斷肢再生對(duì)中華絨螯蟹蛻殼、生長及相關(guān)基因表達(dá)的影響

岳武成， 陳嬌， 慈元吉， 黃姝， 王軍， 王成輝

(上海海洋大學(xué)農(nóng)業(yè)部淡水水產(chǎn)種質(zhì)資源重點(diǎn)實(shí)驗(yàn)室，上海 201306)

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斷肢再生對(duì)中華絨螯蟹蛻殼、生長及相關(guān)基因表達(dá)的影響

岳武成， 陳嬌， 慈元吉， 黃姝， 王軍， 王成輝*

(上海海洋大學(xué)農(nóng)業(yè)部淡水水產(chǎn)種質(zhì)資源重點(diǎn)實(shí)驗(yàn)室，上海 201306)

以中華絨螯蟹(Eriocheirsinensis)幼蟹為材料，研究人工壓力法切斷其4個(gè)步足(左側(cè)第1、3步足和右側(cè)第2、4步足)后的蛻殼、生長與相關(guān)基因的表達(dá)規(guī)律。結(jié)果表明：斷肢蟹當(dāng)期蛻殼的步足再生率為7.6%，第2次蛻殼后的再生率為91.6%；斷肢蟹與正常蟹的成活率相當(dāng)；斷肢蟹在斷肢當(dāng)期的蛻殼周期顯著長于正常蟹(P<0.01)，但蛻殼后增質(zhì)量率顯著高于正常蟹(P<0.05)，而第2次的蛻殼周期顯著短于正常蟹(P<0.01)，但蛻殼后增質(zhì)量率與正常蟹差異無統(tǒng)計(jì)學(xué)意義(P>0.05)；經(jīng)2次蛻殼后，斷肢蟹與正常蟹的整體蛻殼周期差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。相關(guān)基因表達(dá)差異分析表明：斷肢蟹與正常蟹在胰島素樣生長因子2基因IGF2、維甲類X受體基因RXR、蛻皮抑制激素基因MIH的表達(dá)上差異無統(tǒng)計(jì)學(xué)意義(P>0.05)；但斷肢蟹的蛻皮激素受體基因EcR的表達(dá)量顯著高于正常蟹(P<0.05)，而肌肉生長抑制素基因MSTN的表達(dá)量卻顯著低于正常蟹(P<0.05)。綜上表明：中華絨螯蟹幼蟹發(fā)生斷肢后能在2個(gè)蛻殼周期內(nèi)完成再生，且與正常蟹在成活率、蛻殼周期和蛻殼后增質(zhì)量率上差異無統(tǒng)計(jì)學(xué)意義；EcR和MSTN基因?qū)Υ龠M(jìn)中華絨螯蟹再生肢體的生長發(fā)育有重要作用；斷肢蟹可在生產(chǎn)上繼續(xù)使用。

中華絨螯蟹； 斷肢再生； 增質(zhì)量率； 蛻殼周期； 基因表達(dá)

Summary Limb regeneration is a regrowth phenomenon when limbs of animals are truncated by external forces or self-cut in response to predation, which is a self-protection mechanism formed during the long-term evolution process. Currently, amphibians with tails, and crustaceans belonging to arthropods were intensively studied. The results indicated that the feeding ability, growth and development were greatly affected by regeneration. Molting could be accelerated and the ovary development could be promoted by limb regeneration, inducing expression of the related genes. Chinese mitten crab (Eriocheirsinensis) has the ability of limb regeneration like other crustaceans in its life cycle. However, the molecular mechanism of regeneration forE.sinensiswas still unclear. Therefore, study on limb regeneration ofE.sinensiscan supplement basic biological knowledge for Chinese mitten crab, and provide practical guidance for development of aquaculture industries.

A total of 80 juvenile Chinese mitten crabs (40 males and 40 females) were randomly sampled to autotomy treatments with four walking legs (the first and third legs on the left, the second and fourth legs on the right), and the same number of male and female individuals were employed as the control group without any treatment (intact crab). They were separately reared in forty tanks with each of 60 L for two molt cycle, and two crabs with cut limbs (one male, one female) and two intact crabs (one male, one female) were kept in each tank. Body mass, carapace length and carapace width were measured and recorded at the initial stocking stage, and 48 h after the first and second molting, respectively. Meanwhile, the eyes and muscle tissues (regeneration limbs and intact limbs) were collected quickly after the second molt and were stored at -80 ℃ for quantitative real-time polymerase chain reaction (qRT-PCR) analysis of myostatin geneMSTN, insulin-like growth factor-2 geneIGF2, molting hormone receptor geneEcR, retinoid X receptor geneRXR, and molt-inhibiting hormone geneMIH.

The results showed that limb regeneration rate was only 7.6% after the first molt, but reached 91.6% after the second molt; the survival rates of the amputated crabs and intact crabs were roughly equal. Longer inter-molting days (P<0.05) were observed for the amputated crabs than intact crabs, and the body mass-gaining rate was higher than intact crabs (P<0.05) after the first molting. Shorter inter-molting days (P<0.05) were observed for amputated crabs than intact crabs, and no significant difference (P>0.05) was observed for the body mass-gaining rate of the amputated and intact crabs after the second molting. Overall, there was no significant difference of average inter-molting days between the amputated and intact crabs after the two molting. The related gene expression analysis indicated that there were no significant difference (P>0.05) between the amputated and intact crabs inIGF2,RXRandMIHgenes. However, there were significant higher expression (P<0.05) ofEcRgene and lower expression ofMSTNgene for amputated crabs (P<0.05) than the intact crabs.

In conclusion, Chinese mitten crab can regenerate new legs in two molt cycles; there are no significant differences on the survival rate, mass-gaining rate and molt cycles between the amputated and intact crabs. TheEcRandMSTNgenes play important roles in promoting the leg regeneration, and the amputated crab can be kept for further aquaculture.

斷肢再生是動(dòng)物肢體在受到外力截?cái)嗷蛟谔颖軘澈r(shí)發(fā)生自切后重新長出的現(xiàn)象，是動(dòng)物在長期進(jìn)化過程中形成的自我保護(hù)機(jī)制[1]。斷肢再生一直是再生科學(xué)研究的重要課題。目前，斷肢再生研究較多的物種為有尾兩棲類、節(jié)肢動(dòng)物門以及其下屬的甲殼類。基礎(chǔ)生物學(xué)研究表明，斷肢再生過程對(duì)動(dòng)物的攝食能力、生長、發(fā)育等有較大影響[2-4]。動(dòng)物發(fā)生斷肢后機(jī)體處于高應(yīng)激狀態(tài)，其社會(huì)行為會(huì)發(fā)生巨大改變，機(jī)體的生長發(fā)育會(huì)受到較大影響。對(duì)節(jié)肢動(dòng)物門蜘蛛類[5]和蟹類[6]的斷肢再生研究表明，機(jī)體在發(fā)生斷肢后，生長減緩，發(fā)育時(shí)間(蛻皮或蛻皮周期)延長，用以機(jī)體修復(fù)。斷肢再生還會(huì)加速甲殼類蛻殼，促進(jìn)卵巢發(fā)育，誘導(dǎo)相關(guān)基因表達(dá)(如蛻皮激素受體基因EcR的高表達(dá))[6-7]。一些研究還發(fā)現(xiàn)，動(dòng)物肢體或肌肉再生時(shí)，肌肉生長抑制基因MSTN會(huì)表達(dá)下調(diào)，進(jìn)而使再生肢芽呈現(xiàn)出爆發(fā)式的生長現(xiàn)象[8-10]。由此可見，斷肢再生過程和動(dòng)物的生長發(fā)育是相互作用的。

目前，國際上對(duì)鋸緣青蟹(Scyllaserrata)、陸地蟹(Gecarcinuslateralis)、擬穴青蟹(Scyllaparamamosain)等相關(guān)蟹類的斷肢再生開展了研究，發(fā)現(xiàn)斷肢再生受到激素、溫度、光周期以及動(dòng)物發(fā)育程度等多種因素的調(diào)控[6,11]，而相關(guān)分子機(jī)制研究也揭示了斷肢再生、蛻殼與生長相關(guān)基因(MIH、EcR、RXR)的關(guān)系[6-7,12]。中華絨螯蟹(Eriocheirsinensis)與其他蟹類一樣都具有較強(qiáng)的斷肢再生能力，而目前尚無中華絨螯蟹斷肢再生研究的相關(guān)報(bào)道。在生產(chǎn)中，許多斷肢蟹特別是在蟹種放養(yǎng)選擇時(shí)常被拋棄，從而造成種質(zhì)浪費(fèi)和經(jīng)濟(jì)損失。開展中華絨螯蟹斷肢與再生研究，不僅對(duì)豐富中華絨螯蟹基礎(chǔ)生物學(xué)研究具有重要意義，而且對(duì)其產(chǎn)業(yè)發(fā)展也具有重要的實(shí)踐指導(dǎo)作用。本文以中華絨螯蟹幼蟹為試驗(yàn)材料，通過人工壓力迫使幼蟹斷肢來研究其肢體再生現(xiàn)象，探討中華絨螯蟹斷肢后對(duì)其生長和相關(guān)基因表達(dá)的影響，了解新肢體再生所需的時(shí)間或蛻殼周期。

1　材料與方法

1.1試驗(yàn)材料與飼養(yǎng)管理

取本實(shí)驗(yàn)室中華絨螯蟹配套選育系A(chǔ)的同一家系Ⅴ期仔蟹160只，隨機(jī)分為2組：斷肢組(試驗(yàn)組)和正常組(對(duì)照組)，每組雌雄各40只。斷肢前測(cè)量試驗(yàn)蟹體質(zhì)量、殼長、殼寬等性狀指標(biāo)，然后對(duì)斷肢組采用壓力法迫使其自切掉左側(cè)第1、3步足和右側(cè)第2、4步足，共4個(gè)步足。將斷肢組與對(duì)照組分別放養(yǎng)在40個(gè)容積為60 L的水族箱中，每個(gè)水族箱放養(yǎng)4只中華絨螯蟹(斷肢組和正常組雌雄各1只)。在水族箱內(nèi)種植伊樂藻(Elodeanuttallii)，每天早晚各投喂1次飼料，每3天換水1次，試驗(yàn)期間保持各個(gè)水族箱的養(yǎng)殖環(huán)境基本一致。

1.2生長測(cè)定與組織采集

試驗(yàn)期間，每天觀察中華絨螯蟹的蛻殼情況，記錄蛻殼時(shí)間。蛻殼2 d(48 h)后測(cè)量中華絨螯蟹的體質(zhì)量、殼長、殼寬3個(gè)生長性狀。試驗(yàn)時(shí)間為2個(gè)蛻殼周期，即供試中華絨螯蟹在水族箱內(nèi)完成2次蛻殼后結(jié)束。

當(dāng)全部供試中華絨螯蟹個(gè)體完成第2次蛻殼并測(cè)定其生長性狀后，立即采集每只中華絨螯蟹個(gè)體的眼柄和肌肉組織，經(jīng)液氮速凍后保存于-80 ℃冰箱中，備用。

1.3相關(guān)基因表達(dá)分析

隨機(jī)挑選斷肢組和正常組各5只雄蟹，用RNA提取試劑盒(美國Axygen公司)提取其眼柄、正常蟹步足肌肉、斷肢蟹再生步足肌肉的總RNA。使用大連寶生物公司的反轉(zhuǎn)錄試劑盒(PrimeScriptTMRT-PCR Kit)進(jìn)行反轉(zhuǎn)錄。根據(jù)本實(shí)驗(yàn)室已有的肌肉生長抑制素基因MSTN、蛻皮激素受體基因EcR、維甲類X受體基因RXR、蛻皮抑制激素基因MIH、胰島素樣生長因子2基因IGF2的定量實(shí)時(shí)聚合酶鏈?zhǔn)椒磻?yīng)(quantitative real-time polymerase chain reaction, qRT-PCR)引物(表1)進(jìn)行PCR擴(kuò)增。

表1　定量實(shí)時(shí)聚合酶鏈?zhǔn)椒磻?yīng)引物信息

1.4數(shù)據(jù)處理

利用SPSS 20.0、Excel 2013等軟件對(duì)生長性狀進(jìn)行單因素方差分析和顯著性檢驗(yàn)。采用2-ΔΔCT方法[13]對(duì)定量實(shí)時(shí)PCR數(shù)據(jù)進(jìn)行基因表達(dá)差異檢驗(yàn)。

2　結(jié)果

2.1中華絨螯蟹增質(zhì)量率差異

斷肢中華絨螯蟹在每次蛻殼后的步足再生與體質(zhì)量增長情況如表2所示。第1次蛻殼后的斷肢再生率平均僅為7.6%，第2次蛻殼后斷肢再生率達(dá)91.6%。表明中華絨螯蟹須經(jīng)歷2次蛻殼后才能完成斷肢再生過程，而有極個(gè)別個(gè)體經(jīng)2次蛻殼后也未完成斷肢再生。斷肢組由于切除了4個(gè)步足，其起始體質(zhì)量與正常組存在顯著差異，因而每次蛻殼后的體質(zhì)量也與正常組存在統(tǒng)計(jì)學(xué)上的顯著差異(P<0.05)。但從增質(zhì)量率看，斷肢組在第1次蛻殼后的增質(zhì)量率顯著高于正常組，特別是斷肢雄蟹的增質(zhì)量率(58.6%)顯著高于正常雄蟹(47.7%)(P<0.05)，而斷肢雌蟹與正常雌蟹的增質(zhì)量率不存在統(tǒng)計(jì)學(xué)上的顯著差異(P>0.05)。第2次蛻殼后，斷肢組再生后的步足小于正常蟹的步足，其平均體質(zhì)量也顯著小于正常蟹(P<0.05)，但蛻殼后增質(zhì)量率不存在統(tǒng)計(jì)學(xué)上的顯著差異(P>0.05)。經(jīng)2次蛻殼后，斷肢蟹與正常蟹的成活率較為接近，均在60%以上。

表2　斷肢蟹和正常蟹的斷肢再生率與蛻殼后增質(zhì)量率差異

2.2中華絨螯蟹蛻殼周期差異

通過對(duì)平均蛻殼周期(中華絨螯蟹所有個(gè)體完成一次蛻殼所經(jīng)歷的平均天數(shù))分析發(fā)現(xiàn)，斷肢組在去步足后到完成第1次蛻殼所需的平均天數(shù)(10.38 d)顯著高于正常組(6.73 d)(P<0.01，圖1A)，而斷肢組第2次平均蛻殼周期(18.25 d)顯著短于正常組(23.47 d)(P<0.01，圖1B)，但前后2次蛻殼整體上不存在統(tǒng)計(jì)學(xué)上的顯著差異(P>0.05，圖1C)。以上結(jié)果表明，中華絨螯蟹在受到斷肢刺激后會(huì)延長當(dāng)期的蛻殼時(shí)間，隨著斷肢再生和步足的重新生長，則會(huì)顯著縮短蛻殼周期，即附肢再生有刺激蛻殼的作用。

A:第1次蛻殼周期差異；B:第2次蛻殼周期差異；C:2次蛻殼周期平均差異?！猓捍菩?；：雄性。短?hào)派系牟煌髮懟蛐懽帜阜謩e表示在P<0.01或P<0.05水平差異有統(tǒng)計(jì)學(xué)意義。A: Difference in the first inter-molting period; B: Difference in the second inter-molting period; C: Mean difference in the two molting periods. ♀: Female; : Male. Different capital or lowercase letters above bars show statistically significant differences at the 0.01 or 0.05 probability level, respectively.圖1　斷肢蟹和正常蟹的蛻殼周期差異Fig.1　Molt cycle difference between leg-amputated crabs and intact crabs

2.3中華絨螯蟹基因表達(dá)差異

短?hào)派系牟煌懽帜副硎驹赑<0.05水平差異有統(tǒng)計(jì)學(xué)意義。Different lowercase letters above bars show statistically significant differences at the 0.05 probability level.圖2　斷肢蟹和正常蟹的相關(guān)基因表達(dá)差異Fig.2　Gene expression difference between leg-amputated crabs and intact crabs

從圖2可以發(fā)現(xiàn)：在斷肢蟹與正常蟹的步足肌肉中，胰島素樣生長因子2基因IGF2、維甲類X受體基因RXR和眼柄中的蛻皮抑制激素基因MIH的表達(dá)在統(tǒng)計(jì)學(xué)上無顯著差異(P>0.05)；而斷肢蟹步足肌肉的蛻皮激素受體基因EcR的表達(dá)顯著高于正常蟹(P<0.05)，肌肉生長抑制素基因MSTN的表達(dá)顯著低于正常蟹(P<0.05)。該結(jié)果初步表明，斷肢具有促進(jìn)中華絨螯蟹蛻殼和再生肢體肌肉生長的作用。

3　討論

生長與蛻殼是中華絨螯蟹的重要生物學(xué)特性，而斷肢再生以及再生肢體的生長發(fā)育都離不開蛻殼。本研究表明，中華絨螯蟹幼蟹在斷肢后經(jīng)歷2次蛻殼可完成再生，再生肢體的外部形態(tài)接近正常肢體水平，但能觀察到顯著的大小差異，也存在個(gè)別中華絨螯蟹在第1次蛻殼后肢體發(fā)生再生或第2次蛻殼后肢體也無法完成再生的情況。YASUDA等[14]研究發(fā)現(xiàn)，寄居蟹(Pagurusmiddendorffii)的大螯能在一次蛻殼后完成再生；HOPKINS[15]對(duì)不同蛻殼時(shí)期的大西洋砂招潮蟹(Ucapugilator)進(jìn)行了肢體再生研究，發(fā)現(xiàn)肢芽生長是在蛻殼間期而不是蛻殼前期和后期進(jìn)行的。說明甲殼類動(dòng)物完成肢體再生需要的蛻殼周期存在差異。肢體再生所經(jīng)歷的蛻殼周期，一方面可能由斷掉肢體的生理功能決定，另一方面還和蟹本身所處的蛻殼時(shí)期有關(guān)。本研究選取的是蛻殼前期的中華絨螯蟹，斷肢再生所需蛻殼周期符合一般規(guī)律，當(dāng)然也不排除因?yàn)椴蓸诱`差導(dǎo)致的個(gè)別中華絨螯蟹出現(xiàn)提前再生和延遲再生的情況。

從本試驗(yàn)中華絨螯蟹的蛻殼周期與增質(zhì)量率可以看出，斷肢再生過程對(duì)2次蛻殼周期及2次蛻殼后增質(zhì)量率的影響存在差異。斷肢處理對(duì)中華絨螯蟹當(dāng)期蛻殼具有抑制作用，此階段蛻殼周期延長，蛻殼后增質(zhì)量率較正常組高，且肢體未再生。然而，中華絨螯蟹在第2個(gè)蛻殼周期時(shí)，斷肢蟹蛻殼周期顯著縮短，蛻殼后正常蟹和斷肢蟹增質(zhì)量率無顯著差異，且出現(xiàn)肢體再生。因而，從試驗(yàn)起始到完成2次蛻殼的總經(jīng)歷時(shí)間無顯著差異，即在第2次蛻殼內(nèi)，斷肢蟹和正常蟹的蛻殼是基本同步的。這一現(xiàn)象與JOHNSON等[16]對(duì)幽靈蛛(Holocnemuspluchei)的研究結(jié)果相似。此外，WRINN等[5]對(duì)狼蛛(Schizocosaocreata)的研究表明，斷肢刺激會(huì)改變動(dòng)物機(jī)體的能量分配，造成機(jī)體生長延緩和肢芽爆發(fā)式生長以維護(hù)動(dòng)物的生存機(jī)制。綜合上述研究結(jié)果說明，中華絨螯蟹在斷肢再生過程中機(jī)體具有調(diào)整能量收支的能力，延長蛻殼周期有利于動(dòng)物機(jī)體的修復(fù)，同時(shí)再次儲(chǔ)存能量以備下一次蛻殼及肢體再生；而第2次蛻殼周期縮短，是機(jī)體自我調(diào)整以適應(yīng)群體蛻殼活動(dòng)從而提高生存概率的表現(xiàn)。

本研究表明，眼柄中的蛻皮抑制激素基因MIH以及肌肉中的胰島素樣生長因子基因IGF2、維甲類X受體基因RXR在斷肢蟹和正常蟹中表達(dá)無顯著差異，而蛻皮激素受體基因EcR和肌肉生長抑制素基因MSTN的表達(dá)存在顯著差異，即EcR基因在再生肢體中高表達(dá)，而MSTN基因在再生肢體中低表達(dá)。EcR基因是甲殼類蛻殼活動(dòng)調(diào)控的重要因子，隨著研究的逐漸深入，越來越多的證據(jù)顯示EcR還與甲殼類的肢體再生相關(guān)[12,17-19]。DAS等[18]通過RNA干擾技術(shù)證明EcR/RXR在肢芽形成早期對(duì)芽基細(xì)胞增殖具有重要作用；而CHUNG等[20]發(fā)現(xiàn)EcR基因在大西洋砂招潮蟹(U.pugilator)蛻殼周期不同階段的非再生腹肢中穩(wěn)定表達(dá)，而在再生腹肢的肢芽中高表達(dá)。這說明EcR不僅參與了蛻殼調(diào)控，而且還參與了芽基的形成和肢芽的發(fā)育。本研究發(fā)現(xiàn)EcR在再生肢體中仍然高表達(dá)，說明EcR基因還對(duì)再生肢體的生長發(fā)育具有重要作用。MSTN基因是動(dòng)物肌肉形態(tài)發(fā)生的負(fù)調(diào)控因子，與肌肉再生也存在重要聯(lián)系[8,10]。甲殼動(dòng)物在蛻殼過程中肌肉形態(tài)和結(jié)實(shí)程度會(huì)發(fā)生巨大變化。相關(guān)研究發(fā)現(xiàn)，MSTN在蛻殼前期(D4)肌肉中顯著下調(diào)，蛻殼完成后逐漸恢復(fù)到正常水平[21]。本研究中華絨螯蟹第2次蛻殼后再生肢體還未能完全達(dá)到正常肢體的形態(tài)和功能，再生肢體需要繼續(xù)生長發(fā)育，MSTN在再生肢體中特異的低表達(dá)符合動(dòng)物再生肢體在后續(xù)生長蛻殼中逐漸完善的生物學(xué)特性。

4　結(jié)論

中華絨螯蟹幼蟹發(fā)生斷肢后能在2個(gè)蛻殼周期內(nèi)完成再生；斷肢對(duì)中華絨螯蟹的成活率無顯著影響；與正常蟹相比，斷肢蟹第1次蛻殼后增質(zhì)量率升高而第2次蛻殼后其值間差異無統(tǒng)計(jì)學(xué)意義；斷肢蟹第1次蛻殼周期延長而第2次蛻殼周期縮短，正常蟹與斷肢蟹的總蛻殼周期間差異無統(tǒng)計(jì)學(xué)意義；EcR和MSTN基因?qū)Υ龠M(jìn)中華絨螯蟹再生肢體的生長發(fā)育有重要作用。綜上表明，斷肢蟹在生產(chǎn)上可以用于后續(xù)養(yǎng)殖生產(chǎn)。

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Effects of limb regeneration on molt, growth and related gene expression in Chinese mitten crab (Eriocheir sinensis).JournalofZhejiangUniversity(Agric. &LifeSci.), 2016,42(4):502-508

YUE Wucheng, CHEN Jiao, CI Yuanji, HUANG Shu, WANG Jun, WANG Chenghui*

(KeyLaboratoryofFreshwaterFisheriesGermplasmResources,MinistryofAgriculture,ShanghaiOceanUniversity,Shanghai201306,China)

Chinese mitten crab (Eriocheirsinensis); limb regeneration; mass-gaining rate; molt cycle; gene expression

上海市中華絨螯蟹產(chǎn)業(yè)技術(shù)體系項(xiàng)目[滬農(nóng)科(產(chǎn))字2010-4號(hào)]；上海市科委崇明科技專項(xiàng)(13391912102)；上海市工程技術(shù)中心建設(shè)項(xiàng)目(03DZ2251800)。

Corresponding author)：王成輝(http://orcid.org/0000-0002-6523-7610)，Tel：+86-21-61900439， E-mail:wangch@shou.edu.cn

聯(lián)系方式：岳武成(http://orcid.org/0000-0001-6965-3788)，E-mail:547203691@qq.com

2015-09-10；接受日期(Accepted)：2015-12-16；網(wǎng)絡(luò)出版日期(Published online)：2016-07-18

S 917.4

A

URL:http://www.cnki.net/kcms/detail/33.1247.S.20160718.2054.020.html