陸潮峰,郝培應(yīng),俞曉平

(中國計量學(xué)院 生命科學(xué)學(xué)院,浙江省生物計量及檢驗(yàn)檢疫技術(shù)重點(diǎn)實(shí)驗(yàn)室,浙江 杭州 310018)

褐飛虱核糖體蛋白S6激酶基因NlRPS6KA2的克隆及其表達(dá)分析

陸潮峰,郝培應(yīng),俞曉平

(中國計量學(xué)院 生命科學(xué)學(xué)院,浙江省生物計量及檢驗(yàn)檢疫技術(shù)重點(diǎn)實(shí)驗(yàn)室,浙江 杭州 310018)

根據(jù)轉(zhuǎn)錄組提供的RPS6KA2核心序列信息,應(yīng)用RACE技術(shù)獲得了一個編碼褐飛虱核糖體蛋白S6激酶的基因NlRPS6KA2的全長cDNA,編碼的蛋白含706個氨基酸,具有保守的S_TKc和S_TK_X結(jié)構(gòu)域.熒光定量PCR測定結(jié)果表明,NlRPS6KA2基因在褐飛虱若蟲和雄蟲中表達(dá)量均較低,但在懷卵雌蟲中大量表達(dá).同時,褐飛虱從感性水稻品種TN1到抗性水稻品種RHT的適應(yīng)過程中,該基因表達(dá)量呈現(xiàn)明顯的下降趨勢,在適應(yīng)后有所回升.研究結(jié)果為進(jìn)一步研究NlRPS6KA2基因在褐飛虱中的功能和闡明褐飛虱致害性變異機(jī)制提供了依據(jù).

褐飛虱;NlRPS6KA2基因;熒光定量PCR;致害性變異

褐飛虱NilaparvatalugensSt?l(Hemiptera: Delphacidae)以水稻為食,嚴(yán)重威脅亞洲國家的水稻產(chǎn)量[1-2].褐飛虱不僅能吸食水稻韌皮部中的營養(yǎng)導(dǎo)致水稻枯萎,嚴(yán)重時還會產(chǎn)生虱燒;褐飛虱會傳播病毒導(dǎo)致水稻減產(chǎn),如草狀矮縮病、齒葉矮縮病等;同時,褐飛虱產(chǎn)卵會破壞水稻疏導(dǎo)組織導(dǎo)致水分流失[3-4].目前,種植抗性水稻品種作為一種防治褐飛虱的有效手段已被廣泛推廣應(yīng)用[5],但持續(xù)大規(guī)模種植帶來了褐飛虱致害性變異的問題,導(dǎo)致抗性水稻品種抗性失效[6].因此,研究褐飛虱如何適應(yīng)抗性水稻對闡明褐飛虱致害性變異機(jī)制至關(guān)重要.

胰島素信號通路在生物體中的作用保守且非常重要,能調(diào)節(jié)細(xì)胞的生長、代謝、分裂、分化以及細(xì)胞壽命等,還能調(diào)節(jié)生物體中蛋白質(zhì)和脂類等的新陳代謝[7-10].該通路被證實(shí)能調(diào)控昆蟲器官的大小、細(xì)胞大小及生長、激活蛻化類固醇生產(chǎn)等[11-13].TATAR[14]研究發(fā)現(xiàn),該通路在受到抑制時能夠使果蠅生長發(fā)育受到影響,使其體型變小,卵巢發(fā)育推遲和壽命延長.TELEMAN[15]研究發(fā)現(xiàn)該通路受到抑制時會使果蠅體重降低或引起死亡.在蚊子中,SIM和DENLINGER[16]發(fā)現(xiàn)該通路受到抑制時能使蚊子的初級卵泡停止發(fā)育,使蚊子處于類似于滯育的狀態(tài).這些研究說明,在胰島素信號通路受到抑制時昆蟲的生長和發(fā)育也受到抑制,同時能導(dǎo)致死亡.在前期研究中發(fā)現(xiàn),當(dāng)褐飛虱從感性水稻品種TN1(Taichung native 1)到抗性水稻品種Rathu Heenati(RHT)上時,褐飛虱的生長發(fā)育受到抑制,具體表現(xiàn)在生物量合成減少、生長緩慢和存活率降低[17].因此,我們推測胰島素信號通路與褐飛虱致害性變異間有可能存在著某些聯(lián)系,該通路的研究對闡明褐飛虱致害性變異會有所幫助.

本研究通過分析課題組前期獲得的褐飛虱轉(zhuǎn)錄組數(shù)據(jù),經(jīng)RACE法克隆全長得到一個編碼核糖體蛋白S6激酶的基因,Blast發(fā)現(xiàn)該基因?yàn)镽PS6KA2,命名為NlRPS6KA2(KP890676);

通過熒光定量PCR測定NlRPS6KA2在褐飛虱不同齡期和不同性別中的表達(dá),分析NlRPS6KA2在褐飛虱生長發(fā)育過程中的作用.研究結(jié)果為進(jìn)一步研究NlRPS6KA2基因在褐飛虱中的功能奠定了基礎(chǔ).

1 材料和方法

1.1 供試褐飛虱

供試褐飛虱采集于浙江省杭州稻田(30°N,120°E),種群分別在感蟲水稻品種TN1和抗性水稻品種RHT上連續(xù)飼養(yǎng)50代以上,恒溫室飼養(yǎng)溫度(26±2)℃,相對濕度為(80±5)%,光周期為16 L:8 D.本研究飼養(yǎng)了7個褐飛虱種群用于相關(guān)實(shí)驗(yàn):Tn表示長期飼養(yǎng)在TN1上的褐飛虱;RF1表示從TN1轉(zhuǎn)移到RHT上的第1代褐飛虱;RF2表示第2代;RF3表示第3代;RF4表示第4代;RF5表示第5代;Rh表示長期飼養(yǎng)在RHT上的褐飛虱.

1.2 褐飛虱NlRPS6KA2基因的克隆

根據(jù)前期褐飛虱轉(zhuǎn)錄組序列信息獲得一個編碼核糖體蛋白S6激酶的基因RPS6KA2基因核心序列信息,命名為NlRPS6KA2,并采用RACE法克隆基因全長.分別設(shè)計外引物5O-RPS6KA2和3O-RPS6KA2(表1),以及內(nèi)引物5I-RPS6KA2和3I-RPS6KA2(表1).用Trizol法提取褐飛虱的總RNA,根據(jù)BD SMARTTMRACE cDNA Amplification Kit(Clontech, USA)試劑盒說明書合成5′-RACE和3′-RACE的模板.采用巢式PCR對目的基因的2端進(jìn)行擴(kuò)增,反應(yīng)體系50 μL: PCR Mix 25 μL,10 μmol/L正反引物各2 μL,模板2 μL,ddH2O 19 μL.外圍PCR反應(yīng)條件:94 ℃ 4 min;94 ℃ 30 s,60 ℃ 30 s,72 ℃ 3 min,30個循環(huán);72 ℃ 10 min;4 ℃保存.內(nèi)圍PCR所用的模板為第一輪PCR得到的產(chǎn)物.內(nèi)圍PCR反應(yīng)條件:94 ℃ 4 min;94 ℃ 30 s,58 ℃ 30 s,72 ℃ 3 min,30個循環(huán);72 ℃ 10 min;4 ℃保存.PCR擴(kuò)增得到的產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳檢測,割膠回收,連接到PMD-18T(TaKaRa, Japan),轉(zhuǎn)化到JM109感受態(tài)細(xì)胞中,PCR鑒定陽性克隆菌株送到上海桑尼生物科技有限公司測序.測序結(jié)果用DNAMAN軟件和原序列比對驗(yàn)證.

表1 基因克隆和熒光定量PCR的引物

1.3 褐飛虱NlRPS6KA2基因表達(dá)分析

分別對上述所提到7個種群褐飛虱按照1-2齡若蟲、3-4齡若蟲、5齡若蟲、初羽化雌成蟲、懷卵雌成蟲、雄成蟲等進(jìn)行分別取樣,用熒光定量PCR進(jìn)行NlRPS6KA2基因表達(dá)量分析.用Trizol法提取總RNA,用PrimeScript RT Reagent Kit with gDNA Eraser(Takara, Japan)反轉(zhuǎn)錄試劑盒合成用于熒光定量PCR的cDNA模板.熒光定量PCR特異性引物為QRPS6KA2-F和QRPS6KA2-R(表1),熒光片段經(jīng)過測序和原序列比對,以β-actin基因?yàn)閮?nèi)參[18],以1～2齡的Tn褐飛虱種群的NlRPS6KA2基因表達(dá)量為參照,檢測NlRPS6KA2基因在不同褐飛虱種群的相對表達(dá)量.熒光定量PCR參考馬艷等[19]的反應(yīng)體系及方法,其中退火溫度改為51 ℃.

2 結(jié)果與分析

2.1 褐飛虱NlRPS6KA2基因的cDNA全長克隆及序列分析

根據(jù)所得到的5端和3端測序結(jié)果,用DNAMAN進(jìn)行序列拼接得到NlRPS6KA2基因全長為2491 bp,該基因3端具有典型的polyA結(jié)構(gòu).Blast X(http://blast.ncbi.nlm. nih. gov/Blast.cgi)比對發(fā)現(xiàn),克隆所得到的基因與其他物種的編碼核糖體蛋白S6激酶RPS6KA2同源性很高(80%以上),因此命名為NlRPS6KA2.經(jīng)ORF Finder(http://www.ncbi.nlm.nih.gov/projects/gorf/)分析發(fā)現(xiàn),該基因編碼一個長度為2121 bp的開放閱讀框(ORF),編碼706個氨基酸(圖1).經(jīng)蛋白相對分子質(zhì)量及等電點(diǎn)預(yù)測工具(http://web.expasy.org/compute_pi/)預(yù)測發(fā)現(xiàn)該蛋白相對分子質(zhì)量為79630 u(79.63 kDa),等電點(diǎn)為8.49.通過蛋白結(jié)構(gòu)域預(yù)測站網(wǎng)(http://smart. embl-heidelberg.de/)預(yù)測發(fā)現(xiàn)該蛋白具2個絲氨酸蘇氨酸蛋白激酶接觸催化位點(diǎn)(S_TKc)結(jié)構(gòu)和1個絲氨酸/蘇氨酸型蛋白激酶S_TK_X結(jié)構(gòu),與豌豆蚜(Acyrthosiphonpisum),人體虱(Pediculushumanuscorporis)以及柑橘木虱(Diaphorinacitri)等昆蟲具有相同的結(jié)構(gòu)域(圖2).

圖1 褐飛虱NlRPS6KA2基因全長cDNA序列及氨基酸序列Figure 1 Nucleotide sequence and amino acid sequence of NlRPS6KA2 from N. lugens

圖2 褐飛虱、豌豆芽、人體虱和柑橘木虱核糖體蛋白S6激酶結(jié)構(gòu)圖Figure 2 Second structure of Ribosomal protein S6 kinase from N. lugens, A. pisum, P. humanus corporis and D. citri.

利用NCBI上已公布的同翅目、膜翅目、虱目、雙甲目等相近的9種生物核糖體S6激酶氨基酸序列,應(yīng)用MRGA6軟件構(gòu)建系統(tǒng)進(jìn)化樹.進(jìn)化樹分析表明褐飛虱與豌豆芽和柑橘木虱的同源性比較近.

注:生物名稱及其RPS6KA2的GenBank登錄號:褐飛虱Nilaparvata lugens(KP890676);碗豆蚜Acyrthosiphon pisum(XP_008185477.1);人體虱Pediculus humanus corporis(XP_002432758.1);柑橘木虱Diaphorina citri(XP_008475555.1);蚤狀溞Daphnia pulex(EFX78935.1);印度跳蟻Harpegnathos saltator(XP_011138915.1);大蜜蜂Apis dorsata(XP_006614005.1);意蜂Apis mellifera(XP_394955.3);佛羅里達(dá)弓背蟻Camponotus floridanus(XP_01 1251733.1);蠅蛹金小蜂Nasonia vitripennis(XP_001601498.1)圖3 褐飛虱及其他生物核糖體蛋白S6激酶的進(jìn)化樹Figure 3 Phylogenetic relationship of Ribosomal protein S6 kinase from N. lugens and other organisms

2.2 褐飛虱NlRPS6KA2基因表達(dá)變化分析

Tn褐飛虱種群的熒光定量PCR檢測結(jié)果表明,NlRPS6KA2基因在褐飛虱雌雄蟲的所有齡期均有不同程度表達(dá),在懷卵雌蟲中的表達(dá)量最高,在其他發(fā)育階段和雄成蟲中表達(dá)量都很低(圖4).這一結(jié)果與RIEHLE和BROWN[12]在研究埃及伊蚊(Aedesaegypti)中的結(jié)果類似,他們發(fā)現(xiàn)該通路中的另一個基因Akt(蛋白激酶B)只在胚胎早期和卵巢中表達(dá).

圖4 不同發(fā)育階段和不同性別褐飛虱NlRPS6KA2表達(dá)量的變化Figure 4 NlRPS6KA2 expression level in different developmental stages and different genders in N. lugens

對7個種群懷卵雌性褐飛虱的熒光定量PCR檢測表明,NlRPS6KA2基因在褐飛虱從感性水稻品種TN1到抗性水稻品種RHT上處于下調(diào)的過程,而在褐飛虱適應(yīng)這種抗性后該基因表達(dá)量表現(xiàn)為上調(diào)(圖5).這與前面所說該通路受到抑制時會影響昆蟲的生長、發(fā)育、體型和存活等,而在褐飛虱從TN1到RHT適應(yīng)的過程中正好與此相吻合,因此推遲胰島素信道通路可能與褐飛虱的致害性變異相關(guān).

圖5 7個種群懷卵褐飛虱NlRPS6KA2表達(dá)量的變化Figure 5 NlRPS6KA2 expression level of seven populations in gravid N. lugens

3 結(jié) 語

本研究在褐飛虱中克隆了一個合成核糖體蛋白S6激酶的基因NlRPS6KA2,發(fā)現(xiàn)其結(jié)構(gòu)在許多生物中都具有相同的結(jié)構(gòu)域,并且同源性很高,達(dá)到了80%以上.經(jīng)熒光定量PCR檢測發(fā)現(xiàn),該基因在褐飛虱雌雄蟲的各個時期均有表達(dá),其中在懷卵褐飛虱中表達(dá)量最高,是其他時期的23～82倍,證明該基因可能與褐飛虱卵巢的發(fā)育有關(guān).研究結(jié)果為進(jìn)一步研究NlRPS6KA2基因在褐飛虱中的功能奠定了基礎(chǔ),為了解褐飛虱致害性變異機(jī)制提供了新思路.

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Cloning and expression analysis of a ribosomal protein s6 kinase geneNlRPS6KA2 fromNilaparvatalugensSt?l(Hemiptera: Delphacidae)

LU Chaofeng, HAO Peiying, YU Xiaoping

(Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China)

The full-length cDNA ends ofNlRPS6KA2 gene to code ribosomal protein s6 kinase inNilaparvatalugenswere amplified by RT-PCR according to the RNA-seq analysis of transcriptome; and it encoded a protein of 706 amino acid residues, with two S_TKc and one S_TK_X conservative domains. The real-time quantitative PCR analysis showed that theNlRPS6KA2 gene was slightly expressed in nymphs and males, but was strongly expressed in gravid females. It also showed that the expression level ofNlRPS6KA2 was down-regulated in the progress of brown planthopper (BPH) adaption from variety TN1 to variety RHT, and the expression level ofNlRPS6KA2 was slightly up-regulated when BPH was adjusted to RHT. This study was contributes to the further study about this gene’s function inNilaparvatalugens. It also puts forward new ideas to explain the virulence changes.

Nilaparvatalugens;NlRPS6KA2; real-time quantitative PCR; the virulence changes

1004-1540(2015)02-0216-05

10.3969/j.issn.1004-1540.2015.02.017

2015-03-05 《中國計量學(xué)院學(xué)報》網(wǎng)址：zgjl.cbpt.cnki.net

國家自然科學(xué)基金資助項(xiàng)目(No.31171860,30900944),國家重點(diǎn)基礎(chǔ)研究發(fā)展計劃(“973”計劃)項(xiàng)目(No.2012CB114100,2010CB126200),浙江省公益性技術(shù)應(yīng)用研究計劃項(xiàng)目(No.2012C22041).

Q965

A