最大的亞家族, Rab7蛋白作為Rab家族一員, 能特異識(shí)別晚期胞內(nèi)體等囊泡, 介導(dǎo)晚期胞內(nèi)體與溶酶體的膜融合, 以完成溶酶體轉(zhuǎn)運(yùn)過(guò)程。當(dāng)病毒、菌病體等進(jìn)入宿主細(xì)胞后會(huì)與Rab7蛋白結(jié)合, 避開(kāi)溶酶體的消化, 導(dǎo)致宿主感染。因此, Rab7在病原體入侵宿主過(guò)程中起著重要的作用。例如, 斑節(jié)對(duì)蝦的Rab7可以與VP28結(jié)合, 在WSSV侵染對(duì)蝦的過(guò)程中發(fā)揮作用[17]。Ongvarrasopone等[18]在斑節(jié)對(duì)蝦中通過(guò)注射Rab7-dsRNA降低 Rab7基因的表達(dá)量, 從而有效抑制YHV和WSSV感染對(duì)蝦, 推測(cè)Rab7蛋白可能是參與了病毒復(fù)制過(guò)程中的內(nèi)吞運(yùn)輸。在TSV侵染蝦體的研究中也獲得了相似的結(jié)果, Ongvarrasopone等[19]在TSV侵染凡納濱對(duì)蝦48h之后注射Rab7-dsRNA, 有效地抑制TSV的復(fù)制, 提高了蝦的成活率。RNAi在YHV和TSV傳播途徑中其他相關(guān)基因的研究在表 1中列出。

上述研究表明, RNAi作為廣泛存在的抗病毒機(jī)制在甲殼動(dòng)物抵御外來(lái)病毒的侵染過(guò)程中發(fā)揮重要作用。近年來(lái), 有關(guān)甲殼動(dòng)物RNAi抗病毒的分子機(jī)制也取得了較大進(jìn)展。現(xiàn)在公認(rèn)的觀點(diǎn)為:病毒侵染甲殼動(dòng)物有機(jī)體, 并將外源基因整合到寄主的基因組內(nèi)。當(dāng)宿主細(xì)胞進(jìn)行轉(zhuǎn)錄時(shí), 會(huì)產(chǎn)生相應(yīng)的dsRNA。宿主細(xì)胞內(nèi)的核酸內(nèi)切酶Dicer (DCR2)[41,42]與Arsenite resistance蛋白2(Ars2)[43]和HIV-1反式激活應(yīng)答元件RNA結(jié)合蛋白(HIV-1 transactivating response (TAR) RNA-binding protein, TRBP)[44]組成三者復(fù)合物, 將上述dsRNA分割成siRNA, siRNA與宿主細(xì)胞內(nèi)的核酸內(nèi)切酶Argonautes 2(Ago2)結(jié)合組成沉默復(fù)合物, 從而對(duì)病毒的mRNA進(jìn)行切割降解[45], 從而抑制病毒基因的表達(dá),起到防御病毒侵染的目的。

表 1 RNAi在三種病毒中的研究Tab. 1 Studies examining three viruses using RNAi in shrimp

綜上所述, RNAi作為一種對(duì)抗病毒入侵的新技術(shù)手段, 具有高效、完全、無(wú)毒的特點(diǎn), 在治療甲殼動(dòng)物病毒病的過(guò)程中發(fā)揮積極作用。但現(xiàn)有的研究尚處在理論階段, 如何針對(duì)不同的病毒開(kāi)發(fā)長(zhǎng)期性表達(dá)的特異RNAi體系, 并大規(guī)模應(yīng)用到甲殼動(dòng)物養(yǎng)殖中是當(dāng)務(wù)之急。

2.2 生長(zhǎng)發(fā)育

作為有力的分子生物學(xué)工具, RNAi技術(shù)被廣泛應(yīng)用在研究線蟲(chóng)、昆蟲(chóng)和脊椎動(dòng)物的生長(zhǎng)發(fā)育方面[46,47]。近來(lái)RNAi也在研究甲殼動(dòng)物生長(zhǎng)發(fā)育方面取得了長(zhǎng)足進(jìn)步。

肌肉生成抑制素(Myostatin, MSTN), 在無(wú)脊椎動(dòng)物中又稱生長(zhǎng)分化因子(Growth differentiation factor 11, GDF-11), 屬于轉(zhuǎn)化生長(zhǎng)因子-β(Ttransforming growth factor-β, TGF-β)家族, 是控制肌肉生長(zhǎng)的調(diào)控因子[48]。在日本沼蝦(Macrobrachium nipponense)中, MSTN/GDF-11基因在蛻皮的早期階段高豐度表達(dá), 而在隨后的蛻皮期表達(dá)量則逐漸下降, 表明MSTN/GDF-11基因參與了日本沼蝦的生長(zhǎng)發(fā)育過(guò)程[49]。在斑節(jié)對(duì)蝦中, 通過(guò)45d的長(zhǎng)期干擾MSTN/GDF-11基因, 結(jié)果顯示干擾組斑節(jié)對(duì)蝦的增重僅為對(duì)照組的32%[50]。在凡納濱對(duì)蝦中, 經(jīng)過(guò)8周的干擾, 增重率顯著低于對(duì)照組, 且出現(xiàn)較高的死亡率(71%)[51]。上述結(jié)果揭示MSTN/GDF-11基因可以促進(jìn)甲殼動(dòng)物的生長(zhǎng)發(fā)育。

表皮生長(zhǎng)因子受體(Epidermal Growth Factor Receptor, EGFR)是具有酪氨酸激酶活性的跨膜蛋白分子, 可以和配體結(jié)合啟動(dòng)胞內(nèi)信號(hào)傳導(dǎo)途徑,促進(jìn)細(xì)胞的分裂增殖, 從而調(diào)控生長(zhǎng)發(fā)育。羅氏沼蝦(Macrobrachium rosenbergii)的研究表明, 干擾Mr-EGFR基因可使羅氏沼蝦生長(zhǎng)變緩, 13周之后干擾實(shí)驗(yàn)組體重僅為對(duì)照組的37%[52], 表明Mr-EGFR是羅氏沼蝦重要的生長(zhǎng)調(diào)控因子。

Hox基因(Omeobox genes)在節(jié)肢動(dòng)物軀體發(fā)育過(guò)程發(fā)揮重要作用, 其表達(dá)具有嚴(yán)格的組織特異性和胚胎發(fā)育的程序性[53]。Spalt在不同的Hox同源異型盒充當(dāng)輔酶因子和受體的角色, 在不同的物種中, Spalt基因所體現(xiàn)的功能各不相同。Copf等[54]在鹵蟲(chóng)(Artemia saline)中用RNAi方法研究了Spalt基因的功能。結(jié)果顯示, 降低Spalt基因的表達(dá)可以導(dǎo)致鹵蟲(chóng)體節(jié)發(fā)育異常, 據(jù)此推測(cè)Spalt基因可能是Hox的調(diào)節(jié)因子之一。Hox同源異型盒中的Ubx基因在明鉤蝦(Parhyale hawaiensis)附肢的發(fā)育過(guò)程中發(fā)揮作用, 通過(guò)干擾減少Ubx基因的表達(dá)量可以導(dǎo)致附肢的表型發(fā)生變化[55], 表明Ubx基因很可能參與了明鉤蝦附肢的發(fā)育過(guò)程。另一個(gè)參與附肢發(fā)育的基因是Distal-less (Dll), Kato等[56]在水溞(Daphnia magna) Dll基因的不同位置構(gòu)建了兩條dsRNA, 分別注射這兩條dsRNA到水蚤的受精卵中,結(jié)果都導(dǎo)致Dll基因的表達(dá)量明顯下降, 并且孵化后的幼體都發(fā)現(xiàn)觸角變得更為鈍平。表明Dll基因參與了水蚤觸角的發(fā)育過(guò)程。RNAi在其他生長(zhǎng)發(fā)育相關(guān)基因的研究在表 2中列出。

動(dòng)物體的生長(zhǎng)發(fā)育是一個(gè)嚴(yán)謹(jǐn)而復(fù)雜的生物學(xué)過(guò)程, 多個(gè)基因和調(diào)控通路涉及其中。有關(guān)甲殼動(dòng)物生長(zhǎng)發(fā)育的分子機(jī)制, 雖然取得了一定的研究成果, 但已經(jīng)明顯落后于同屬節(jié)肢動(dòng)物門的昆蟲(chóng)綱。生長(zhǎng)發(fā)育作為甲殼動(dòng)物最基本的生物學(xué)過(guò)程,其中尚有許多關(guān)鍵環(huán)節(jié)等待研究人員借助RNAi和其他技術(shù)對(duì)其進(jìn)行解讀。

2.3 蛻皮

甲殼動(dòng)物由于獨(dú)特的身體構(gòu)造, 在個(gè)體發(fā)育過(guò)程中存在蛻皮現(xiàn)象。研究表明, 甲殼動(dòng)物的蛻皮受多種因素的參與, 蛻皮抑制激素(Molt Inhibiting Hormone, MIH)、蛻皮激素受體(Ecdysone receptor, EcR)、維甲酸X受體(Retinoid X receptor, RXR)及轉(zhuǎn)錄因子調(diào)控蛻殼響應(yīng)基因E75、幾丁質(zhì)酶均在其中發(fā)揮用[57,58]。

Pamuru等[59]在紅螯螯蝦(Cherax quadricarinatus)用dsRNA沉默Cq-MIH基因, 結(jié)果導(dǎo)致蛻皮周期加快, 蛻皮的間隔期也相應(yīng)縮短到原來(lái)的32%。江豐偉[60]通過(guò)連續(xù)6周干擾日本沼蝦的Mn-MIH基因, 顯著增加日本沼蝦的蛻皮頻次, 實(shí)驗(yàn)組雄蝦和雌蝦分別蛻皮17次、12次, 而對(duì)照組雄、雌蝦則僅為2次、5次。表明MIH是甲殼動(dòng)物蛻皮周期中的負(fù)調(diào)控因子。最近在藍(lán)蟹(Callinectes sapidus)中的研究表明, EcR正調(diào)控MIH基因的表達(dá), 是MIH基因上游調(diào)控因子[61]。Techa[62]在藍(lán)蟹中通過(guò)RNAi證實(shí), 蛻皮激素(Ecdysteroids, E)通過(guò)EcR刺激MIH基因的表達(dá), 證實(shí)甲殼動(dòng)物的蛻皮機(jī)制中存在“E→EcR→MIH”的信號(hào)通路。

RXR是節(jié)肢動(dòng)物在蛻皮過(guò)程中重要調(diào)控基因之一, RXR與EcR形成二聚體結(jié)構(gòu), 調(diào)控下游基因E75的表達(dá)。Priya等[63]注射RXR-dsRNA到中國(guó)對(duì)蝦幼蝦體內(nèi)導(dǎo)致下游的兩種幾丁質(zhì)酶(Chi和Chi-1)基因和E75基因的表達(dá)豐度下調(diào), 表明RXR也參與了甲殼動(dòng)物的蛻皮信號(hào)途徑。接著Priya等[64]通過(guò)持續(xù)注射沉默E75基因, 嚴(yán)重阻礙中國(guó)對(duì)蝦的蛻皮并最終導(dǎo)致其死亡, 說(shuō)明 E75基因與甲殼動(dòng)物的蛻皮調(diào)控密切相關(guān)且是其生長(zhǎng)過(guò)程中必需基因。最近在凡納濱對(duì)蝦中的研究表明, 干擾RXR、EcR和E75基因中任意一個(gè)都會(huì)引起其他兩個(gè)基因的表達(dá)量發(fā)生變化, 進(jìn)而會(huì)影響一系列的蛻皮和生長(zhǎng)相關(guān)基因的變化[65]。上述結(jié)果進(jìn)一步證實(shí)了RXR、EcR和E75基因調(diào)控甲殼動(dòng)物的蛻皮過(guò)程。

甲基法尼酯(Methyl farnesoate, MF)是甲殼動(dòng)物一種重要的生長(zhǎng)調(diào)控激素, 主要負(fù)責(zé)調(diào)節(jié)體節(jié)發(fā)育、生長(zhǎng)和生殖[66,67]。法尼酸甲基轉(zhuǎn)移酶(Farnesoic acid O-methyltransferase, FAMeT)在MF的形成過(guò)程中起著重要作用, 負(fù)責(zé)把法尼酸(Farnesoic acid, FA)轉(zhuǎn)化為MF。在凡納濱對(duì)蝦中, Hui等[68]研究了FAMeT基因在蛻皮中的作用, 通過(guò)注射dsRNA降低了FAMeT基因的表達(dá)量, 從而推遲了蛻皮, 而且與蛻皮相關(guān)的血藍(lán)蛋白基因和組織蛋白酶基因的表達(dá)量也會(huì)下降。最后注射dsRNA的實(shí)驗(yàn)組(因蛻皮阻滯)全部死亡, 而與之相對(duì)應(yīng)的對(duì)照組則無(wú)一死亡。這些結(jié)果表明, FAMeT基因在甲殼動(dòng)物的生長(zhǎng)和蛻皮周期中發(fā)揮作用。RNAi在其他蛻皮相關(guān)基因的研究在表 2中列出。

蛻皮是甲殼動(dòng)物生長(zhǎng)和發(fā)育的標(biāo)志性特征, 是一個(gè)復(fù)雜有序的生物學(xué)過(guò)程。它貫穿于甲殼動(dòng)物個(gè)體發(fā)育的始終, 受MIH信號(hào)通路、Ca2+信號(hào)通路以及NO信號(hào)通路的共同調(diào)節(jié)[57]。目前對(duì)蛻皮機(jī)制的認(rèn)識(shí)還相當(dāng)有限, 各信號(hào)通路直接是否存在關(guān)聯(lián),以及每條信號(hào)通路都存在哪些具體的調(diào)控因子, 這些都需要進(jìn)一步研究。

表 2 RNAi在三種病毒中的研究Tab. 2 Studies examining three viruses using RNAi in shrimp

2.4 生殖

甲殼動(dòng)物的生殖發(fā)育是神經(jīng)多肽、激素及多種環(huán)境因子綜合調(diào)控的結(jié)果。涉及性腺的發(fā)育、生殖細(xì)胞的發(fā)生和受精等多個(gè)過(guò)程。卵黃蛋白原(Vitellogenin, Vg)是卵生動(dòng)物雌性卵黃蛋白的前體, Vg的合成和積累對(duì)于雌性個(gè)體卵母細(xì)胞的發(fā)育和卵子的數(shù)量和質(zhì)量至關(guān)重要[100]; 卵黃蛋白原受體(Vitellogenin receptor, VgR)是低脂蛋白受體超家族中的一員, 在卵巢發(fā)育過(guò)程中與Vg結(jié)合, 起到受體介導(dǎo)的作用, 在胚胎成熟的過(guò)程中發(fā)揮作用。因此Vg和VgR就成為研究雌性水產(chǎn)動(dòng)物卵巢發(fā)育的重要靶標(biāo)基因。

甲殼動(dòng)物Vg的合成部位一直存在內(nèi)源性(卵巢)和外源性(肝臟)兩種觀點(diǎn)。Tiu等[73]在斑節(jié)對(duì)蝦和Bai等[74]在日本沼蝦中通過(guò)干擾VgR基因, 推測(cè)Vg可能是在肝臟中合成后運(yùn)輸?shù)铰殉仓械穆涯讣?xì)胞內(nèi)。而且Bai等[72,74]進(jìn)一步干擾了日本沼蝦的Vg和VgR基因發(fā)現(xiàn), 日本沼蝦的性腺指數(shù)明顯低于對(duì)照組, 表明Vg和VgR參與了甲殼動(dòng)物的性腺發(fā)育過(guò)程。

甲殼動(dòng)物眼柄中的X-器竇腺?gòu)?fù)合體(X-organsinus gland, XO-SG)合成并分泌甲殼動(dòng)物高血糖激素(Crustacean hyplyeemic hormone, CHH)家族激素,包括性腺抑制激素(Gonad inhibiting hormones, GIH)、CHH和MIH等多種神經(jīng)多肽激素, 調(diào)控甲殼動(dòng)物的糖代謝、蛻皮及生殖等多個(gè)生理過(guò)程[101,102]。Treerattrakool等[76]通過(guò)向馴養(yǎng)和野生的雌性斑節(jié)對(duì)蝦注射GIH-dsRNA, 結(jié)果導(dǎo)致卵巢發(fā)育提前, 干擾的馴養(yǎng)組和野生組產(chǎn)卵率分別為14%和63%, 而作為對(duì)照的馴養(yǎng)組和野生組產(chǎn)卵率則分別6%和20%,表明GIH抑制斑節(jié)對(duì)蝦卵巢的發(fā)育。已有多個(gè)研究證實(shí)(表 2), 沉默GIH[75,77,78]和MIH-B[79]可以提高Vg基因的表達(dá)水平, 表明GIH和MIH-B是卵巢發(fā)育的負(fù)調(diào)控因子。

此外, 核受體RXR和EcR作為信號(hào)通路的重要成員也在甲殼動(dòng)物性腺的發(fā)育過(guò)程中起調(diào)控作用。Gong等[80]在擬穴青蟹(Scylla paramamosain)中沉默EcR基因, Nagaraju等[81]在濱蟹(Carcinus maenas)中沉默RXR基因均發(fā)現(xiàn)Vg基因的表達(dá)量顯著下降, 表明EcR和RXR參與了卵巢的發(fā)育過(guò)程, 并且后者的研究進(jìn)一步表明MF和RXR組成復(fù)合物, 調(diào)控卵巢的發(fā)育。另有研究表明, 蝗抗利尿肽(Neuroparsin)基因[82]、腫瘤抑制因子P53基因[83]和熱激蛋白(Heat Shock Cognate 70, HSC70)[84]也在卵巢發(fā)育過(guò)程中發(fā)揮作用。

精子明膠酶(Spermgelatinase, SG)最先發(fā)現(xiàn)于雄性羅氏沼蝦的生殖道中, 是一種可以水解明膠的蛋白, 在受精過(guò)程中發(fā)揮作用。Yang等[85]在羅氏沼蝦中通過(guò)沉默SG基因, 發(fā)現(xiàn)羅氏沼蝦的精子在外形上未見(jiàn)異常, 但其明膠的水解活性明顯下降, 并推測(cè)SG在精子的蛋白水解活性方面起到了很重要的作用。Ma等[86]通過(guò)干擾壺腹多肽(Terminal ampullae peptide, TAP)降低了SG的活性, 進(jìn)一步的研究表明, TAP參與卵膜蛋白降解相關(guān)酶的活力調(diào)節(jié),但不影響精子入卵和受精過(guò)程。

迄今有關(guān)甲殼動(dòng)物生殖方面的研究主要集中在生殖細(xì)胞的發(fā)生、成熟及受精機(jī)制的形態(tài)觀察和生理生化分析水平上, 在分子水平上探討上述機(jī)制的報(bào)道還為數(shù)不多[103]。眾所周知, 甲殼動(dòng)物在養(yǎng)殖過(guò)程中會(huì)出現(xiàn)“性早熟”現(xiàn)象, 其中的分子調(diào)控機(jī)制還不甚明了。RNAi技術(shù)的應(yīng)用將有助于揭示甲殼動(dòng)物“性早熟”的發(fā)生機(jī)制, 進(jìn)而豐富甲殼動(dòng)物的生殖生物學(xué), 為建立高產(chǎn)、穩(wěn)產(chǎn)的甲殼動(dòng)物養(yǎng)殖體系奠定理論基礎(chǔ)。

2.5 性別調(diào)控

動(dòng)物的性別決定存在多種機(jī)制, 例如高等哺乳動(dòng)物的性別由性染色體決定, 而甲殼動(dòng)物缺乏相應(yīng)的性染色體, 與脊椎動(dòng)物相比, 其性別決定機(jī)制具有原始性和可塑性的特點(diǎn)。研究表明, 相關(guān)基因在甲殼動(dòng)物的性別分化過(guò)程中起決定作用。

Dmrt (Doble-sex and Mab-3 Relatated Transcription factor)是參與性別決定最古老的基因家族, Doublesex基因就屬于此家族。Kato等[87]在水溞中克隆得到兩種Dsx 基因DapmaDsx1和DapmaDsx2,其中DapmaDsx1編碼兩種不同的亞型DapmaDsx1-α和DapmaDsx1-β。DapmaDsx1展現(xiàn)出明顯的性別二態(tài)性表達(dá), 在雄性胚胎的形成過(guò)程中, DapmaD-sx1表達(dá)量明顯升高, 而在雌性胚胎中則沒(méi)有這種現(xiàn)象。進(jìn)一步在雄性胚胎中沉默DapmaDsx1, 可誘導(dǎo)其卵巢成熟, 產(chǎn)生雌性特征; 相反, 在雌性胚胎中異位表達(dá)DapmaDsx1, 可使其產(chǎn)生雄性特性。表明DapmaDsx1雄性性別決定中起著關(guān)鍵作用。因此, DapmaDsx1被認(rèn)為是水溞的性別決定基因[104]。

促雄腺(Androgenic gland, AG)是雄性甲殼動(dòng)物特有的內(nèi)分泌器官, 其分泌的胰島素樣促雄腺激素(Insulin-like androgenic gland hormone, IAG)迄今為止是唯一被證明直接參與了甲殼動(dòng)物性別分化的蛋白類激素。Ventura等[89]在成體羅氏沼蝦(變態(tài)后70—80天, 體重為0.25—1.6 g)中, 通過(guò)長(zhǎng)期(55d)干擾IAG基因抑制了雄性性征的發(fā)展。Rosen等[88]在紅螯螯蝦中, 通過(guò)干擾沉默IAG基因, 使雄性個(gè)體出現(xiàn)了雌性化特征、精子發(fā)生受到抑制、Vg基因開(kāi)始表達(dá)、卵母細(xì)胞中的卵黃大量積累等現(xiàn)象。以上結(jié)果表明, IAG基因在甲殼動(dòng)物的性別分化方面發(fā)揮重要作用。2012年在甲殼動(dòng)物性別分化方面取得了突破性進(jìn)展, Ventura等[90]通過(guò)長(zhǎng)時(shí)間干擾雄性羅氏沼蝦幼體(變態(tài)后30d, 體重30—70 mg), 獲得了完全性逆轉(zhuǎn)的“新雌蝦”。隨后應(yīng)用“新雌蝦”與正常的雄蝦交配產(chǎn)生了全雄的后代[91], 從而實(shí)現(xiàn)了羅氏沼蝦的單性化養(yǎng)殖。

近來(lái)RNAi技術(shù)也用在研究IAG基因的調(diào)控機(jī)制方面。我們前期通過(guò)RNAi技術(shù)在日本沼蝦中論證了IAG和胰島素樣促雄腺激素結(jié)合蛋白(Insulinlike androgenic gland hormone binding protein, IAGBP)存在彼此的調(diào)控關(guān)系[92]; 而且我們進(jìn)一步確認(rèn)MIH和GIH是IAG基因的負(fù)調(diào)控因子[105]。在羅氏沼蝦中, Yu等[106]通過(guò)RNAi確認(rèn)Dmrt11E基因是IAG基因的正調(diào)控因子。

羅氏沼蝦的干擾實(shí)驗(yàn)標(biāo)志著第一次通過(guò)沉默一個(gè)基因而實(shí)現(xiàn)甲殼動(dòng)物的單性化養(yǎng)殖。在此過(guò)程中沒(méi)有改變機(jī)體的基因序列, 因此具有廣闊的應(yīng)用前景[107]。

2.6 滲透壓調(diào)節(jié)

甲殼動(dòng)物多廣鹽性物種, 鹽度的變化必然會(huì)引起血糖濃度、滲透壓發(fā)生改變等一系列生理調(diào)控過(guò)程, 神經(jīng)多肽激素便在其中發(fā)揮作用。Lugo等[93]將CHH-dsRNA通過(guò)腹部淋巴腺注射進(jìn)入南方濱對(duì)蝦(Litopenaeus schmitti)體內(nèi), 24h之內(nèi)沒(méi)有檢測(cè)到CHH-mRNA, 其血淋巴內(nèi)的血糖水平也發(fā)生了下降, 表明CHH可使血糖升高, 從而調(diào)節(jié)甲殼動(dòng)物的滲透壓。Manfrin等[94]在克氏原螯蝦中通過(guò)長(zhǎng)時(shí)間干擾CHH基因, 實(shí)驗(yàn)組克氏原螯蝦的死亡率達(dá)到47%, 并且死亡個(gè)體呈現(xiàn)明顯的滲透壓失調(diào)癥狀(頭胸甲和腹部出現(xiàn)明顯的分離)。此外大劑量干擾離子轉(zhuǎn)運(yùn)肽(Ion transport peptide, ITP)[98]和Na+/K+-ATPase (NKA)[99]基因可使實(shí)驗(yàn)蝦出現(xiàn)死亡, 表明上述兩個(gè)基因也在調(diào)控滲透壓方面發(fā)揮各自的作用。

我國(guó)幅員遼闊, 鹽堿地區(qū)眾多, 這些地區(qū)多不適宜農(nóng)作物種植, 因此荒廢。對(duì)鹽堿地的水質(zhì)、離子成分進(jìn)行分析, 針對(duì)具體物種的滲透壓調(diào)節(jié)機(jī)制,必要時(shí)補(bǔ)充相關(guān)離子, 進(jìn)行水產(chǎn)養(yǎng)殖可以提高鹽堿地區(qū)的經(jīng)濟(jì)效益。因此闡明甲殼動(dòng)物的滲透壓調(diào)節(jié)機(jī)制, 就顯得尤為關(guān)鍵。

2.7 代謝

缺氧誘導(dǎo)因子-1 (Hypoxia inducible factor 1, HIF-1)是由α和β兩個(gè)亞基組成的異源蛋白二聚體,作為轉(zhuǎn)錄因子, HIF-1調(diào)控缺氧應(yīng)激下的多種生理活動(dòng)。So?anez等[95]在缺氧條件下分別干擾凡納濱對(duì)蝦HIF-1-α和HIF-1-β, 結(jié)果顯示血淋巴中葡萄糖的濃度出現(xiàn)明顯變化; 繼續(xù)干擾發(fā)現(xiàn), HIF-1通過(guò)己糖激酶(Hexokinase, HK)[96]、磷酸果糖激酶(Phosphofructokinase, PFK)和果糖-1, 6-二磷酸酶(Fructose -1, 6-bisphosphatase, FBP)[97]途徑來(lái)調(diào)控葡萄糖的變化。

目前為止, 有關(guān)甲殼動(dòng)物代謝途徑中相關(guān)基因的研究還偏少, 作為基礎(chǔ)的代謝過(guò)程, 應(yīng)用RNAi技術(shù)開(kāi)展此方面的研究有助于全面解析甲殼動(dòng)物的營(yíng)養(yǎng)代謝途徑。

3 展望

技術(shù)優(yōu)勢(shì): 基因組編輯技術(shù)(ZFN, TALEN, CRISPR/Cas9h)和RNAi作為研究基因功能的兩種有力工具, 已經(jīng)在水產(chǎn)動(dòng)物中得以廣泛運(yùn)用?；蚪M編輯技術(shù)的操作對(duì)象是基因組DNA, 且主要是通過(guò)顯微注射對(duì)細(xì)胞系或胚胎進(jìn)行操作。而目前為止, 甲殼動(dòng)物還缺乏成熟的細(xì)胞系構(gòu)建體系, 且卵殼較硬易碎。因此, 有關(guān)應(yīng)用基因組編輯技術(shù)研究甲殼動(dòng)物基因功能的文章還鮮見(jiàn)報(bào)道。雖然基因組編輯技術(shù)有望成為研究基因功能的主流方法, 但在上述問(wèn)題未解決之前, RNAi仍然在甲殼動(dòng)物中發(fā)揮自己獨(dú)特的技術(shù)優(yōu)勢(shì)。從技術(shù)層面分析, RNAi在甲殼動(dòng)物中也存在脫靶和效應(yīng)劑量等方面的問(wèn)題。但這些問(wèn)題可以通過(guò)對(duì)不同靶點(diǎn)和不同劑量dsRNA的篩選來(lái)解決。而且相比脊椎動(dòng)物, 甲殼動(dòng)物體型較小, dsRNA注射用量也較小, 現(xiàn)有成熟的RNAi試劑盒完全可以滿足實(shí)驗(yàn)要求, 因此成本較低。作為內(nèi)生性的調(diào)控機(jī)制, 人工合成的dsRNA可以持續(xù)在甲殼動(dòng)物體內(nèi)發(fā)揮作用, 如研究人員發(fā)現(xiàn)GIH-dsRNA在斑節(jié)對(duì)蝦體內(nèi)發(fā)揮作用的有效時(shí)間最少可以達(dá)到30天[76]。注射法是目前甲殼動(dòng)物RNAi的主要轉(zhuǎn)染方式, 近來(lái)在探索RNAi的其他轉(zhuǎn)染途徑方面也取得了突破性進(jìn)展。在斑節(jié)對(duì)蝦中, 研究人員將GIH-dsRNA構(gòu)建到大腸桿菌中, 然后用鹵蟲(chóng)濾食大腸桿菌從而“富集”GIH-dsRNA, 最后將鹵蟲(chóng)飼喂斑節(jié)對(duì)蝦可有效降低GIH基因的表達(dá)水平[108]。這種方法簡(jiǎn)單有效, 更為重要的是, 對(duì)于不適用注射法的甲殼動(dòng)物幼體來(lái)說(shuō), 鹵蟲(chóng)飼喂法介導(dǎo)的RNAi為研究甲殼動(dòng)物幼體階段相關(guān)基因的功能開(kāi)辟了一條新路。因此可以肯定的是, RNAi作為一項(xiàng)成熟的技術(shù), 在未來(lái)一段時(shí)間內(nèi)還將是研究甲殼動(dòng)物基因功能的熱門工具。

免疫機(jī)制研究: RNAi作為一種抵抗外源病毒的重要天然免疫反應(yīng), 迄今為止, 甲殼動(dòng)物在免疫過(guò)程中的具體分子機(jī)制還不完全清楚。在昆蟲(chóng)中,病毒誘導(dǎo)的siRNA和miRNA是獨(dú)立生成的, 各自在免疫系統(tǒng)中發(fā)揮作用[45,109]。已有研究表明, 甲殼動(dòng)物的miRNA也在抵御病毒入侵的過(guò)程發(fā)揮作用[110,111]。那么, 甲殼動(dòng)物是否和昆蟲(chóng)一樣也存在類似的獨(dú)立作用機(jī)制, 還需進(jìn)一步探索。上述問(wèn)題的闡明, 將有助于揭開(kāi)當(dāng)下危害甲殼動(dòng)物養(yǎng)殖過(guò)程中出現(xiàn)的病毒性疾病的發(fā)病原理, 進(jìn)而開(kāi)發(fā)高效實(shí)用的防治藥物, 服務(wù)于水產(chǎn)養(yǎng)殖業(yè)。

基因功能與信號(hào)通路: 甲殼動(dòng)物的各個(gè)生物學(xué)過(guò)程從來(lái)都不是獨(dú)立存在的, 如甲殼動(dòng)物的生長(zhǎng)和蛻皮總是相輔相成的, 甲殼動(dòng)物有機(jī)體的生長(zhǎng)導(dǎo)致蛻皮, 蛻皮又可以促進(jìn)甲殼動(dòng)物的生長(zhǎng); 再如, 甲殼動(dòng)物“性早熟”也與蛻皮密切相關(guān), 原因在于早期幼體性腺發(fā)育過(guò)快, 蛻皮次數(shù)過(guò)頻。因此, 性腺的發(fā)育與蛻皮機(jī)制必然存在交叉點(diǎn)。換而言之, 甲殼動(dòng)物的各個(gè)基因調(diào)控通路相互連接, 形成一個(gè)復(fù)雜的調(diào)控網(wǎng)絡(luò)。因此有關(guān)甲殼動(dòng)物基因組學(xué)的研究勢(shì)必將從單個(gè)基因功能的確定過(guò)渡到信號(hào)通路的研究。如前文所述, 基因測(cè)序技術(shù)的發(fā)展為我們獲取大規(guī)模的基因信息提供了有力的技術(shù)保障。RNAi的技術(shù)特點(diǎn)使它可以精確地靶向敲降目的基因, 進(jìn)而可以檢測(cè)出下游基因表達(dá)量的變化, 在轉(zhuǎn)錄水平為信號(hào)通路的研究提供基本的數(shù)據(jù), 進(jìn)而通過(guò)相關(guān)的技術(shù)手段解讀其調(diào)控網(wǎng)絡(luò)。

綜上所述, 作為一項(xiàng)快速發(fā)展并取得廣泛應(yīng)用的技術(shù), RNAi在甲殼動(dòng)物研究中已經(jīng)展示了廣闊的用途和前景, 由此我們相信隨著對(duì)甲殼動(dòng)物RNAi信號(hào)傳遞機(jī)制的深入研究, 必將有助于全面闡釋甲殼動(dòng)物的基因功能及其調(diào)控網(wǎng)絡(luò), 進(jìn)而推動(dòng)甲殼動(dòng)物基因組學(xué)的研究, 同時(shí)也可為甲殼動(dòng)物的健康養(yǎng)殖提供更多的理論指導(dǎo), 從而實(shí)現(xiàn)理論研究和生產(chǎn)應(yīng)用的統(tǒng)一。

[1]Song L, Bian C, Luo Y, et al. Draft genome of the Chinese mitten crab, Eriocheir sinensis [J]. Giga Science, 2016, 5(1): 1—3

[2]Guo S, Kemphues K J. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed [J]. Cell, 1995, 81(4): 611—620

[3]Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J]. Nature, 1998, 391(6669): 806—811

[4]Wang P H, Weng S P, He J G. Nucleic acid-induced antiviral immunity in invertebrates: An evolutionary perspective [J]. Developmental & Comparative Immunology, 2015, 48(2): 291—296

[5]He Y, Ju C, Zhang X. Roles of small RNAs in the immune defense mechanisms of crustaceans [J]. Molecular Immunology, 2015, 68(2): 399—403

[6]Robalino J, Browdy C L, Prior S, et al. Induction of antiviral immunity by double-stranded RNA in a marine invertebrate [J]. Journal of Virology, 2004, 78(19): 10442—10448

[7]Escobedo Bonilla C M. Application of RNA interference (RNAi) against viral infections in shrimp: A review [J]. Journal of Antivirals & Antiretrovirals, 2013, 5(3): 1—12

[8]Seok S H, Park J H, Cho S A, et al. Cloning and sequencing of envelope proteins (VP19, VP28) and nucleocapsid proteins (VP15, VP35) of a white spot syndrome virus isolate from Korean shrimp [J]. Diseases of Aquatic Organisms, 2004, 60(1): 85—88

[9]Sindhupriya M, Saravanan P, Otta S, et al. White spot syndrome virus (WSSV) genome stability maintained over six passages through three different penaeid shrimp species [J]. Diseases of Aquatic Organisms, 2014, 111(1): 23—29

[10]Kulkarni A, Rombout J, Singh I, et al. Truncated VP28 as oral vaccine candidate against WSSV infection in shrimp: an uptake and processing study in the midgut of Penaeus monodon [J]. Fish & Shellfish Immunology, 2013, 34(1): 159—166

[11]Kim C S, Kosuke Z, Nam Y K, et al. Protection of shrimp (Penaeus chinensis) against white spot syndrome virus (WSSV) challenge by double-stranded RNA [J]. Fish & Shellfish Immunology, 2007, 23(1): 242—246

[12]Xu J, Han F, Zhang X. Silencing shrimp white spot syndrome virus (WSSV) genes by siRNA [J]. Antiviral Research, 2007, 73(2): 126—131

[13]Thammasorn T, Sangsuriya P, Meemetta W, et al. Large-scale production and antiviral efficacy of multi target double stranded RNA for the prevention of white spot syndrome virus (WSSV) in shrimp [J]. Bmc Biotechnology, 2015, 15(1): 1—7

[14]Mouillesseaux K P, Klimpel K R, Dhar A K. Improvement in the specificity and sensitivity of detection for the Taura syndrome virus and yellow head virus of penaeid shrimp by increasing the amplicon size in SYBR Green real time RT-PCR [J]. Journal of Virological Methods, 2003, 111(2): 121—127

[15]Weili Y, Sihua Z, Zhiqin Y, et al. Development of a Liquid Chip Technique to Simultaneously Detect Taura Syndrome Virus (TSV) and Yellow Head Disease Virus (YHDV) [J]. Animal Husbandry and Feed Science, 2014, 6(5): 256—260

[16]Tirasophon W, Roshorm Y, Panyim S. Silencing of yellow head virus replication in penaeid shrimp cells by dsRNA q [J]. Biochemical and Biophysical Research Communications, 2005, 334(1): 102—107

[17]Sritunyalucksana K, Wannapapho W, Lo C F, et al. PmRab7 is a VP28-binding protein involved in white spot syndrome virus infection in shrimp [J]. Journal of Virology, 2006, 80(21): 10734—10742

[18]Ongvarrasopone C, Chanasakulniyom M, Sritunyalucksana K, et al. Suppression of PmRab7 by dsRNA inhibits WSSV or YHV infection in shrimp [J]. Marine Biotechnology, 2008, 10(4): 374—381

[19]Ongvarrasopone C, Saejia P, Chanasakulniyom M, et al. Inhibition of Taura syndrome virus replication in Litopenaeus vannamei through silencing the LvRab7 gene using double-stranded RNA [J]. Archives of Virology, 2011, 156(7): 1117—1123

[20]Robalino J, Bartlett T, Shepard E, et al. Double-stranded RNA induces sequence-specific antiviral silencing in addition to nonspecific immunity in a marine shrimp: convergence of RNA interference and innate immunity in the invertebrate antiviral response [J]？Journal of Virology, 2005, 79(21): 13561—13571

[21]Yue W, Ling L, Yang L S, et al. Inhibition of white spot syndrome virus in Litopenaeus vannamei shrimp by sequence-specific siRNA [J]. Aquaculture, 2007, 271(4): 21—30

[22]Mejía C H, Vega S, Alvarez P, et al. Double-stranded RNA against white spot syndrome virus (WSSV) vp28 or vp26 reduced susceptibility of Litopenaeus vannamei to WSSV, and survivors exhibited decreased susceptibility in subsequent reinfections [J]. Journal of Invertebrate Pathology, 2011, 107(1): 65—68

[23]Rijiravanich A, Browdy C L, Withyachumnarnkul B. Knocking down caspase-3 by RNAi reduces mortality in Pacific white shrimp Penaeus Litopenaeus vannamei challenged with a low dose of white spot syndrome virus [J]. Fish & Shellfish Immunology, 2008, 24(3): 308—313

[24]Wang K H C, Tseng C W, Lin H Y, et al. RNAi knock down of the Litopenaeus vannamei Toll gene (LvToll) significantly increases mortality and reduces bacterial clearance after challenge with Vibrio harveyi [J]. Developmental & Comparative Immunology, 2010, 34(1): 49—58

[25]Westenberg M, Heinhuis B, Zuidema D, et al. siRNA injection induces sequence-independent protection in Penaeus monodon against white spot syndrome virus [J]. Virus Research, 2005, 114(1): 133—139

[26]Sarathi M, Simon M C, Venkatesan C, et al. Oral administration of bacterially expressed VP28dsRNA to protect Penaeus monodon from white spot syndrome virus [J]. Marine Biotechnology, 2008, 10(3): 242—249

[27]He F, Syed S M, Hameed A S, et al. Viral ubiquitin ligase WSSV222 is required for efficient white spot syndrome virus replication in shrimp [J]. Journal of General Virology, 2009, 90(6): 1483—1490

[28]Sarathi M, Simon M, Venkatesan C, et al. Efficacy of bacterially expressed dsRNA specific to different structural genes of white spot syndrome virus (WSSV) in protection of shrimp from WSSV infection [J]. Journal of Fish Diseases, 2010, 33(7): 603—607

[29]Attasart P, Kaewkhaw R, Chimwai C, et al. Inhibition of white spot syndrome virus replication in Penaeus monodon by combined silencing of viral rr2 and shrimp PmRab7 [J]. Virus Research, 2009, 145(1): 127—133

[30]Kulkarni A D, Caipang C M, Kiron V, et al. Evaluation of immune and apoptosis related gene responses using an RNAi approach in vaccinated Penaeus monodon during oral WSSV infection [J]. Marine Genomics, 2014, 18: 55—65

[31]Zhu F, Zhang X. The antiviral vp28-siRNA expressed in bacteria protects shrimp against white spot syndrome virus (WSSV) [J]. Aquaculture, 2011, 319(3): 311—314

[32]Sudhakaran R, Mekata T, Kono T, et al. Double stranded RNA mediated silencing of the white spot syndrome virus VP28 gene in kuruma shrimp, Marsupenaeus japonicus [J]. Aquaculture Research, 2011, 42(8): 1153—1162

[33]Ye T, Zong R, Zhang X. Involvement of interaction between viral VP466 and host tropomyosin proteins in virus infection in shrimp [J]. Gene, 2012, 505(2): 254—258

[34]Alenton R R R, Kondo H, Hirono I, et al. Gene silencing of VP9 gene impairs WSSV infectivity on Macrobrachium rosenbergii [J]. Virus Research, 2016, 214: 65—70

[35]Heng Z, Li G, Feng Y. RNA interference on vp15 gene of shrimp white spot syndrome virus (WSSV) [J]. Journal of Oceanography in Taiwan Strait, 2012, 31(1): 47—52

[36]Tirasophona W, Yodmuanga S, Chinnirunvonga W, et al. Therapeutic inhibition of yellow head virus multiplication in infected shrimps by YHV-protease dsRNA [J]. Antiviral Research, 2007, 74: 150—155

[37]Yodmuang S, Tirasophon W, Roshorm Y, et al. YHV-protease dsRNA inhibits YHV replication in Penaeus monodon and prevents mortality [J]. Biochemical and Biophysical Research Communications, 2006, 341(2): 351—356

[38]Posiri P, Ongvarrasopone C, Panyim S. Improved preventive and curative effects of YHV infection in Penaeus monodon by a combination of two double stranded RNAs [J]. Aquaculture, 2011, 314(1): 34—38

[39]Jatuyosporn T, Supungul P, Tassanakajon A, et al. The essential role of clathrin mediated endocytosis in yellow head virus propagation in the black tiger shrimp Penaeus monodon [J]. Developmental & Comparative Immunology, 2014, 44(1): 100—110

[40]Senapin S, Phiwsaiya K, Anantasomboon G, et al. Knocking down a Taura syndrome virus (TSV) binding protein Lamr is lethal for the whiteleg shrimp Penaeus vannamei [J]. Fish & Shellfish Immunology, 2010, 29(3): 422—429

[41]Yao X, Wang L, Song L, et al. A Dicer-1 gene from white shrimp Litopenaeus vannamei: expression pattern in the processes of immune response and larval development [J]. Fish & Shellfish Immunology, 2010, 29(4): 565—570

[42]Chen Y H, Jia X T, Zhao L, et al. Identification and functional characterization of Dicer2 and five single VWC domain proteins of Litopenaeus vannamei [J]. Developmental & Comparative Immunology, 2011,35(6): 661—671

[43]Chen Y H, Zhao L, Jia X T, et al. Isolation and characterization of cDNAs encoding Ars2 and Pasha homologues, two components of the RNA interference pathway in Litopenaeus vannamei [J]. Fish & Shellfish Immunology, 2012, 32(2): 373—380

[44]Wang S, Chen A J, Shi L J, et al. TRBP and eIF6 homologue in Marsupenaeus japonicus play crucial roles in antiviral response [J]. Plos One, 2012, 7(1): e30057

[45]Labreuche Y, Warr G W. Insights into the antiviral functions of the RNAi machinery in penaeid shrimp [J]. Fish & Shellfish Immunology, 2013, 34(4): 1002—1010

[46]Xiao R, Chun L, Ronan E A, et al. RNAi interrogation of dietary modulation of development, metabolism, behavior, and aging in C. elegans [J]. Cell Reports, 2015, 11(7): 1123—1133

[47]Chikate Y R, Dawkar V V, Barbole R S, et al. RNAi of selected candidate genes interrupts growth and development of Helicoverpa armigera [J]. Pesticide Biochemistry and Physiology, 2016, DOI: 10.1016/j.pestbp. 2016.03.006

[48]Bhassu S, Maningas M B, Othman R Y. Myostatin: a potential growth regulating gene in giant river prawn, Macrobrachium rosenbergii [J]. Journal of the World Aquaculture Society, 2015, 46(6): 624—634

[49]Shen W, Ren G, Zhu Y, et al. Characterization of MSTN/GDF11 gene from shrimp Macrobrachium nipponense and its expression profiles during molt cycle and after eyestalk ablation [J]. Genes & Genomics, 2015, 37(5): 441—449

[50]De Santis C, Wade N M, Jerry D R, et al. Growing backwards: an inverted role for the shrimp ortholog of vertebrate myostatin and GDF11 [J]. Journal of Experimental Biology, 2011, 214(16): 2671—2677

[51]Lee J H, Momani J, Kim Y M, et al. Effective RNA silencing strategy of Lv-MSTN/GDF11 gene and its effects on the growth in shrimp, Litopenaeus vannamei [J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2015, 179: 9—16

[52]Sharabi O, Ventura T, Manor R, et al. Epidermal growth factor receptor in the prawn Macrobrachium rosenbergii: Function and putative signaling cascade [J]. Endocrinology, 2013, 154(9): 3188—3196

[53]Hughes C L, Kaufman T C. Hox genes and the evolution of the arthropod body plan [J]. Evolution & Development, 2002, 4(6): 459—499

[54]Copf T, Rabet N, Averof M. Knockdown of spalt function by RNAi causes derepression of Hox genes and homeotic transformations in the crustacean Artemia franciscana [J]. Developmental Biology, 2006, 298(1): 87—94

[55]Liubicich D M, Serano J M, Pavlopoulos A, et al. Knockdown of Parhyale Ultrabithorax recapitulates evolutionary changes in crustacean appendage morphology [J]. Proceedings of the National Academy of Sciences, 2009, 106(33): 13892—13896

[56]Kato Y, Shiga Y, Kobayashi K, et al. Development of an RNA interference method in the cladoceran crustacean Daphnia magna [J]. Development Genes and Evolution, 2011, 220(12): 337—345

[57]Chang E S, Mykles D L. Regulation of crustacean molting: a review and our perspectives [J]. General and Comparative Endocrinology, 2011, 172(3): 323—330

[58]Shen H, Zhou X, Bai A, et al. Ecdysone receptor gene from the freshwater prawn Macrobrachium nipponense: identification of different splice variants and sexually dimorphic expression, fluctuation of expression in the molt cycle and effect of eyestalk ablation [J]. General and Comparative Endocrinology, 2013, 193(1): 86—94

[59]Pamuru R R, Rosen O, Manor R, et al. Stimulation of molt by RNA interference of the molt inhibiting hormone in the crayfish Cherax quadricarinatus [J]. General and Comparative Endocrinology, 2012, 178(2): 227—236

[60]Jiang F W. The study of RNAi technology and its application in function study of molt-inhibiting hormone gene (MIH) in Macrobrachium nipponense [D]. Nanjing Agriculture University. Nanjing. 2014 [江豐偉. 青蝦RNAi技術(shù)的研究及其在蛻皮抑制激素(MIH)基因功能研究中的應(yīng)用. 南京農(nóng)業(yè)大學(xué), 南京. 2014]

[61]Techa S, Chung J S. Ecdysteroids regulate the levels of molt inhibiting hormone (MIH) expression in the Blue Crab, Callinectes sapidus [J]. Plos One, 2015, 10(4): e0117278

[62]Techa S. The functional importance and significance of ecdysteroids in molt cycle regulation of the blue crab, Callinectes sapidus [D]. Maryland, The University of Maryland. 2014

[63]Priya T J, Li F, Zhang J, et al. Molecular characterization and effect of RNA interference of retinoid X receptor (RXR) on E75 and chitinase gene expression in Chinese shrimp Fenneropenaeus chinensis [J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2009, 153(1): 121—129

[64]Priya T, Li F, Zhang J, et al. Molecular characterization of an ecdysone inducible gene E75 of Chinese shrimp Fenneropenaeus chinensis and elucidation of its role in molting by RNA interference [J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2010, 156(3): 149—157

[65]Qian Z, He S, Liu T, et al. Identification of ecdysteroid signaling late response genes from different tissues of the Pacific white shrimp, Litopenaeus vannamei [J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2014, 172(6): 10—30

[66]Wen D, Rivera-Perez C, Abdou M, et al. Methyl farnesoate plays a dual role in regulating Drosophila metamorphosis [J]. Plos Genet, 2015, 11(3): e1005038

[67]Xie X, Tao T, Liu M, et al. The potential role of juvenile hormone acid methyltransferase in methyl farnesoate (MF) biosynthesis in the swimming crab, Portunus trituberculatus [J]. Animal Reproduction Science, 2016, 168: 40—49

[68]Hui J H L, Tobe S S, Chan S M. Characterization of the putative farnesoic acid methyltransferase (Lv-FAMeT) cDNA from white shrimp, Litopenaeus vannamei: Evidence for its role in molting [J]. Peptides, 2008, 29(2): 252—260

[69]Das S, Durica D S. Ecdysteroid receptor signaling disruption obstructs blastemal cell proliferation during limb regeneration in the fiddler crab, Uca pugilator [J]. Molecular and Cellular Endocrinology, 2013, 365(2): 249—259

[70]Peng T, Wang D, Yu Y, et al. Identification and expression of an ecdysteroid-responsive amylase from red crayfish Procambarus clarkii [J]. Fisheries science, 2015, 81(2): 345—352

[71]Shen H, Hu Y, Zhang Y, et al. Calcium calmodulin dependent protein kinase I from Macrobrachium nipponense: cDNA cloning and involvement in molting [J]. Gene, 2014, 538(2): 235—243

[72]Bai H, Qiao H, Li F, et al. Molecular characterization and developmental expression of vitellogenin in the oriental river prawn Macrobrachium nipponense and the effects of RNA interference and eyestalk ablation on ovarian maturation [J]. Gene, 2015, 562(1): 22—31

[73]Tiu S H K, Benzie J, Chan S M. From hepatopancreas to ovary: molecular characterization of a shrimp vitellogenin receptor involved in the processing of vitellogenin [J]. Biology of Reproduction, 2008, 79(1): 66—74

[74]Bai H, Qiao H, Li F, et al. Molecular and functional characterization of the vitellogenin receptor in oriental river prawn, Macrobrachium nipponense [J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2016, 194: 45—55

[75]Treerattrakool S, Panyim S, Chan S M, et al. Molecular characterization of gonad-inhibiting hormone of Penaeus monodon and elucidation of its inhibitory role in vitellogenin expression by RNA interference [J]. FEBS Journal, 2008, 275(5): 970—980

[76]Treerattrakool S, Panyim S, Udomkit A. Induction of ovarian maturation and spawning in Penaeus monodon broodstock by double-stranded RNA [J]. Marine Biotechnology, 2011, 13(2): 163—169

[77]So K Y. Gene organization of the lobster (Homarus americanus) Gonad inhibiting hormone, and its functional analysis in relation to vitellogenesis byRNA interference [D]. the University of Hong Kong, Hong Kong. 2008

[78]Feijó R G, Braga A L, Lanes C F, et al. Silencing of gonad inhibiting hormone transcripts in Litopenaeus vannamei females by use of the RNA interference technology [J]. Marine Biotechnology, 2016, 18(1): 117—123

[79]Tiu S H K, Chan S M. The use of recombinant protein and RNA interference approaches to study the reproductive functions of a gonad stimulating hormone from the shrimp Metapenaeus ensis [J]. FEBS Journal, 2007, 274(17): 4385—4395

[80]Gong J, Ye H, Xie Y, et al. Ecdysone receptor in the mud crab Scylla paramamosain: a possible role in promoting ovarian development [J]. Journal of Endocrinology, 2015, 224(3): 273—287

[81]Nagaraju G P C, Rajitha B, Borst D W. Molecular cloning and sequence of retinoid X receptor in the green crab Carcinus maenas: a possible role in female reproduction [J]. Journal of Endocrinology, 2011, 210(3): 379—390

[82]Yang S P, He J G, Sun C B, et al. Characterization of the shrimp neuroparsin (MeNPLP): RNAi silencing resulted in inhibition of vitellogenesis [J]. FEBS Open Bio, 2014, 4(1): 976—986

[83]Dai W, Qiu L, Zhao C, et al. Characterization, expression and silencing by RNAi of p53 from Penaeus monodon [J]. Molecular Biology Reports, 2016, 43(6): 549—561

[84]Chan S F, He J G, Chu K H, et al. The shrimp heat shock cognate 70 functions as a negative regulator in vitellogenin gene expression [J]. Biology of Reproduction, 2014, 91(1): 14

[85]Yang F, Qian Y, Ma W, et al. MSG is involved in sperm gelatinolytic activity in the prawn, Macrobrachium rosenbergii [J]. Chinese Science Bulletin, 2013, 58(18): 2113—2118

[86]Ma W M, Qian Y Q, Wang M R, et al. A novel terminal ampullae peptide is involved in the proteolytic activity of sperm in the prawn, Macrobrachium rosenbergii [J]. Reproduction, 2010, 140(2): 235—245

[87]Kato Y, Kobayashi K, Watanabe H, et al. Environmental sex determination in the branchiopod crustacean Daphnia magna: deep conservation of a Doublesex gene in the sex determining pathway [J]. Plos Genet, 2011, 7(3): e1001345

[88]Rosen O, Manor R, Weil S, et al. A sexual shift in-duced by silencing of a single insulin like gene in crayfish: ovarian upregulation and testicular degeneration [J]. Plos One, 2010, 5(12): e15281

[89]Ventura T, Manor R, Aflalo E D, et al. Expression of an androgenic gland specific insulin like peptide during the course of prawn sexual and morphotypic differentiation [J]. ISRN Endocrinology, 2011, 476283: 1—13

[90]Ventura T, Manor R, Aflalo E, et al. Timing sexual differentiation: full functional sex reversal achieved in Macrobrachium rosenbergii through silencing of a single insulin like gene [J]. Biology of Reproduction, 2012, 86(3): 81—89

[91]Lezer Y, Aflalo E D, Manor R, et al. On the safety of RNAi usage in aquaculture: The case of all male prawn stocks generated through manipulation of the insulin like androgenic gland hormone [J]. Aquaculture, 2015, 435: 157—166

[92]Li F, Bai H, Xiong Y, et al. Molecular characterization of insulin like androgenic gland hormone-binding protein gene from the oriental river prawn Macrobrachium nipponense and investigation of its transcriptional relationship with the insulin like androgenic gland hormone gene [J]. General and Comparative Endocrinology, 2015, 216: 152—160

[93]Lugo J M, Morera Y, Rodríguez T, et al. Molecular cloning and characterization of the crustacean hyperglycemic hormone cDNA from Litopenaeus schmitti [J]. FEBS Journal, 2006, 273(24): 5669—5677

[94]Manfrin C, Peruzza L, Bonzi L, et al. Silencing two main isoforms of crustacean hyperglycemic hormone (CHH) induces compensatory expression of two CHH like transcripts in the red swamp crayfish Procambarus clarkii [J]. Invertebrate Survival Journal, 2015, 12(17): 29—37

[95]So?anez J G, Racotta I S, Yepiz G. Silencing of the hypoxia inducible factor 1(HIF-1) obliterates the effects of hypoxia on glucose and lactate concentrations in a tissue-specific manner in the shrimp Litopenaeus vannamei [J]. Journal of Experimental Marine Biology and Ecology, 2010, 393(1): 51—58

[96]So?anez-Organis J G, Peregrino-Uriarte A B, Sotelo-Mundo R R, et al. Hexokinase from the white shrimp Litopenaeus vannamei: cDNA sequence, structural protein model and regulation via HIF-1 in response to hypoxia [J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2011, 158(3): 242—249

[97]Cota-Ruiz K, Leyva-Carrillo L, Peregrino-Uriarte A B, et al. Role of HIF-1 on phosphofructokinase and fructose 1, 6 bisphosphatase expression during hypoxia in the white shrimp Litopenaeus vannamei [J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2016, 198: 1—7

[98]Tiu S H, He J G, Chan S M. The LvCHH-ITP gene of the shrimp (Litopenaeus vannamei) produces a widely expressed putative ion transport peptide (LvITP) for osmo regulation [J]. Gene, 2007, 396(2): 226—235

[99]Pham D, Charmantier G, Boulo V, et al. Ontogeny of osmoregulation in the Pacific blue shrimp, Litopenaeus stylirostris (Decapoda, Penaeidae): Deciphering the role of the Na+/K+-ATPase [J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2016, 196: 27—37

[100]Tian H F, Meng Y, Xiao H B. Advances in vitellogenin research of aquatic animals [J]. South China Fisheries Science, 2014, 10(4): 91—96 [田海峰, 孟彥, 肖漢兵. 水生動(dòng)物卵黃蛋白原研究新進(jìn)展. 南方水產(chǎn)科學(xué), 2014, 10(4): 91—96]

[101]Webster S G, Keller R, Dircksen H. The CHH-superfamily of multifunctional peptide hormones controlling crustacean metabolism, osmoregulation, moulting, and reproduction [J]. General and Comparative Endocrinology, 2012, 175(2): 217—233

[102]Hopkins P M. The eyes have it: A brief history of crustacean neuroendocrinology [J]. General and Comparative Endocrinology, 2012, 175(3): 357—366

[103]Han K H, Zhang Z P, Wang Y L, et al. Cyclin-CDKCKI and UPP participate in the regulation of reproduction and the progression of gonad development in crustacean [J]. Biotechnology Bulletin, 2010, 7: 48—54 [韓坤煌, 張子平, 王藝?yán)? 等. Cyclin-CDK-CKI 及 UPP參與生殖調(diào)控及在甲殼動(dòng)物性腺發(fā)育中的研究進(jìn)展.生物技術(shù)通報(bào), 2010, 7: 48—54]

[104]Picard A L, Cosseau C, Mouahid G, et al. The roles of Dmrt (Double sex/Male-abnormal-3 Related Transcription factor) genes in sex determination and differentiation mechanisms: Ubiquity and diversity across the animal kingdom [J]. Comptes Rendus Biologies, 2015, 338: 451—462

[105]Li F, Bai H, Zhang W, et al. Cloning of genomic sequences of three crustacean hyperglycemic hormone superfamily genes and elucidation of their roles of regulating insulin like androgenic gland hormone gene [J]. Gene, 2015, 561(1): 68—75

[106]Yu Y Q, Ma W M, Zeng Q G, et al. Molecular cloning and sexually dimorphic expression of two dmrt genes in the fiant freshwater prawn, Macrobrachium rosenbergii [J]. Agricultural Research, 2014, 3(2): 181—191

[107]Stein A J, Rodríguez-Cerezo E. International trade and the global pipeline of new GM crops [J]. Nature Biotechnology, 2010, 28(1): 23—25

[108]Treerattrakool S, Chartthai C, Phromma-in N, et al. Silencing of gonad-inhibiting hormone gene expression in Penaeus monodon by feeding with GIH dsRNA-enriched Artemia [J]. Aquaculture, 2013, 404: 116—121

[109]F?rstemann K, Horwich M D, Wee L, et al. Drosophila microRNAs are sorted into functionally distinct argonaute complexes after production by dicer-1 [J]. Cell, 2007, 130(2): 287—297

[110]Huang T, Xu D, Zhang X. Characterization of host microRNAs that respond to DNA virus infection in a crustacean [J]. BMC Genomics, 2012, 13(1): 159—169

[111]Huang T, Zhang X. Functional analysis of a crustacean microRNA in host-virus interactions [J]. Journal of Virology, 2012, 86(23): 12997—13004

RESEARCH PROGRESS OF RNA INTERFERENCE IN CRUSTACEANS

LI Fa-Jun1,2, FU Chun-Peng1, LI Ming-Shuang3, LI Qun-Feng1and FU Hong-Tuo2
(1. Weifang University of Science and Technology, Shouguang 262700, China; 2. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China; 3. National Fisheries Technical Extension Center, Beijing 100125, China)

RNA interference (RNAi) is a post-transcriptional gene regulatory mechanism induced by the specific double-stranded RNA (dsRNA) in eukaryotes. RNAi technology is widely used in the genomic studies of nematode, fruit fly, zebra fish and mice. Recently, significant advancement has also been made in the crustacean research by using the RNAi technology. This review summarizes recent discovery and progress focusing on immunity, growth and development, molting, reproduction, sex differentiation, osmoregulation and metabolism. Finally, the development prospect of RNAi technology usage in crustacean is previewed. The aim of this review is to provide basic scope for studying gene functions and regulatory mechanisms in crustacean.

RNA interference; Gene silencing; Crustaceans; Gene function; Regulatory mechanism

Q137

A

1000-3207(2017)02-0460-13

10.7541/2017.58

2016-06-07;

2016-08-21

國(guó)家自然科學(xué)基金(31572617); 國(guó)家“十二五”科技支撐計(jì)劃(2012BAD2604-05); 山東省自然科學(xué)基金面上項(xiàng)目(ZR2016CM12);中央級(jí)基本科研業(yè)務(wù)費(fèi)專項(xiàng)(2015JBFM11); 江蘇省農(nóng)業(yè)科技自主創(chuàng)新資金(CX(15)1012-4); 江蘇省水產(chǎn)三新工程(D2015-16);無(wú)錫市科技發(fā)展資金(CLE02N1514)資助 [Supported by the National Natural Science Foundation of China (Grant No. 31572617); the National Science & Technology Supporting Program of the 12th Five-year Plan of China (2012BAD26B04-05); Shandong Provincial Natural Science Foundation (ZR2016CM12); the Freshwater Fisheries Research Center, China Central Governmental Research Institutional Basic Special Research Project from the Public Welfare Fund (2015JBFM11), Fund of Independent Innovation of Agricultural Sciences of Jiangsu Province (CX (15)1012-4); the three aquatic projects of Jiangsu Province (D2015-16); the Science and Technology Development Fund of Wuxi (CLE02N1514)]

李法君(1976—), 男, 山東壽光人; 博士; 主要研究方向?yàn)樗a(chǎn)動(dòng)物遺傳育種。E-mail: lifajun1976@163.com

傅洪拓(1964—), 男, 博士, 研究員; 研究方向?yàn)樗a(chǎn)動(dòng)物遺傳育種。E-mail: fuht@ffrc.cn