RNAi在甲殼動物中的研究進展

李法君付春鵬李明爽李群峰傅洪拓

(1. 濰坊科技學院，壽光 262700; 2. 中國水產(chǎn)科學研究院淡水漁業(yè)研究中心，農(nóng)業(yè)部淡水漁業(yè)和種質資源利用重點實驗室，無錫 214081; 3. 全國水產(chǎn)技術推廣總站，北京 100125)

RNAi在甲殼動物中的研究進展

李法君1,2付春鵬1李明爽3李群峰1傅洪拓2

(1. 濰坊科技學院，壽光 262700; 2. 中國水產(chǎn)科學研究院淡水漁業(yè)研究中心，農(nóng)業(yè)部淡水漁業(yè)和種質資源利用重點實驗室，無錫 214081; 3. 全國水產(chǎn)技術推廣總站，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介導的轉錄后基因沉默機制。作為一項研究基因功能的有力工具, RNAi技術已經(jīng)被廣泛應用在線蟲、果蠅、斑馬魚和小鼠等生物的基因組學研究中。近來在甲殼動物中, 通過RNAi技術取得了眾多的科研成果。文章從免疫、生長發(fā)育、蛻皮、生殖、性別調控、滲透壓調節(jié)和代謝等幾個方面進行了綜述。進而對RNAi技術在甲殼動物中的研究前景進行了展望, 旨在為以后更好地研究甲殼動物的基因功能和調控網(wǎng)絡提供參考。

RNA干擾; 基因沉默; 甲殼動物; 基因功能; 調控機制

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

雙鏈RNA (Double-stranded RNA, dsRNA)介導的RNA干擾(RNA interference, RNAi)現(xiàn)象是普遍存在線蟲、果蠅、斑馬魚和小鼠等真核生物中的轉錄后調控機制, 在生物體的生長發(fā)育、穩(wěn)定轉座子和抵御外來病毒的入侵等方面發(fā)揮重要作用。RNAi技術于2001年被《Science》雜志評為十大科學進展之一, 2002年更是位居十大科學進展之首。作為研究基因功能的重要工具, 近年來RNAi技術也在甲殼動物中得以廣泛應用, 并取得了眾多研究成果。鑒于此, 本文對RNAi在甲殼動物中的研究進展進行了綜述, 旨在為應用此技術解析甲殼動物重要基因的功能及調控機制提供理論參考。

1 RNAi簡介

RNAi是指在進化過程中高度保守的、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 (Caenorhabditis ele-gans) 的part-1基因表達時, 意外發(fā)現(xiàn)作為對照組的正義RNA也可以抑制part-1基因的表達; 1998年, Fire等[3]分別將part-1基因的正義、反義和dsRNA導入線蟲, 發(fā)現(xiàn)dsRNA的沉默效果明顯高于單鏈RNA。于是將這種由dsRNA抑制特定基因表達的現(xiàn)象稱為RNAi。隨后, RNAi以其獨有的高效性、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作用機制分兩步進行: 起始階段和效應階段。起始階段: 外源或內(nèi)源的dsRNA進入細胞后, 被Dicer酶劈成21—23個堿基的小片段, 稱為小分子干擾RNA (Small interfering RNA, siRNA); 效應階段: siRNA和RNA誘導沉默復合物(RNA Induced Silencing Complex, RISC)相結合, 結合后的復合物具有核酸酶的作用,能識別并降解目標RNA。

2 RNAi技術在甲殼動物中的研究

甲殼動物隸屬節(jié)肢動物門, 處在由無脊椎動物向脊椎動物進化的特殊階段, 獨特的進化地位也決定了發(fā)育過程和組織構造的特殊性。RNAi作為真核生物普遍存在的機制, 也在甲殼動物中調控多個生物學過程。

2.1 免疫

病毒病已經(jīng)給對蝦養(yǎng)殖業(yè)造成重大損失, 對蝦白斑綜合癥病毒(White spot syndrome virus, WSSV)、黃頭病毒(Yellow head virus, YHV)和桃拉病毒(Taura syndrome virus, TSV)是危害我國對蝦養(yǎng)殖業(yè)的三種主要病毒。其中以WSSV的危害最為嚴重, 已經(jīng)給對蝦養(yǎng)殖業(yè)造成巨大的經(jīng)濟損失。RNAi被認為是一種古老的抗病毒機制, 是生物體中先天性存在的免疫因子, 甲殼動物也不例外。研究表明,免疫系統(tǒng)是后生動物普遍存在的抵抗外來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技術最早應用在甲殼動物也是在此方面。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。結果顯示, 注射異源物種dsRNA的實驗組死亡率下降到對照組的50%—70%, 由于凡納濱對蝦體內(nèi)不存在上述脊椎動物的免疫球蛋白基因, 所以脊椎動物的dsRNA分子不可能參與到凡納濱對蝦的抗病毒干擾體系中去。引起染病凡納濱對蝦死亡率降低的原因在于, 脊椎動物免疫球蛋白基因dsRNA作為外源性物質注射到凡納濱對蝦體內(nèi), 從而激活了凡納濱對蝦先天性的免疫反應。

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、包裝和釋放等過程, 對病毒的感染起著至關重要的作用[9,10]。由于WSSV屬于雙鏈DNA病毒, 因此科研人員通常利用RNAi技術定向沉默編碼上述蛋白的mRNA來抑制WSSV的繁殖。Kim等[11]將VP28和VP281的長鏈dsRNA注射到感染W(wǎng)SSV的中國對蝦(Fenneropenaeus chinensis)體內(nèi), 有效提高存活率。Xu等[12]將短鏈VP28的dsRNA注射到日本囊對蝦(Penaeus japonicus)體內(nèi), 也發(fā)現(xiàn)有類似的結果。近來Thammasorn等[13]在凡納濱對蝦中利用基因重組技術, 將VP28和WSSV051 (WSSV的極早期基因之一)兩個基因干擾位點構建到同一條dsRNA中, 并將其飼喂凡納濱對蝦。7d后此實驗組的死亡率為40%, 顯著低于VP28和WSSV051的單獨干擾組,表現(xiàn)出良好的治療效果。RNAi在甲殼動物不同WSSV結構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對其結構基因的研究僅限于YHV的gp116和gp64兩個功能基因, 而且是在體外細胞中進行的[16]。有關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基因方面。Rab是GTP結合蛋白家族中最大的亞家族, Rab7蛋白作為Rab家族一員, 能特異識別晚期胞內(nèi)體等囊泡, 介導晚期胞內(nèi)體與溶酶體的膜融合, 以完成溶酶體轉運過程。當病毒、菌病體等進入宿主細胞后會與Rab7蛋白結合, 避開溶酶體的消化, 導致宿主感染。因此, Rab7在病原體入侵宿主過程中起著重要的作用。例如, 斑節(jié)對蝦的Rab7可以與VP28結合, 在WSSV侵染對蝦的過程中發(fā)揮作用[17]。Ongvarrasopone等[18]在斑節(jié)對蝦中通過注射Rab7-dsRNA降低 Rab7基因的表達量, 從而有效抑制YHV和WSSV感染對蝦, 推測Rab7蛋白可能是參與了病毒復制過程中的內(nèi)吞運輸。在TSV侵染蝦體的研究中也獲得了相似的結果, Ongvarrasopone等[19]在TSV侵染凡納濱對蝦48h之后注射Rab7-dsRNA, 有效地抑制TSV的復制, 提高了蝦的成活率。RNAi在YHV和TSV傳播途徑中其他相關基因的研究在表 1中列出。

上述研究表明, RNAi作為廣泛存在的抗病毒機制在甲殼動物抵御外來病毒的侵染過程中發(fā)揮重要作用。近年來, 有關甲殼動物RNAi抗病毒的分子機制也取得了較大進展。現(xiàn)在公認的觀點為:病毒侵染甲殼動物有機體, 并將外源基因整合到寄主的基因組內(nèi)。當宿主細胞進行轉錄時, 會產(chǎn)生相應的dsRNA。宿主細胞內(nèi)的核酸內(nèi)切酶Dicer (DCR2)[41,42]與Arsenite resistance蛋白2(Ars2)[43]和HIV-1反式激活應答元件RNA結合蛋白(HIV-1 transactivating response (TAR) RNA-binding protein, TRBP)[44]組成三者復合物, 將上述dsRNA分割成siRNA, siRNA與宿主細胞內(nèi)的核酸內(nèi)切酶Argonautes 2(Ago2)結合組成沉默復合物, 從而對病毒的mRNA進行切割降解[45], 從而抑制病毒基因的表達,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作為一種對抗病毒入侵的新技術手段, 具有高效、完全、無毒的特點, 在治療甲殼動物病毒病的過程中發(fā)揮積極作用。但現(xiàn)有的研究尚處在理論階段, 如何針對不同的病毒開發(fā)長期性表達的特異RNAi體系, 并大規(guī)模應用到甲殼動物養(yǎng)殖中是當務之急。

2.2 生長發(fā)育

作為有力的分子生物學工具, RNAi技術被廣泛應用在研究線蟲、昆蟲和脊椎動物的生長發(fā)育方面[46,47]。近來RNAi也在研究甲殼動物生長發(fā)育方面取得了長足進步。

肌肉生成抑制素(Myostatin, MSTN), 在無脊椎動物中又稱生長分化因子(Growth differentiation factor 11, GDF-11), 屬于轉化生長因子-β(Ttransforming growth factor-β, TGF-β)家族, 是控制肌肉生長的調控因子[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, 而在隨后的蛻皮期表達量則逐漸下降, 表明MSTN/GDF-11基因參與了日本沼蝦的生長發(fā)育過程[49]。在斑節(jié)對蝦中, 通過45d的長期干擾MSTN/GDF-11基因, 結果顯示干擾組斑節(jié)對蝦的增重僅為對照組的32%[50]。在凡納濱對蝦中, 經(jīng)過8周的干擾, 增重率顯著低于對照組, 且出現(xiàn)較高的死亡率(71%)[51]。上述結果揭示MSTN/GDF-11基因可以促進甲殼動物的生長發(fā)育。

表皮生長因子受體(Epidermal Growth Factor Receptor, EGFR)是具有酪氨酸激酶活性的跨膜蛋白分子, 可以和配體結合啟動胞內(nèi)信號傳導途徑,促進細胞的分裂增殖, 從而調控生長發(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變緩, 13周之后干擾實驗組體重僅為對照組的37%[52], 表明Mr-EGFR是羅氏沼蝦重要的生長調控因子。

Hox基因(Omeobox genes)在節(jié)肢動物軀體發(fā)育過程發(fā)揮重要作用, 其表達具有嚴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(Artemia saline)中用RNAi方法研究了Spalt基因的功能。結果顯示, 降低Spalt基因的表達可以導致鹵蟲體節(jié)發(fā)育異常, 據(jù)此推測Spalt基因可能是Hox的調節(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)育過程中發(fā)揮作用, 通過干擾減少Ubx基因的表達量可以導致附肢的表型發(fā)生變化[55], 表明Ubx基因很可能參與了明鉤蝦附肢的發(fā)育過程。另一個參與附肢發(fā)育的基因是Distal-less (Dll), Kato等[56]在水溞(Daphnia magna) Dll基因的不同位置構建了兩條dsRNA, 分別注射這兩條dsRNA到水蚤的受精卵中,結果都導致Dll基因的表達量明顯下降, 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)育過程。RNAi在其他生長發(fā)育相關基因的研究在表 2中列出。

動物體的生長發(fā)育是一個嚴謹而復雜的生物學過程, 多個基因和調控通路涉及其中。有關甲殼動物生長發(fā)育的分子機制, 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)肢動物門的昆蟲綱。生長發(fā)育作為甲殼動物最基本的生物學過程,其中尚有許多關鍵環(huán)節(jié)等待研究人員借助RNAi和其他技術對其進行解讀。

2.3 蛻皮

甲殼動物由于獨特的身體構造, 在個體發(fā)育過程中存在蛻皮現(xiàn)象。研究表明, 甲殼動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)及轉錄因子調控蛻殼響應基因E75、幾丁質酶均在其中發(fā)揮用[57,58]。

Pamuru等[59]在紅螯螯蝦(Cherax quadricarinatus)用dsRNA沉默Cq-MIH基因, 結果導致蛻皮周期加快, 蛻皮的間隔期也相應縮短到原來的32%。江豐偉[60]通過連續(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, 實驗組雄蝦和雌蝦分別蛻皮17次、12次, 而對照組雄、雌蝦則僅為2次、5次。表明MIH是甲殼動物蛻皮周期中的負調控因子。最近在藍蟹(Callinectes sapidus)中的研究表明, EcR正調控MIH基因的表達, 是MIH基因上游調控因子[61]。Techa[62]在藍蟹中通過RNAi證實, 蛻皮激素(Ecdysteroids, E)通過EcR刺激MIH基因的表達, 證實甲殼動物的蛻皮機制中存在“E→EcR→MIH”的信號通路。

RXR是節(jié)肢動物在蛻皮過程中重要調控基因之一, RXR與EcR形成二聚體結構, 調控下游基因E75的表達。Priya等[63]注射RXR-dsRNA到中國對蝦幼蝦體內(nèi)導致下游的兩種幾丁質酶(Chi和Chi-1)基因和E75基因的表達豐度下調, 表明RXR也參與了甲殼動物的蛻皮信號途徑。接著Priya等[64]通過持續(xù)注射沉默E75基因, 嚴重阻礙中國對蝦的蛻皮并最終導致其死亡, 說明 E75基因與甲殼動物的蛻皮調控密切相關且是其生長過程中必需基因。最近在凡納濱對蝦中的研究表明, 干擾RXR、EcR和E75基因中任意一個都會引起其他兩個基因的表達量發(fā)生變化, 進而會影響一系列的蛻皮和生長相關基因的變化[65]。上述結果進一步證實了RXR、EcR和E75基因調控甲殼動物的蛻皮過程。

甲基法尼酯(Methyl farnesoate, MF)是甲殼動物一種重要的生長調控激素, 主要負責調節(jié)體節(jié)發(fā)育、生長和生殖[66,67]。法尼酸甲基轉移酶(Farnesoic acid O-methyltransferase, FAMeT)在MF的形成過程中起著重要作用, 負責把法尼酸(Farnesoic acid, FA)轉化為MF。在凡納濱對蝦中, Hui等[68]研究了FAMeT基因在蛻皮中的作用, 通過注射dsRNA降低了FAMeT基因的表達量, 從而推遲了蛻皮, 而且與蛻皮相關的血藍蛋白基因和組織蛋白酶基因的表達量也會下降。最后注射dsRNA的實驗組(因蛻皮阻滯)全部死亡, 而與之相對應的對照組則無一死亡。這些結果表明, FAMeT基因在甲殼動物的生長和蛻皮周期中發(fā)揮作用。RNAi在其他蛻皮相關基因的研究在表 2中列出。

蛻皮是甲殼動物生長和發(fā)育的標志性特征, 是一個復雜有序的生物學過程。它貫穿于甲殼動物個體發(fā)育的始終, 受MIH信號通路、Ca2+信號通路以及NO信號通路的共同調節(jié)[57]。目前對蛻皮機制的認識還相當有限, 各信號通路直接是否存在關聯(lián),以及每條信號通路都存在哪些具體的調控因子, 這些都需要進一步研究。

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)境因子綜合調控的結果。涉及性腺的發(fā)育、生殖細胞的發(fā)生和受精等多個過程。卵黃蛋白原(Vitellogenin, Vg)是卵生動物雌性卵黃蛋白的前體, Vg的合成和積累對于雌性個體卵母細胞的發(fā)育和卵子的數(shù)量和質量至關重要[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)育過程中與Vg結合, 起到受體介導的作用, 在胚胎成熟的過程中發(fā)揮作用。因此Vg和VgR就成為研究雌性水產(chǎn)動物卵巢發(fā)育的重要靶標基因。

甲殼動物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。Tiu等[73]在斑節(jié)對蝦和Bai等[74]在日本沼蝦中通過干擾VgR基因, 推測Vg可能是在肝臟中合成后運輸?shù)铰殉仓械穆涯讣毎麅?nèi)。而且Bai等[72,74]進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)明顯低于對照組, 表明Vg和VgR參與了甲殼動物的性腺發(fā)育過程。

甲殼動物眼柄中的X-器竇腺復合體(X-organsinus gland, XO-SG)合成并分泌甲殼動物高血糖激素(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, 調控甲殼動物的糖代謝、蛻皮及生殖等多個生理過程[101,102]。Treerattrakool等[76]通過向馴養(yǎng)和野生的雌性斑節(jié)對蝦注射GIH-dsRNA, 結果導致卵巢發(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)對蝦卵巢的發(fā)育。已有多個研究證實(表 2), 沉默GIH[75,77,78]和MIH-B[79]可以提高Vg基因的表達水平, 表明GIH和MIH-B是卵巢發(fā)育的負調控因子。

此外, 核受體RXR和EcR作為信號通路的重要成員也在甲殼動物性腺的發(fā)育過程中起調控作用。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基因的表達量顯著下降, 表明EcR和RXR參與了卵巢的發(fā)育過程, 并且后者的研究進一步表明MF和RXR組成復合物, 調控卵巢的發(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, 在受精過程中發(fā)揮作用。Yang等[85]在羅氏沼蝦中通過沉默SG基因, 發(fā)現(xiàn)羅氏沼蝦的精子在外形上未見異常, 但其明膠的水解活性明顯下降, 并推測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]通過干擾壺腹多肽(Terminal ampullae peptide, TAP)降低了SG的活性, 進一步的研究表明, TAP參與卵膜蛋白降解相關酶的活力調節(jié),但不影響精子入卵和受精過程。

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

2.5 性別調控

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

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, 在雄性胚胎的形成過程中, DapmaD-sx1表達量明顯升高, 而在雌性胚胎中則沒有這種現(xiàn)象。進一步在雄性胚胎中沉默DapmaDsx1, 可誘導其卵巢成熟, 產(chǎn)生雌性特征; 相反, 在雌性胚胎中異位表達DapmaDsx1, 可使其產(chǎn)生雄性特性。表明DapmaDsx1雄性性別決定中起著關鍵作用。因此, DapmaDsx1被認為是水溞的性別決定基因[104]。

促雄腺(Androgenic gland, AG)是雄性甲殼動物特有的內(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, 通過長期(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, 通過干擾沉默IAG基因, 使雄性個體出現(xiàn)了雌性化特征、精子發(fā)生受到抑制、Vg基因開始表達、卵母細胞中的卵黃大量積累等現(xiàn)象。以上結果表明, IAG基因在甲殼動物的性別分化方面發(fā)揮重要作用。2012年在甲殼動物性別分化方面取得了突破性進展, Ventura等[90]通過長時間干擾雄性羅氏沼蝦幼體(變態(tài)后30d, 體重30—70 mg), 獲得了完全性逆轉的“新雌蝦”。隨后應用“新雌蝦”與正常的雄蝦交配產(chǎn)生了全雄的后代[91], 從而實現(xiàn)了羅氏沼蝦的單性化養(yǎng)殖。

近來RNAi技術也用在研究IAG基因的調控機制方面。我們前期通過RNAi技術在日本沼蝦中論證了IAG和胰島素樣促雄腺激素結合蛋白(Insulinlike androgenic gland hormone binding protein, IAGBP)存在彼此的調控關系[92]; 而且我們進一步確認MIH和GIH是IAG基因的負調控因子[105]。在羅氏沼蝦中, Yu等[106]通過RNAi確認Dmrt11E基因是IAG基因的正調控因子。

羅氏沼蝦的干擾實驗標志著第一次通過沉默一個基因而實現(xiàn)甲殼動物的單性化養(yǎng)殖。在此過程中沒有改變機體的基因序列, 因此具有廣闊的應用前景[107]。

2.6 滲透壓調節(jié)

甲殼動物多廣鹽性物種, 鹽度的變化必然會引起血糖濃度、滲透壓發(fā)生改變等一系列生理調控過程, 神經(jīng)多肽激素便在其中發(fā)揮作用。Lugo等[93]將CHH-dsRNA通過腹部淋巴腺注射進入南方濱對蝦(Litopenaeus schmitti)體內(nèi), 24h之內(nèi)沒有檢測到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, 從而調節(jié)甲殼動物的滲透壓。Manfrin等[94]在克氏原螯蝦中通過長時間干擾CHH基因, 實驗組克氏原螯蝦的死亡率達到47%, 并且死亡個體呈現(xiàn)明顯的滲透壓失調癥狀(頭胸甲和腹部出現(xiàn)明顯的分離)。此外大劑量干擾離子轉運肽(Ion transport peptide, ITP)[98]和Na+/K+-ATPase (NKA)[99]基因可使實驗蝦出現(xiàn)死亡, 表明上述兩個基因也在調控滲透壓方面發(fā)揮各自的作用。

我國幅員遼闊, 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。對鹽堿地的水質、離子成分進行分析, 針對具體物種的滲透壓調節(jié)機制,必要時補充相關離子, 進行水產(chǎn)養(yǎng)殖可以提高鹽堿地區(qū)的經(jīng)濟效益。因此闡明甲殼動物的滲透壓調節(jié)機制, 就顯得尤為關鍵。

2.7 代謝

缺氧誘導因子-1 (Hypoxia inducible factor 1, HIF-1)是由α和β兩個亞基組成的異源蛋白二聚體,作為轉錄因子, HIF-1調控缺氧應激下的多種生理活動。So?anez等[95]在缺氧條件下分別干擾凡納濱對蝦HIF-1-α和HIF-1-β, 結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]途徑來調控葡萄糖的變化。

目前為止, 有關甲殼動物代謝途徑中相關基因的研究還偏少, 作為基礎的代謝過程, 應用RNAi技術開展此方面的研究有助于全面解析甲殼動物的營養(yǎng)代謝途徑。

3 展望

技術優(yōu)勢: 基因組編輯技術(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)動物中得以廣泛運用。基因組編輯技術的操作對象是基因組DNA, 且主要是通過顯微注射對細胞系或胚胎進行操作。而目前為止, 甲殼動物還缺乏成熟的細胞系構建體系, 且卵殼較硬易碎。因此, 有關應用基因組編輯技術研究甲殼動物基因功能的文章還鮮見報道。雖然基因組編輯技術有望成為研究基因功能的主流方法, 但在上述問題未解決之前, RNAi仍然在甲殼動物中發(fā)揮自己獨特的技術優(yōu)勢。從技術層面分析, RNAi在甲殼動物中也存在脫靶和效應劑量等方面的問題。但這些問題可以通過對不同靶點和不同劑量dsRNA的篩選來解決。而且相比脊椎動物, 甲殼動物體型較小, 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驗要求, 因此成本較低。作為內(nèi)生性的調控機制, 人工合成的dsRNA可以持續(xù)在甲殼動物體內(nèi)發(fā)揮作用, 如研究人員發(fā)現(xiàn)GIH-dsRNA在斑節(jié)對蝦體內(nèi)發(fā)揮作用的有效時間最少可以達到30天[76]。注射法是目前甲殼動物RNAi的主要轉染方式, 近來在探索RNAi的其他轉染途徑方面也取得了突破性進展。在斑節(jié)對蝦中, 研究人員將GIH-dsRNA構建到大腸桿菌中, 然后用鹵蟲濾食大腸桿菌從而“富集”GIH-dsRNA, 最后將鹵蟲飼喂斑節(jié)對蝦可有效降低GIH基因的表達水平[108]。這種方法簡單有效, 更為重要的是, 對于不適用注射法的甲殼動物幼體來說, 鹵蟲飼喂法介導的RNAi為研究甲殼動物幼體階段相關基因的功能開辟了一條新路。因此可以肯定的是, RNAi作為一項成熟的技術, 在未來一段時間內(nèi)還將是研究甲殼動物基因功能的熱門工具。

免疫機制研究: RNAi作為一種抵抗外源病毒的重要天然免疫反應, 迄今為止, 甲殼動物在免疫過程中的具體分子機制還不完全清楚。在昆蟲中,病毒誘導的siRNA和miRNA是獨立生成的, 各自在免疫系統(tǒng)中發(fā)揮作用[45,109]。已有研究表明, 甲殼動物的miRNA也在抵御病毒入侵的過程發(fā)揮作用[110,111]。那么, 甲殼動物是否和昆蟲一樣也存在類似的獨立作用機制, 還需進一步探索。上述問題的闡明, 將有助于揭開當下危害甲殼動物養(yǎng)殖過程中出現(xiàn)的病毒性疾病的發(fā)病原理, 進而開發(fā)高效實用的防治藥物, 服務于水產(chǎn)養(yǎng)殖業(yè)。

基因功能與信號通路: 甲殼動物的各個生物學過程從來都不是獨立存在的, 如甲殼動物的生長和蛻皮總是相輔相成的, 甲殼動物有機體的生長導致蛻皮, 蛻皮又可以促進甲殼動物的生長; 再如, 甲殼動物“性早熟”也與蛻皮密切相關, 原因在于早期幼體性腺發(fā)育過快, 蛻皮次數(shù)過頻。因此, 性腺的發(fā)育與蛻皮機制必然存在交叉點。換而言之, 甲殼動物的各個基因調控通路相互連接, 形成一個復雜的調控網(wǎng)絡。因此有關甲殼動物基因組學的研究勢必將從單個基因功能的確定過渡到信號通路的研究。如前文所述, 基因測序技術的發(fā)展為我們獲取大規(guī)模的基因信息提供了有力的技術保障。RNAi的技術特點使它可以精確地靶向敲降目的基因, 進而可以檢測出下游基因表達量的變化, 在轉錄水平為信號通路的研究提供基本的數(shù)據(jù), 進而通過相關的技術手段解讀其調控網(wǎng)絡。

綜上所述, 作為一項快速發(fā)展并取得廣泛應用的技術, RNAi在甲殼動物研究中已經(jīng)展示了廣闊的用途和前景, 由此我們相信隨著對甲殼動物RNAi信號傳遞機制的深入研究, 必將有助于全面闡釋甲殼動物的基因功能及其調控網(wǎng)絡, 進而推動甲殼動物基因組學的研究, 同時也可為甲殼動物的健康養(yǎng)殖提供更多的理論指導, 從而實現(xiàn)理論研究和生產(chǎn)應用的統(tǒng)一。

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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

國家自然科學基金(31572617); 國家“十二五”科技支撐計劃(2012BAD2604-05); 山東省自然科學基金面上項目(ZR2016CM12);中央級基本科研業(yè)務費專項(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; 主要研究方向為水產(chǎn)動物遺傳育種。E-mail: lifajun1976@163.com

傅洪拓(1964—), 男, 博士, 研究員; 研究方向為水產(chǎn)動物遺傳育種。E-mail: fuht@ffrc.cn