一個(gè)番茄細(xì)胞分裂素受體類激酶基因的生物信息學(xué)分析

李愛芹 李利斌 劉國(guó)明

摘要:利用生物信息學(xué)的方法從番茄的基因組中解析得到一個(gè)與擬南芥滲透脅迫相關(guān)的細(xì)胞分裂素受體類激酶HK1高度同源的基因,命名為L(zhǎng)eHK1,并對(duì)它的序列結(jié)構(gòu)、蛋白基序、啟動(dòng)子區(qū)的順式元件和基因的遺傳進(jìn)化進(jìn)行了分析。 結(jié)果顯示,番茄基因LeHK1編碼的蛋白具有組氨酸蛋白激酶的結(jié)構(gòu)特征,而且在進(jìn)化上與擬南芥的滲透脅迫感受基因AtHK1分屬同一個(gè)類群。從基因順式反應(yīng)元件分析來看,番茄基因LeHK1具有多個(gè)響應(yīng)不同環(huán)境信號(hào)如熱、干旱、激素信號(hào)的順式元件,預(yù)示這個(gè)基因在環(huán)境響應(yīng)和植物生長(zhǎng)發(fā)育過程中具有重要功能。

關(guān)鍵詞:番茄; HK1; 基因和蛋白結(jié)構(gòu); 順式反應(yīng)元件; 遺傳進(jìn)化

中圖分類號(hào):Q75文獻(xiàn)標(biāo)識(shí)碼:ADOI編碼:10.3969/j.issn.1006-6500.2009.05.001

Bioinformatics of A Cytokinin Receptor Like Kinase Gene in Tomato(Lycopersicum esculentum L.)

LI Ai-qin1, LI Li-bin2, LIU Guo-ming3

(1. High and New Technology Center of Shandong Academy of Agricultural Sciences,Jinan, Shandong 250100, China; 2. Vegetable Research Institute, Shandong Academy of Agricultural Sciences, Jinan, Shandong250100, China; 3.Taitou Town Comprehensive Agricultural Servicce Station of Shouguang City, Shouguang , Shandong 262735,China)

Abstract:Through bioinformatic method and datamining, a tomato homolog of arabidopsis histidine kinase gene HK1 which encodes a cytokinin recepter like protein, was isolated and named LeHK1. The structure ofLeHK1 sequence and its putative coding protein, aslo its cis-elements in promoter regioin and phylogenetic relasionship with other plants HKs was analyzed. The results showed that LeHK1 possesed the character of histidine kinase, and it was clusteredin the same group with AtHK1. According to the result of cis-elements analysis,tomato HK1 has muptiple cis-elements responsive to different environmental factors such as heat, drought, and hormones. This suggests that LeHK1 may play key roles in response to stresses and development.

Key words: Lycopersicum esculentum; HK1; gene and protein strtuctre ; cis-element; phylogeny

組氨酸蛋白激酶家族在植物乙烯、細(xì)胞分裂素和光信號(hào)響應(yīng)過程中具有重要功能,參與和調(diào)節(jié)植物生長(zhǎng)發(fā)育和對(duì)環(huán)境因子響應(yīng)的眾多過程。組氨酸蛋白激酶基因在模式植物擬南芥和水稻中得到了廣泛和深入的研究[1-12], 但是在西紅柿中的研究,尤其是對(duì)與細(xì)胞分裂素信號(hào)相關(guān)的組氨酸激酶基因的研究還很不充分?，F(xiàn)有研究表明,細(xì)胞分裂素受體類蛋白在植物生長(zhǎng)發(fā)育和逆境響應(yīng)過程中具有極其重要的功能[1-3,5-15]。番茄作為世界上種植面積最大、也是我國(guó)最重要的蔬菜之一,在農(nóng)業(yè)生產(chǎn)和人們的生活中具有不可替代的作用。因此,研究番茄細(xì)胞分裂素受體類蛋白基因?qū)τ诟牧挤训目鼓嫘?以適應(yīng)全球氣候變暖和水資源日益匱乏,以及由于各種人為原因造成的土壤鹽漬化等局面,力求生產(chǎn)更多的蔬菜,滿足人們的生活需求具有重要意義。擬南芥滲透脅迫感受基因AtHK1(細(xì)胞分裂素受體類蛋白基因)已被克隆并得到功能驗(yàn)證[1,7],其直系同源基因在楊樹、長(zhǎng)春花中都已得到克隆和研究[13,16], 但在西紅柿中還未見報(bào)道。因此,本研究擬利用擬南芥基因AtHK1的信息來解析番茄的同源基因序列。

1材料和方法

利用擬南芥的滲透脅迫感受基因AtHK1序列在Genbank中搜索相應(yīng)的番茄基因組序列[17],根據(jù)內(nèi)含子的邊界特征解析得到編碼區(qū)序列,利用編碼區(qū)和基因組序列進(jìn)行結(jié)構(gòu)展示[18],并對(duì)預(yù)測(cè)編碼的蛋白進(jìn)行結(jié)構(gòu)特征分析[19]和亞細(xì)胞定位預(yù)測(cè)[20]。根據(jù)基因組序列得到上游啟動(dòng)子區(qū)的序列,并對(duì)啟動(dòng)子序列中的順式反應(yīng)元件進(jìn)行預(yù)測(cè)分析[21]。遺傳進(jìn)化分析利用MEGA4.1來進(jìn)行,采用Bootstrap test-Neighbor Joining 方法,重復(fù)500次運(yùn)算。

2結(jié)果與分析

2.1番茄LeHK1的序列和結(jié)構(gòu)特征

本研究通過比較基因組學(xué)和生物信息學(xué)的方法從番茄基因組中得到一個(gè)與擬南芥HK1同源的基因組序列,命名為L(zhǎng)eHK1。其預(yù)測(cè)編碼的蛋白與擬南芥HK1的序列一致性為60.81%。LeHK1基因組序列長(zhǎng)為5 261 bp,編碼區(qū)為3 738 bp,預(yù)測(cè)編碼一個(gè)含有1 245個(gè)氨基酸殘基,分子量為138.86 kD的蛋白(表1)。這個(gè)基因位于番茄2號(hào)染色體的BAC克隆AC215474中,含有13個(gè)外顯子,其外顯子和內(nèi)含子的結(jié)構(gòu)如圖1所示。

2.2 番茄LeHK1的進(jìn)化分析

番茄LeHK1與擬南芥細(xì)胞分裂素受體相關(guān)的蛋白基因家族的進(jìn)化分析表明(圖2),LeHK1與AtHK1的關(guān)系最近, 推測(cè)與AtHK1有類似的功能,在滲透脅迫反應(yīng)過程中具有重要作用。另外,LeHK1類基因在苔蘚、楊樹、長(zhǎng)春花中都已得到克隆,而且在進(jìn)化上分為同一個(gè)類群。長(zhǎng)春花的CIHK1基因受冷脅迫誘導(dǎo)[16],因此,推測(cè)LeHK1可能在冷脅迫響應(yīng)中也具有某種功能。功能性失活和獲得研究表明,AtHK1能夠正向調(diào)節(jié)植物對(duì)干旱、鹽的響應(yīng)和ABA的信號(hào)轉(zhuǎn)導(dǎo)[1,2,7]?；蛐酒治龅慕Y(jié)果顯示,突變體athk1中多個(gè)受逆境/ABA誘導(dǎo)表達(dá)的基因表達(dá)明顯下降,這包括AREB1、ANAC和DREB2A及其調(diào)控的下游基因[1]。這說明,AtHK1基因作用于AREB1、ANAC和DREB2A的上游,可以通過依賴和不依賴ABA的信號(hào)途徑正向調(diào)節(jié)逆境響應(yīng)。另外,研究表明AtHK1在植物生長(zhǎng)和種子成熟中也具有重要功能[1,7]。

2.3番茄LeHK1基因的順式反應(yīng)元件

通過分析啟動(dòng)子及其上游序列的順式反應(yīng)元件可以預(yù)測(cè)基因的功能。因此,本研究在得到LeHK1的基因組序列以后,取起始密碼子上游2 000 bp的序列進(jìn)行順式元件分析,結(jié)果見表2。在LeHK1起始密碼子上游2 000 bp的范圍內(nèi)共有10類順式反應(yīng)元件,包括赤霉素響應(yīng)元件GARE和TATC-box,熱激響應(yīng)元件HSE,能和Myb蛋白結(jié)合的干旱誘導(dǎo)的順式反應(yīng)元件mybs,富含TC的逆境響應(yīng)元件,晝夜節(jié)律響應(yīng)元件,胚乳特異表達(dá)的順式元件Skn-1,與防衛(wèi)反應(yīng)有關(guān)的順式元件W-box和EIRE,還有Box-W1。這些結(jié)果表明,LeHK1基因可能不僅能夠響應(yīng)某些激素、熱等非生物因子,而且與防衛(wèi)反應(yīng)和種子發(fā)育也有關(guān)系,值得深入探討。

2.4番茄LeHK1蛋白的結(jié)構(gòu)預(yù)測(cè)

為了研究LeHK1的功能,在解析得到它的編碼序列后,對(duì)其預(yù)測(cè)編碼的蛋白進(jìn)行了結(jié)構(gòu)分析。結(jié)果顯示,LeHK1蛋白含有磷酸結(jié)合結(jié)構(gòu)域、反應(yīng)調(diào)節(jié)結(jié)構(gòu)域、組氨酸激酶結(jié)構(gòu)域、跨膜螺旋結(jié)構(gòu)域和潛在的豆蔻?；稽c(diǎn)。這說明LeHK1蛋白具有組氨酸激酶的結(jié)構(gòu)特征,而且有可能被豆蔻?；?。蛋白豆蔻?；欣谂c細(xì)胞膜的結(jié)合。亞細(xì)胞定位預(yù)測(cè)表明,LeHK1蛋白可能定位在細(xì)胞膜上,這與結(jié)構(gòu)分析的結(jié)果相一致。

3討論

細(xì)胞分裂素受體類激酶在植物逆境響應(yīng)和生長(zhǎng)發(fā)育過程具有重要功能。本研究解析的番茄基因LeHK1編碼的蛋白具有組氨酸蛋白激酶的結(jié)構(gòu)特征,而且在進(jìn)化上與擬南芥的滲透脅迫感受基因HK1分屬同一個(gè)類群,二者蛋白序列一致性達(dá)60.81%,而且亞細(xì)胞定位預(yù)測(cè)的結(jié)果表明LeHK1蛋白可能定位在細(xì)胞膜上。從這些分析結(jié)果來看,番茄基因LeHK1應(yīng)為細(xì)胞分裂素受體類激酶基因,可能與擬南芥的滲透脅迫感受基因AtHK1具有相同或者相似的功能。另外,從基因順式反應(yīng)元件分析來看,番茄基因LeHK1 具有多個(gè)能響應(yīng)不同環(huán)境信號(hào)如熱、干旱、晝夜節(jié)律、病原物侵染,以及激素信號(hào)如赤霉素等的順式反應(yīng)元件,可對(duì)多種信號(hào)作出響應(yīng),預(yù)示這個(gè)基因在環(huán)境信號(hào)響應(yīng)和植物生長(zhǎng)發(fā)育過程中具有重要功能。至于番茄基因LeHK1對(duì)各種環(huán)境和激素信號(hào)的響應(yīng)模式,在逆境響應(yīng)和生長(zhǎng)發(fā)育中的具體功能以及與細(xì)胞分裂素信號(hào)的關(guān)系需要深入探討。本研究為進(jìn)一歩研究番茄LeHK1基因的功能奠定了基礎(chǔ)。

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