松毛虫赤眼蜂脂肪酸合酶基因的克隆、表达谱及其在滞育中的功能分析

【目的】本研究旨在克隆松毛虫赤眼蜂Trichogramma dendrolimi脂肪酸合酶(fattty acid synthase, Fas)基因(TdFas),并分析其在滞育中的调控功能,为松毛虫赤眼蜂滞育研究提供新依据。【方法】基于松毛虫赤眼蜂转录组数据,克隆TdFas的cDNA全长序列并进行生物信息学分析。利用RT-qPCR检测TdFas在松毛虫赤眼蜂滞育与非滞育预蛹、蛹和成虫及滞育诱导维持期预蛹和滞育解除期预蛹中的表达量。通过以1 000, 2 000和3 000 ng/μL dsTdFas溶液浸泡松毛虫赤眼蜂幼虫的RNAi方式干扰TdFas的表达,探究其对松毛虫赤眼蜂滞育率及甘油三酯相对含量的影响。【结果】克隆得到松毛虫赤眼蜂TdFas的cDNA序列全长为8 601 bp(GenBank登录号：OP146440),其开放阅读框长7 278 bp,推测编码一个2 426个氨基酸的蛋白,含有一个典型的脂肪酸合酶的β-酮酰还原酶结构域。系统发育分析结果表明,TdFas与膜翅目其他昆虫的FASs亲缘关系较近,其中与短管赤眼蜂Trichogramma pretiosum FAS亲缘关...     

松毛虫赤眼蜂滞育诱导及解除条件研究

【目的】以柞蚕Antheraea pernyi卵为繁殖寄主,对松毛虫赤眼蜂Trichogramma dendrolim滞育诱导及解除条件进行研究,以解决赤眼蜂工厂化生产和大面积应用中面临的的中、长期储存问题。【方法】通过观测不同发育阶段(寄生柞蚕卵在26℃培养40、96和144 h)、滞育诱导温度(10、13和16℃)和诱导时间对松毛虫赤眼蜂滞育的影响,确定松毛虫赤眼蜂滞育诱导条件;通过观测滞育诱导温度和滞育后的贮藏温度对滞育解除的影响,确定松毛虫赤眼蜂滞育解除条件。【结果】在松毛虫赤眼蜂的不同发育阶段对其进行持续的低温刺激均能使其导入滞育,但以小幼阶段(26℃培养40 h)开始效果最佳,寄生卵在26℃培养40 h后,转入10℃和13℃下连续诱导31 d,滞育率可达100%和99.12%。滞育诱导温度和滞育后的贮藏温度对松毛虫赤眼蜂解除滞育所需时间和解除滞育后的羽化出蜂率有较大影响,10℃诱导滞育后置于1℃冷藏的赤眼蜂解除滞育所需时间最短,解除滞育后的羽化出蜂率和单卵出蜂数更高,更耐储存。此条件下冷藏约30 d开始打破滞育,在正常发育下温度下羽化出蜂,60 d羽化出蜂率达到95.24%,冷藏4个月后羽化出蜂率仍在60%以上,单卵出蜂数高于50头。【结论】松毛虫赤眼蜂最佳滞育诱导条件为26℃培养40 h后,转入10℃连续低温诱导31 d;最佳滞育解除条件为1℃低温储存,但储存期不能超过4个月。     

金钗石斛过氧化物酶基因的克隆及原核表达

为了获得金钗石斛(Dendrobium nobile)过氧化物酶基因(命名为DnPOD)编码序列,分析该基因的分子特征,掌握该基因原核表达数据,本研究采用同源克隆的方法,以金钗石斛cDNA为模板,设计基因特异性引物克隆DnPOD基因,分析了DnPOD基因结构,将重组质粒pET28α-DnPOD在BL21 (DE3)菌株...     

大鼠诱导型一氧化氮合酶基因转录调控区的克隆与鉴定

采用单特异引物ＰＣＲ克隆法,得到大鼠诱导型一氧化氮合酶（ｉＮＯＳ）基因转录调控区ＤＮＡ片段。核酸序列分析证实,大鼠ｉＮＯＳ基因的５＇－侧翼区含有ＩＦＮ－γ和ＴＮＦ－α应答元件及ＮＦ－ｋＢ结合位点的保守序列。这些保守序列的位置及排列显区别于人和小鼠的ｉＮＯＳ基因。电泳迁移率改变分析（ＥＭＳＡ）表达,ＶＳＭＣ受ＩＬ－１和ＩＦＮ－γ刺激后,细胞核内产生某种可与ｉＮＯＳ基因５＇－侧翼区特异结合的核蛋白因     

松墨天牛漆酶基因MaLac1的克隆、原核表达及表达谱分析

【目的】本研究旨在克隆并鉴定松墨天牛Monochamus alternatus内源漆酶基因MaLac1,分析其在松墨天牛不同发育阶段的表达水平,为进一步明确MaLac1功能提供依据。【方法】基于松墨天牛肠道转录组测序数据,通过RACE克隆松墨天牛MaLac1基因的全长cDNA序列,并对其进行生物信息学分析;将该基因与pET-32a载体链接构建表达载体pET-MaLac1,导入大肠杆菌Escherichia coli Rosetta (DE3)使其表达;使用qPCR检测MaLac1基因在松墨天牛不同发育阶段(低龄幼虫、老熟幼虫、蛹、雌成虫和雄成虫)肠道中的表达差异。【结果】克隆获得松墨天牛MaLac1的cDNA全长序列(GenBank登录号:KY073340)。MaLac1开放阅读框全长2 067 bp,编码一个含688个氨基酸的蛋白质,预测分子量为78.34 kD,等电点为5.30。SignalP 4.1 Server预测MaLac1在N端包含一个15个氨基酸的信号肽。序列比对分析表明,MaLac1具有典型的昆虫漆酶基因特征,与赤拟谷盗Tribolium castaneum漆酶基因的氨...     

海藻糖合酶基因的克隆及其植物表达载体的构建

以担子菌灰树花的菌丝体中提取总ＲＮＡ,并纯化出ｍＲＮＡ,ｍＲＮＡ经反转录合成ｃＤＮＡ第一链,以ｃＤＮＡ第一链为模板经ＰＣＲ扩增海藻糖合酶（Ｔｓａｓｅ）基因,获得一长约２．２ｋｂ的片段,把该片段连接一ｐＧＥＭ－Ｔ－ｅａｓｙ ｖｅｃｔｏｒ上进行测序,其全长共２１９９ｂｐ。随后将此片段以正向插入植物表达载体ｐＢＩ１２１的ＨｉｎｄⅢ＋Ｘｂａｌ位点构建ｐＵＢ,再把海藻糖合酶基因以正向插入载体ｐＵＢ的ＢａｍＨ     

蛇白蔹白藜芦醇合酶基因CNRS2的克隆与原核表达

白藜芦醇合酶(resveratrol synthetic enzyme,RS)是植物白藜芦醇合成途径中的关键酶,利用已知的葡萄RS基因(AF274281)序列设计并合成了一对引物,以蛇白蔹基因组DNA为模板,PCR扩增得到包含RS完整基因在内的一段序列,测序与序列分析表明:该克隆片段全长1 536bp,其中包含一个内含子及两个外显子。采用悬挂延伸PCR法克隆了目的基因,命名为CNRS2。序列分析表明该基因的开放读码框1 170 bp,编码389个氨基酸残基。同源性比较发现,CNRS2与已知葡萄RS基因序列的同源性达93%～98%。CNRS2与pET-30a(+)构建原核表达载体,经IPTG诱导后可表达获得相对分子量约为46 kD的外源融合蛋白。以上结果证实CNRS2属葡萄RS基因家族成员,为今后进一步对该基因的研究利用打下基础。     

水母雪莲查尔酮合酶基因的克隆、表达及酶活分析

从水母雪莲Saussurea medusa Maxim. cDNA文库中得到一段查尔酮合酶基因 (SmCHS) 片段,然后通过RT-PCR得到完整的查尔酮合酶基因cDNA。序列分析表明SmCHS全长1 313 bp,其开放阅读框为1 170 bp,编码389个氨基酸,预测表达蛋白的分子量为43 kDa。构建原核表达质粒pET28a(+)-SmCHS,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。经IPTG诱导表达后,对表达产物进行SDS-PAGE分析,结果显示,表达的融合蛋白以部分可溶的形式存在。用Ni-NTA预装柱对融合蛋白进行亲和纯化,对纯化蛋白进行酶活检测,结果表明融合蛋白具有查尔酮合酶活性,可催化底物4-香豆酰辅酶A和丙二酰辅酶A缩合生成产物柚皮素查尔酮。     

榛子产紫杉醇内生真菌Penicillium aurantiogriseum中GGPP合酶基因的克隆与表达

牻牛儿基牻牛儿基焦磷酸合酶是产紫杉醇内生真菌紫杉醇合成下游途径中的关键步骤之一,在榛子产紫杉醇内生真菌中进行紫杉醇生物合成研究时首先要确认GGPP合酶的存在。该研究通过RT-PCR方法克隆得到了榛子产紫杉醇内生真菌Penicillium aurantiogriseum中GGPP合酶基因Pa GGPPS(Gen Bank登录号为KM881430)的c DNA开放阅读框(Open reading frame,ORF)。利用生物信息学方法,我们分析了该基因的序列,并对其编码的氨基酸序列进行了预测。结果发现该基因ORF长度为1 113 bp,预计蛋白分子量为40.98k D,等电点为6.168,表明该蛋白呈酸性。对其进行亲疏水性分析发现肽链整体呈现为亲水性。Pa GGPPS的主要二级结构元件为α-螺旋,并且包含一个异戊二烯类化合物合酶功能域。对榛子产紫杉醇内生真菌与其他物种的GGPPS进行氨基酸同源性分析,发现其与娄地青霉、曲霉菌和费氏新萨托菌的一致性较高,分别为94%、76%和76%。进化树分析表明来自动物、真菌和酵母的GGPPS聚为一类,来自植物的GGPPS聚为一类,其中榛子产紫杉醇内生真菌的GGPPS和青霉菌的进化关系最近,与植物的GGPPS的进化关系最远。对其进行基础生物信息学分析后,我们构建了原核表达载体,成功诱导其表达并得到可溶性蛋白。该研究结果为下一步深入研究GGPPS基因在内生真菌Penicillium aurantiogriseum紫杉醇合成途径中的作用及和构建高产紫杉醇基因工程菌株奠定了一定的基础。     

烟夜蛾几丁质酶基因的克隆与表达

运用RT-PCR技术扩增编码烟夜蛾Helicoverpaassulta(Guen啨e)幼虫几丁质酶基因的cDNA片段,将其克隆至pMD18-T载体,获得该基因的成熟蛋白阅读框序列。将该基因重组到表达型质粒pGEX-4T-2中,并转化入原核细胞中表达,序列测定结果表明,烟夜蛾幼虫几丁质酶基因的成熟蛋白阅读框全长1338bp,编码445个氨基酸残基,预测分子量和等电点分别为50.1kDa和9.26;推导的氨基酸序列与其近缘种棉铃虫几丁质酶氨基酸序列的一致性达99%,与其他6种昆虫几丁质酶的氨基酸序列也高度一致(65%~76%),并具有几丁质酶的典型特征。将该基因克隆到原核表达载体pGEX-4T-2上并转化BL21,SDS-PAGE和Western印迹分析表明,经IPTG诱导,76kDa附近没有特异蛋白条带出现,表明烟夜蛾几丁质酶基因不能在原核表达载体pGEX-4T-2中表达。     

Molecular cloning and expression of nitric oxide synthase gene in chick embryonic muscle cells

The chick skeletal muscle nitric oxide synthase (NOS) gene was cloned in order to further define the involvement of NOS in the differentiation of skeletal muscle cells. The respective cDNA had an open reading frame of 1136 amino acid residues, predicting a protein of 129,709.85 Da, and recognition sites for FAD, FMN, NADPH, and a calmodulin-binding site like those of other mammalian NOS's. Alignment of the deduced amino acid sequence revealed high homology with mammalian inducible NOS (iNOS), but not other NOS isoforms, suggesting chick skeletal muscle NOS may be an iNOS isoform. Immunoblots showed that NOS expression was highly restricted in embryonic muscle, but not in adult skeletal muscle: NOS expression markedly increased from embryonic day 9, reached a maximum by embryonic day 13, and then gradually declined until it was no longer detectable on embryonic day 19. When muscle cells obtained on embryonic day 12 were cultured, NOS expression increased transiently prior to the onset of differentiation and decreased thereafter. Inhibition of NOS expression by PDTC completely prevented muscle cell differentiation, as indicated by the inhibition of expression of myosin heavy chain and creatine kinase. The inhibitory effect of PDTC was completely reversed by addition of sodium nitroprusside, a compound that produces NO. These results clearly indicate that NOS is significantly involved in the differentiation of chick skeletal muscle cells.     

Molecular cloning, prokaryotic expression and expression at different developmental stages of cathepsin B gene in mealybug Phenacoccus solenopsis Tinsley ( Hemiptera: Pseudococcidae)

昆虫组织蛋白酶B在昆虫代谢过程中发挥重要作用.本研究利用RACE技术克隆了扶桑绵粉蚧Phenacoccus solenopsis Tinsley组织蛋白酶B基因的开放阅读框(ORF)序列,命名为PsCb(GenBank登录号:JQ727999).生物信息学分析表明,该基因的开放阅读框包含927 bp的片段,编码308个氨基酸.多序列比对表明,该基因编码的蛋白在N端变异较大,在C端保守性高.组织蛋白酶B基因的系统进化树结果表明扶桑绵粉蚧组织蛋白酶独自成为一支.原核表达电泳检测到一条大约35 kDa的目的条带,与预测的蛋白分子量相符.组织蛋白酶B基因在扶桑绵粉蚧各个虫态均有表达,卵期表达量相对较低,2龄若虫期达到最高峰,然后下降.本研究为进一步研究该基因的功能并开发出组织蛋白酶抑制剂,从而研制出扶桑绵粉蚧杀卵剂和胚胎发育抑制剂等提供理论依据.     

Molecular cloning and expression profiles of nitric oxide synthase (NOS) in mud crab Scylla paramamosain

The importance of the nitric oxide synthase (NOS) gene family is demonstrated by many studies in vertebrates and invertebrates in recent years. However, it keeps unknown of nitric oxide (NO) system and NOS gene family in mud crab Scylla paramamosain, an important cultured commercial crustacean in China and Pacific area. In this report, the cDNA of NOS containing full-length ORF was cloned from mud crab, S. paramamosain. It was of 4424 bp, including a 5′-terminal untranslated region (UTR) of 239 bp, a 3′-terminal UTR of 540 bp, which contained two ATTTA motifs, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids. Structural analysis indicated that NOS contained a typical NO synthase domain at the N-terminal, next to a flavodoxin 1 domain, a flavin adenine dinucleotide (FAD) binding domain, respectively, and a conservative nicotinamide adenine dinucleotide (NAD) binding domain structure at the C-terminal. Quantitative real-time PCR analysis revealed S. paramamosain NOS (SpNOS) to be expressed in all tissues examined, with the highest expression in midintestine and the weakest level in heart and eyestalk. The expression profiles of SpNOS indicated that the NOS expression levels were significantly induced in midintestine, hepatopancrease and hemocytes after challenged with Vibrio Parahaemolyticus, the synthetic double-stranded RNA polyinosinic polycytidylic acid (poly I:C) and lipopolysaccharides (LPS). The NOS activity in hemocytes showed significant increase during at 24 h-48 h time period after immune challenges with V. Parahaemolyticus, poly I:C and LPS. Results here may suggest that the inducible NOS play an important role in mud crab’s defense against pathogenic infection.     

扶桑绵粉蚧组织蛋白酶B基因的克隆、原核表达和不同发育阶段表达分析

昆虫组织蛋白酶B在昆虫代谢过程中发挥重要作用。本研究利用RACE技术克隆了扶桑绵粉蚧Phenacoccus solenopsis Tinsley组织蛋白酶B基因的开放阅读框（ORF）序列, 命名为PsCb （GenBank登录号: JQ727999）。生物信息学分析表明, 该基因的开放阅读框包含927 bp的片段, 编码308个氨基酸。多序列比对表明, 该基因编码的蛋白在N端变异较大, 在C端保守性高。组织蛋白酶B基因的系统进化树结果表明扶桑绵粉蚧组织蛋白酶独自成为一支。原核表达电泳检测到一条大约35 kDa的目的条带, 与预测的蛋白分子量相符。组织蛋白酶B基因在扶桑绵粉蚧各个虫态均有表达, 卵期表达量相对较低, 2龄若虫期达到最高峰, 然后下降。本研究为进一步研究该基因的功能并开发出组织蛋白酶抑制剂, 从而研制出扶桑绵粉蚧杀卵剂和胚胎发育抑制剂等提供理论依据。     

Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase.

Endothelium-derived relaxing factor (EDRF), identified as nitric oxide (NO), is derived from a guanidino nitrogen of L-arginine via its metabolism by nitric oxide synthase (NOS). Herein, we report the molecular cloning of a cDNA encoding the constitutive calcium-calmodulin (Ca2+/CaM)-regulated nitric oxide synthase (ECNOS). A full-length ECNOS clone was isolated by screening a bovine aortic endothelial cell cDNA library using a fragment of rat brain NOS (bNOS) cDNA. This cDNA has an open reading frame of 3615 nucleotides encoding a 1205-amino acid protein. Membranes prepared from COS cells transfected with the ECNOS cDNA demonstrated NADPH- and Ca2+/CaM- dependent conversion of L-, but not D-, arginine to NO and citrulline that was inhibited by NG-nitro-L-arginine methyl ester. Comparison of the deduced amino acid sequence of ECNOS to the bNOS and macrophage NOS (Mac-NOS) sequences revealed 57 and 50% identity, respectively. In addition, ECNOS contains a unique N-myristylation consensus sequence (not shared by bNOS or Mac-NOS) that may explain its membrane localization.     

Molecular cloning,prokaryotic expression and promoter analysis of squalene synthase gene from Schizochytrium Limacinum

Squalene synthase (SQS) is an important enzyme in the steroid biosynthetic pathways which condenses two molecules of farnesyl pyrophosphate into a squalene. In this study, the gene encoding SQS was isolated from Schizochytrium limacinum and characterized. The full-length cDNA of S. limacinum SQS gene (SlSQS) is 1605 bp in length, it contains a 1293 bp ORF encoding a polypeptide of 430 amino acids. Multiple amino acid sequence alignment showed that the SlSQS protein sequence shared 5 conserved signature domains and a hydrophobic carboxy-terminal part with other known SQS protein sequences. C-terminal-truncated SlSQS was constructed into expression vector pGEX and successfully expressed in Escherichia coli cells. The expressed fusion protein was confirmed to have SQS activity. In addition, a 724 bp promoter region of SlSQS was also cloned and several cis-acting elements were predicted. These results might be helpful to understand the structure and expression regulation of SQS in S. limacinum.     

Pattern of Egg Management by Trichogramma cacoeciae and T. dendrolimi (Hymenoptera: Trichogrammatidae)

The effect of temporary host deprivation on parasitization rates of Trichogramma cacoeciae Marchal and T. dendrolimi Matsumura was investigated. The study was conducted with females that we allowed to engage in 3 days of oviposition after various periods of host deprivation. It seems that the production and management of eggs by the two species is completely different. During the first day of oviposition, parasitization by T. cacoeciae was almost unaffected after 1 to 5 days of host deprivation. As deprivation time increased, however, the number of parasitized hosts decreased from an average of 28.6 ±2.0 hosts provided at emergence to an average of 12.5 ±2.3 hosts when the waiting time was 10 days. The number of hosts parasitized on the first day of parasitization by T. dendrolimi were not affected whatever the waiting test period. During the second or third days of oviposition, the lack of suitable hosts for T. cacoeciae did not depress egg-laying potentiality, whereas a strong reduction in parasitization rates by T. dendrolimi occurred in the next 2 days of oviposition whatever was the waiting period. This leads to ca. 50% reduction in total activity of 3 days of oviposition. Only in T. cacoeciae was it possible to distinguish between ageing and host deprivation. The data suggest that T. dendrolimi is a typical proovigenic species, while T. cacoeciae is neither definitely proovigenic nor synovigenic. A slight decrease in rate of emergence of offspring of T. cacoeciae females that had waited 8 to 10 days for their hosts was observed. As far as biological control is concerned, the efficiency of T. dendrolimi females may be more sensitive to host deprivation than T. cacoeciae.     

Maternal thermal effect on diapause in Trichogramma species (Hymenoptera: Trichogrammatidae)

Both direct thermal and maternal photoperiodic effects on diapause induction have been thoroughly investigated in many insect species, while maternal thermal effects have been infrequently studied. We studied the effect of temperature during development of maternal generation on the proportion of diapausing progeny in four species of the genus Trichogramma Westw., minute egg parasitoids, widely used for biological control of lepidopteran pests. The maternal generations were reared at day lengths of 12 and 18 h and temperatures of 17, 20, 25 and 30°C, and their progeny developed under day length of 12 h and temperatures of 13 and 14°C. In T. evanescens and T. piceum, the proportion of diapausing progeny decreased with increasing temperature under all tested photoperiods and thermal regimes of progeny development; the high temperature of 30°C totally averted diapause of progeny. In T. buesi and T. principium, low temperatures of 17 and 20°C resulted in relatively high proportion of diapausing progeny only when the maternal generation developed under short‐day conditions. The threshold of the maternal thermal response varied from 17–18 to 22–23°C. Under field conditions, Trichogramma females are exposed to such high temperatures only during summer, when diapause in their progeny is in any case prevented by the maternal photoperiodic response and by the thermal response of the larvae. We conclude that the maternal thermal effect on diapause induction, although to a different extent, is inherent to Trichogramma species but, at least as suggested by laboratory experiments, it does not play any role in the regulation of seasonal development under natural conditions. However, during mass rearing of Trichogramma wasps, it should be taken into account that high temperature, even when combined with short photoperiod, can avert diapause in the next generation.