中国萤火虫云南窗萤荧光素酶cDNA的克隆表达和序列分析

从一种来自中国日行性萤火虫(云南窗萤)发光器官mRNA中克隆、测序并表达了有功能的荧光素酶.云南窗萤荧光素酶的cDNA序列有1647个碱基,编码548个氨基酸残基.从推测得到的氨基酸序列的比对分析得出:云南窗萤的荧光素酶与来自Lampyris noctiluca,L.turkestanicus和Nyctophila cf.caucasica三种萤火虫的荧光素酶有97.8%的序列一致性.从推测得出的氨基酸序列进行系统发育分析,其结果表明:云南窗萤和Lampyris Nyctophila聚在一起,与同属的发光强夜行性的萤火虫不形成的单系.云南窗萤荧光素酶在大肠杆菌中表达的条带大约70kDa,并且在有荧光素存在时发出黄绿色荧光.对荧光素酶的结构模拟和分析表明,云南窗萤荧光素酶基因的氨基端和羧基端结构域之间的裂沟处存在这5个多肽环,这正是从其他荧光素酶推测得到的催化荧光反应时的底物结合位点.云南窗萤和窗萤属的其他3种萤火虫的荧光素酶卡目比,有13个不同氨基酸位点,位于模拟分子结构的表面.对于这些多肽环、不刚氨基酸残基和晶体结构的进一步研究有利于解释日行和夜行性萤火虫荧光素酶的差异.     

栉角雪萤的荧光酶基因结构及其系统发育分析

短角窗萤属是萤科第四大属，但未见有该属物种荧光酶基因的研究报道。通过对总基因组的PCR扩增，对该属的栉角雪萤荧光酶基因进行了测序分析。基因序列长1 958 bp。与已知荧光酶基因进行同源性比较后推断，栉角雪萤的荧光酶基因由7个外显子和6个内含子组成，编码547个氨基酸残基；由推导的氨基酸序列进行同源性比较后发现，栉角雪萤的荧光酶基因与同一亚科中Lampyrini族和Cratomorphini族分别具有93—94%和92%相似性，而与北美萤火虫Photinus pyralis(Photinini族)的相似性较低(83%)。系统发育分析进一步表明栉角雪萤与Pyrocoelia、Lampyris、Cratomorphus和Photinus同属于萤亚科，且与前3个属的亲缘关系较近。这在一定程度上与形态(Branham & Wenzel, 2003)及线粒体DNA(Li et al, 2006)系统发育分析所得结果一致。     

栉角雪萤的荧光酶基因结构及其系统发育分析

短角窗萤属是萤科第四大属,但未见有该属物种荧光酶基因的研究报道。通过对总基因组的PCR扩增,对该属的栉角雪萤荧光酶基因进行了测序分析。基因序列长1958bp。与已知荧光酶基因进行同源性比较后推断,栉角雪萤的荧光酶基因由7个外显子和6个内含子组成,编码547个氨基酸残基;由推导的氨基酸序列进行同源性比较后发现,栉角雪萤的荧光酶基因与同一亚科中Lampyrini族和Cratomorphini族分别具有93—94％和92％相似性,而与北美萤火虫Photinus pyralis（Photinini族）的相似性较低（83％）。系统发育分析进一步表明栉角雪萤与Pyrocoelia、Lampyris、Cratomorphus和Photinus同属于萤亚科,且与前3个属的亲缘关系较近。这在一定程度上与形态（Branham＆Wenzel,2003）及线粒体DNA（Lietal,2006）系统发育分析所得结果一致。     

不同地域卵黄萤荧光素酶基因的克隆与序列分析

为了比较不同地域萤火虫荧光素酶基因的进化关系,通过GenBank中已知的荧光素酶基因保守区段设计引物,利用5′ RACE（rapid amplification of cDNA ends）和3′ RACE技术克隆了来自云南省文山州和西双版纳州的同种卵黄萤荧光素酶基因cDNA和全基因序列.来自不同地域的2种卵黄萤荧光素酶在基因序列上存在3个不同碱基位点,但是它们编码的荧光素酶只存在1个不同的氨基酸.卵黄萤荧光素酶基因全长（从起始密码子到终止密码子）1998 bp,包含7个外显子,6个内含子,其cDNA序列共1976 bp,包含102 bp 5′ UTR (untranslated region)、1635 bp的荧光素酶基因开放阅读框和239 bp的3′ UTR序列.卵黄萤荧光素酶基因的开放阅读框编码1个544个氨基酸的蛋白质,比同属的其它几种荧光素酶少4个氨基酸. 来自2个不同地域的卵黄萤荧光素酶在进化上是比较保守的,它们与北美萤火虫Photinus pyralis荧光素酶在碱基序列上分别有629%和63%相似性.     

云南窗萤的形态学及其生物学特性（鞘翅目：萤科）

研究了云南窗萤卵到成虫不同发育阶段的形态学。根据野外观察和实验室饲养结果,记录了其生物学特性。卵、幼虫、蛹和雌成虫可发弱光,但雄成虫仅在受刺激时才发出更弱的光,云南窗萤可归属于昼行性萤火虫,而化学信号则是其雌雄在求偶时的主要识别方式。     

千里光肌动蛋白基因（Actin）的生物信息学及功能分析

肌动蛋白（Actin）是广泛存在于高等植物中的组成型表达蛋白质,与细胞分裂、细胞运动、细胞信号转导等功能密切相关。为明确肌动蛋白性质与功能的关系,本研究从千里光全长cDNA文库中分离得到Actin基因。序列分析结果表明：该基因长度为1 481 bp,编码360个氨基酸残基的多肽,与甘草Actin基因编码的氨基酸序列（GenBank登录号：ABW71681.1）的同源性最高,达96%;预测蛋白质的分子量为40.02 kDa,理论等电点为5.85;结构分析发现,螺旋结构和无规则卷曲构成Actin二级结构的主要组分;三级结构预测发现,Actin具有4个结构域;蛋白系统发生树发现,与甘草和鹰嘴豆的亲缘关系较近。本研究认为,高等植物Actin可能参与基因的转录调控,其氨基酸突变位点未对功能结构域产生影响。     

不同寄主植物的烟粉虱（Bemisia tabaci）内共生菌传毒相关GroEL蛋白的一致性

通过对7种寄主植物上B型烟粉虱北京种群的内共生菌传毒相关groEL基因进行PCR扩增和测序,结合已有的相关序列,构建了groEL基因及其编码的GroEL蛋白的分子系统树。结果表明：烟粉虱内共生菌产生的groEL基因是一个非常保守的基因,北京不同寄主植物的烟粉虱内共生菌与Israel B型烟粉虱内共生菌的groEL基因亲源关系非常近,位于同一进化分支,其编码的GroEL蛋白的分子系统树也基本上是一致的。不同物种的groEL基因及其编码的GroEL蛋白分别位于不同的分支,说明groEL基因及其编码的GroEL蛋白的分子系统树可以用于分析物种间的进化关系。氨基酸序列比较表明：烟粉虱内共生菌GroEL具有原核GroEL的保守氨基酸、ATP酶活性位点、多肽结合位点和GroES连接位点,为典型的hsp60。不同来源烟粉虱内共生菌GroEL有少数几个保守氨基酸发生了置换,可能不是GroEL功能的重要位点。说明在容易变异的细菌基因组中,groEL基因为了维持其正常重要的生理功能,会通过保持功能位点的稳定性来应对不同生态因素的影响。     

番茄泛素蛋白基因LeEBF1和LeEBF2的克隆表达分析

通过RACE和RT-PCR方法从番茄中克隆了LeEBF1（EIN3 binding F-box protein 1）和LeEBF2（EIN3 binding F-box protein 2）的全长cDNA序列,两个基因LeEBF1、LeEBF2全长分别是2 866和2 891 bp,对序列的分析表明,它们的开放阅读框分别是1 911和1 995 bp,编码区编码637和665个氨基酸残基,在氨基端含保守的F-box区域和在羧基端有14个亮氨酸重复单位,通过BLAST软件和DNAMAN分析表明这两个基因的氨基酸序列与拟南芥EBF1和EBF2有58.6％相似,同时又与其他物种的EBF蛋白的F-box区域比较有24.4％到73.2％的相近。Northern杂交指出：LeEBF1与LeEBF2在野生型和Nr的幼叶中的表达量高于成熟叶;当在果实发育期,LeEBF1与LeEBF2在青果期的表达量相比其他时期要弱。初步结果表明,LeEBF1与LeEBF2可能在番茄的生长发育中起着重要的作用。     

丹参ACC氧化酶基因(SmACO1)的克隆与序列分析

依据丹参转录组数据库序列信息,采用RT-PCR和染色体步移技术从丹参中首次克隆得到ACC氧化酶基因,命名为SmACO1(GenBank注册号为JQ026111)。该基因gDNA序列长1 347 bp,由3个外显子和2个内含子组成;cDNA全长1 117 bp,包含945 bp的开放阅读框,编码314个氨基酸残基。生物信息学分析显示SmACO1为无信号肽与跨膜结构域,且定位于细胞质的稳定亲水蛋白,含有Fe²⁺依赖的加氧酶结构域。实时荧光定量PCR结果表明,SmACO1基因在丹参不同组织器官中差异表达,花中表达量最高;其表达受到病原菌和茉莉酸甲酯的诱导,表明SmACO1基因可能在植物防御反应中发挥作用。     

甜菜ATP合酶β亚基基因atpB的克隆、序列分析及进化

atpB基因编码ATP合酶β亚基,是光合作用中的重要基因。ATP合酶是生物体内能量代谢的关键酶,参与氧化磷酸化和光合磷酸化反应。利用植物叶绿体基因组在进化过程中高度保守的特点,根据已知植物烟草、水稻和菠菜等的叶绿体基因组全序列,设计并合成了一对引物,以甜菜叶绿体DNA为模板,PCR扩增得到包含atpB 完整基因（GenBank登录号为 DQ067451）在内的一段序列,测序与序列分析表明：该克隆片段全长2 293 bp,其中包括有1 497 bp的编码区序列,推测编码498个氨基酸。同源性比较,该克隆基因与烟草、菠菜、油菜、水稻atpB基因的核苷酸序列同源性分别为90.92%、95.79%、87.71%和86.37%,推测的氨基酸序列同源性分别为94.58%、97.19%、92.17%和91.97%。同时,建立了几种植物的氨基酸序列系统进化树。     

Firefly luciferase genes from the subfamilies Psilocladinae and Ototretinae (Lampyridae, Coleoptera)

Firefly luciferase genes have been isolated from approximately 20 species of Lampyrinae, Luciolinae, and Photurinae. These are mostly nocturnal luminescent species that use light signals for sexual communication. In this study, we isolated three cDNAs for firefly luciferase from Psilocladinae (Cyphonocerus ruficollis) and Ototretinae (Drilaster axillaris and Stenocladius azumai), which are diurnal non-luminescent or weakly luminescent species that may use pheromones for communication. The amino acid sequences deduced from the three cDNAs showed 81-89% identities to each other and 60-81% identities with known firefly luciferases. The three purified recombinant proteins showed luminescence and fatty acyl-CoA synthetic activities, as observed in other firefly luciferases. The emission maxima by the three firefly luciferases (λmax, 545-546 nm) were shorter than those by known luciferases from the nocturnal fireflies (λmax, 550-568 nm). These results suggest that the primary structures and enzymatic properties of luciferases are conserved in Lampyridae, but the luminescence colors were red-shifted in nocturnal species compared to diurnal species.     

Orthologous gene of beetle luciferase in non-luminous click beetle, Agrypnus binodulus (Elateridae), encodes a fatty acyl-CoA synthetase

A homologous gene of beetle luciferase, AbLL (Agrypnus binodulusluciferase-like gene) was isolated from a Japanese non-luminous click beetle, A. binodulus, and its gene product was characterized. The identity of amino acid sequence deduced from AbLL with the click beetle luciferase from the Jamaican luminous click beetle, Pyrophorus plagiophthalmus, is 55%, which is higher than that between click beetle luciferase and firefly luciferase (approximately 48%). Phylogenetic analysis indicated that AbLL places in a clade of beetle luciferases, suggesting that AbLL is an orthologous gene of beetle luciferase. The gene product of AbLL (AbLL) has medium- and long-chain fatty acyl-CoA synthetase activity, but not luciferase activity. The fatty acyl-CoA synthetic activity was slightly inhibited in the presence of beetle luciferin, suggesting that AbLL has poor affinity for beetle luciferin. By comparing the amino acid residues of the catalytic domains in beetle luciferases with AbLL, the key substitutions for the luminescence activity in beetle luciferase will be proposed.     

Cloning and characterization of the cDNA for the Brazilian Cratomorphus distinctus larval firefly luciferase: similarities with European Lampyris noctiluca and Asiatic Pyrocoelia luciferases

Studies on firefly (Lampyridae) luciferases have focused on nearctic species of Photinus and Photuris and Euroasiatic species of Lampyris, Luciola, Hotaria, and Pyrocoelia. Despite accounting for the greatest diversity of fireflies in the world, no molecular studies have been carried out on the highly diverse genera from the neotropical region. Here we report the luciferase cDNA cloning for the larva of the Brazilian firefly Cratomorphus distinctus. The cDNA has 1978 bp and codes for a 547-residue-long polypeptide. Noteworthy, sequence comparison as well as functional properties show the highest degree of similarity with Lampyris noctiluca (93%) and Pyrocoelia spp. (91%) luciferases, suggesting a close phylogenetic relationship despite the geographical distance separating these species. The bioluminescence emission spectrum peaks at 550 nm and, as expected, is sensitive to pH, shifting to 605 nm at pH 6. The kinetic properties of the recombinant luciferase were similar to those of other firefly luciferases.     

Crystal structure of native and a mutant of Lampyris turkestanicus luciferase implicate in bioluminescence color shift

Firefly bioluminescence reaction in the presence of Mg^2 +, ATP and molecular oxygen is carried out by luciferase. The luciferase structure alterations or modifications of assay conditions determine the bioluminescence color of firefly luciferase. Among different beetle luciferases, Phrixothrix hirtus railroad worm emits either yellow or red bioluminescence color. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional arginine residue at 353 that is absent in other firefly luciferases. It was reported that insertion of Arg in an important flexible loop350–359 showed changes in bioluminescence color from green to red and the optimum temperature activity was also increased. To explain the color tuning mechanism of firefly luciferase, the structure of native and a mutant (E354R/356R/H431Y) of Lampyris turkestanicus luciferase is determined at 2.7 Å and 2.2 Å resolutions, respectively. The comparison of structure of both types of Lampyris turkestanicus luciferases reveals that the conformation of this flexible loop is significantly changed by addition of two Arg in this region. Moreover, its surface accessibility is affected considerably and some ionic bonds are made by addition of two positive charge residues. Furthermore, we noticed that the hydrogen bonding pattern of His431 with the flexible loop is changed by replacing this residue with Tyr at this position. Juxtaposition of a flexible loop (residues 351–359) in firefly luciferase and corresponding ionic and hydrogen bonds are essential for color emission.     

The nucleotide sequence of the luxA and luxB genes of Xenorhabdus luminescens HM and a comparison of the amino acid sequences of luciferases from four species of bioluminescent bacteria

The luxA and luxB genes of bioluminescent bacteria encode the alpha and beta subunits of luciferase, respectively. Sequences of the luxA and luxB genes of Xenorhabdus luminescens, the only terrestrial bioluminescent bacterium known, were determined and the amino acid sequence of luciferase deduced. The alpha subunit was found to contain 360 amino acids and has a calculated molecular weight of 41,005 Da, while the beta subunit contains 327 amino acids and has a calculated molecular weight of 37,684 Da. Alignment of this luciferase with the luciferases of three marine bacteria showed 196 (or 55%) conserved residues in the alpha subunit and 114 (or 35%) conserved residues in the beta subunit. The highest degree of homology between any two species was between the luciferases of X. luminescens and Vibrio harveyi with 84% identity in the alpha subunits and 59% identity in the beta subunits.     

Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures.

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of lateral lanterns along the body and red light through two cephalic lanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda max= 542 nm), and for the head lanterns of P. hirtus, which emit the most red-shifted bioluminescence (lambda max= 628 nm) among luminescent beetles, were cloned. Positive clones which emitted green (PvGR: lambda max= 549 nm) and red (PhRE: lambda max= 622 nm) bioluminescence were isolated. The lucifereases coded by PvGR (545 amino acid residues) and PhRE (546 amino acid residues) cDNAs share 71% identity. PvGR and PhRE luciferases showed 50-55% and 46-49% identity with firefly luciferases, respectively, and 47-49% with click-beetle luciferases. PhRE luciferase has some unique residues which replace invariant residues in other beetle luciferases. The additional residue Arg 352 in PhRE, which is deleted in PvGR polypeptide, seems to be another important structural feature associated with red light production. As in the case of other railroad-worms and click-beetle luciferases studied, Phrixothrix luciferases do not undergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shared in common between railroad-worm and click-beetle luciferase.     

Bioluminescence of the insect pathogen Xenorhabdus luminescens

Luminescence of batch cultures of Xenorhabdus luminescens was maximal when cultures approached stationary phase; the onset of in vivo luminescence coincided with a burst of synthesis of bacterial luciferase, the enzyme responsible for luminescence. Expression of luciferase was aldehyde limited at all stages of growth, although more so during the preinduction phase. Luciferase was purified from cultures of X. luminescens Hm to a specific activity of 4.6 x 10(13) guanta/s per mg of protein and found to be similar to other bacterial luciferases. The Xenorhabdus luciferase consisted of two subunits with approximate molecular masses of 39 and 42 kilodaltons. A third protein with a molecular mass of 24 kilodaltons copurified with luciferase, and in its presence, either NADH or NADPH was effective in stimulating luminescence, indicating that this protein is an NAD(P)H oxidoreductase. Luciferases from two other luminous bacteria, Vibrio harveyii (B392) and Vibrio cholerae (L85), were partially purified, and their subunits were separated in 5 M urea and tested for complementation with the subunits prepared from X. luminescens Hb. Positive complementation was seen with luciferase subunits among all three species. The slow decay kinetics of the Xenorhabdus luciferase were attributed to the alpha subunit.     

Bioluminescence of the insect pathogen Xenorhabdus luminescens.

Luminescence of batch cultures of Xenorhabdus luminescens was maximal when cultures approached stationary phase; the onset of in vivo luminescence coincided with a burst of synthesis of bacterial luciferase, the enzyme responsible for luminescence. Expression of luciferase was aldehyde limited at all stages of growth, although more so during the preinduction phase. Luciferase was purified from cultures of X. luminescens Hm to a specific activity of 4.6 x 10(13) guanta/s per mg of protein and found to be similar to other bacterial luciferases. The Xenorhabdus luciferase consisted of two subunits with approximate molecular masses of 39 and 42 kilodaltons. A third protein with a molecular mass of 24 kilodaltons copurified with luciferase, and in its presence, either NADH or NADPH was effective in stimulating luminescence, indicating that this protein is an NAD(P)H oxidoreductase. Luciferases from two other luminous bacteria, Vibrio harveyii (B392) and Vibrio cholerae (L85), were partially purified, and their subunits were separated in 5 M urea and tested for complementation with the subunits prepared from X. luminescens Hb. Positive complementation was seen with luciferase subunits among all three species. The slow decay kinetics of the Xenorhabdus luciferase were attributed to the alpha subunit.