甜椒细胞质小分子量热激蛋白基因(CaHSP18)的cDNA克隆与表达

用RT-PCR和RACE-PCR技术,从热激处理的甜椒叶片总RNA中扩增出了细胞质小分子量热激蛋白(sHSP)全长779 bp的cDNA基因序列,包含一个480 bp开放阅读框,编码159个氨基酸.Southern杂交结果表明在甜椒基因组中有该基因的小的多基因家族.Northern结果显示该基因在甜椒根、茎、叶中的表达受热激和低温的诱导.原核表达分析表明该基因在高温以及低温条件下可以提高大肠杆菌的生存能力.     

叶绿体小分子量热激蛋白介绍

本文对叶绿体小分子量热激蛋白的研究进行了简要的回顾和总结.叶绿体小分子量热激蛋白是热激蛋白超家族的成员,具有3个特殊的保守区域;当植物遇到热胁迫时,叶绿体小分子量热激蛋白能够保护光合系统Ⅱ和类囊体膜;初步分析了叶绿体小分子量热激蛋白与植物的耐热性和耐冷性关系以及其分子伴侣功能.     

叶绿体小分子量热激蛋白介绍

本文对叶绿体小分子量热激蛋白的研究进行了简要的回顾和总结。叶绿体小分子量热激蛋白是热激蛋白超家族的成员,具有3个特殊的保守区域;当植物遇到热胁迫时,叶绿体小分子量热激蛋白能够保护光合系统Ⅱ和类囊体膜;初步分析了叶绿体小分子量热激蛋白与植物的耐热性和耐冷性关系以及其分子伴侣功能。     

昆虫小分子量热激蛋白的研究进展

昆虫小分子量热激蛋白(Small heat shock proteins,s HSPs)是最早被发现的热激蛋白,但是有关它们的研究相对较少。本文对昆虫小分子量热激蛋白的最新研究成果进行了总结,旨在引起人们对该类蛋白的关注,以便进一步研究其功能,探讨其可能的应用前景。目前研究表明:昆虫小分子量热激蛋白是其所有热激蛋白中最不保守的家族。同时,它们通常拥有一个α-晶状体结构域;分子量范围一般在12~43 ku;具有分子伴侣的活性。每种昆虫体内拥有多种s HSPs,而且其功能也各不相同。这些热激蛋白在昆虫的生长发育、生殖以及滞育等重要生命活动中起着重要的作用;同时在抵御不良环境以及适应性进化中也具有重要意义。随着研究的深入,还将会有更多的昆虫s HSPs被鉴定,它们更多的功能也将被逐渐发掘。     

二化螟热休克蛋白70基因的克隆及热胁迫下的表达分析

热休克蛋白70是已知热休克蛋白家族中最重要的一种, 它在细胞内的大量表达可以明显改善细胞的生存能力, 提高对环境胁迫的耐受性。为探讨热胁迫对二化螟Chilo suppressalis幼虫热休克蛋白70表达的影响, 采用RT-PCR及RACE技术从二化螟血淋巴细胞中克隆了热休克蛋白70基因全长cDNA序列。该基因全长2 102 bp, 开放阅读框 (open reading frame, ORF)为1 959 bp, 编码652个氨基酸; 5′非编码区（untranslated region, UTR)为81 bp, 3′UTR为62 bp。从该基因推导的氨基酸序列与其他昆虫的同源序列比较有很高的相似性（73%~97%)。实时定量PCR显示二化螟HSP70基因能被热胁迫诱导表达, 幼虫血淋巴细胞的HSP70基因在36℃时表达量最高。流式细胞术研究发现HSP70在蛋白质水平上的表达变化与在mRNA水平上高度一致, 说明二化螟HSP70基因在转录及翻译水平上受到热应激的调节。     

嗜热古菌S.solfataricus小热激蛋白基因的克隆及表达

目的:从嗜热古菌Sulfolobus solfataricus中克隆一种新的小热激蛋白SsHsp14.1的基因,并研究其表达和生物活性。方法:用PCR技术以S.solfataricus基因组为模板扩增得到目的基因序列片段,并将其克隆到pET-28a(+)中,转化到E coli BL21(DE3)中经IPTG诱导表达,纯化后对产物进行生物活性测定。结果:从菌株S.solfataricus中克隆出目的基因,该基因的编码框由375个碱基组成,编码的蛋白质由124个氨基酸组成。含该质粒的大肠杆菌经诱导表达了一个与预期理论值相符的约14kDa的蛋白,利用亲和层析和凝胶柱分离纯化了重组蛋白。试验证明纯化后的重组SsHsp14.1具有分子伴侣活性,重组蛋白在体内表达时能提高E.coli细胞的耐热性。结论:成功克隆SsHsp14.1基因并表达出蛋白,并明确了其分子伴侣活性,为该热激蛋白的研究和应用奠定基础。     

白及小分子热激蛋白BsHsp17.3基因的克隆与表达分析

为了探索人工栽培白及的适宜条件,该研究以湖北省十堰市野生白及为对象,采用同源克隆和3'RACE技术,从白及(Bletilla striata)中获得与热激蛋白合成有关的BsHsp17.3基因,并分析BsHsp17.3基因对不同胁迫的响应。结果表明:BsHsp17.3基因开放阅读框长度为453 bp,编码150个氨基酸;蛋白的分子量为17.42 kD,等电点为6.33。进化树分析表明BsHSP17.3蛋白与同为兰科的铁皮石斛进化关系较近,同在一分支上。半定量RT-PCR分析显示BsHsp17.3基因在白及根、叶、鳞茎及花组织中的表达具有特异性,且BsHsp17.3基因在叶中的表达量较高,在鳞茎及花中不表达。实时荧光定量PCR检测显示BsHsp17.3对非生物胁迫高温、低温具有明显应答反应,20%PEG模拟干旱胁迫不诱导该基因表达,推测该基因在白及防止倒苗过程中可能发挥一定作用。     

家蚕小热激蛋白家族成员Hsp23.7基因的克隆与分析

Hsp23.7基因是小热激蛋白家族的成员,本文研究了家蚕BombyxmoriL.的Hsp23.7基因,并对其进行了原核表达,获得了相应分子量的表达产物。推导的开放阅读框编码210个氨基酸,分子量为23.7ku,等电点为5.17。同时,利用实时定量PCR技术对Hsp23.7基因在家蚕不同组织的表达谱进行了鉴定。结果显示Hsp23.7基因在5龄幼虫时期的各组织中都有表达,在卵巢中表达量最高,达到3.64×107拷贝数/μg,其次在脂肪体,翅原基,马氏管中表达量也较高,在血淋巴中表达量最低,仅为7.11×103拷贝数/μg。     

苹果蠹蛾热激蛋白Hsp90基因的克隆及热胁迫下的表达分析

世界检疫性害虫苹果蠹蛾Cydia pomonella是一种温度耐受可塑性很高的物种。本研究针对温度波动可能导致其耐热性增强的科学问题, 采用生测法鉴定了苹果蠹蛾实验种群的高温耐受阈值, 采用同源克隆、 RACE和实时荧光定量PCR (RT-qPCR)等方法研究了苹果蠹蛾热激蛋白Hsp90基因的应激表达对耐热性的重要作用。高温耐受阈值研究结果表明, 苹果蠹蛾实验种群的死亡率随温度的升高和时间的延长显著性升高, 1-5龄幼虫分别经50℃和52℃高温处理2, 5和10 min后, 3龄幼虫耐热性最差, 5龄幼虫最强。50℃和52℃分别处理10 min和5 min均可导致1-4龄幼虫全部死亡, 而5龄幼虫在这两种处理下仍有25.0%和11.1%的存活率。以35℃处理的5龄雌幼虫为材料克隆苹果蠹蛾Hsp90基因全长cDNA, 结果显示该基因全长为2 470 bp, 完整开放阅读框为2 148 bp, 共编码716个氨基酸, 预测分子量为82.07 kDa, 命名为Cphsp90 (GenBank登录号JN624775)。该基因编码的氨基酸序列与亚洲玉米螟Ostrinia furnacalis和甘蓝夜蛾Mamestra brassicae等昆虫的Hsp90的氨基酸序列一致性高达96%, 表明了Hsp90家族的保守特性。Cphsp90 mRNA的相对表达量在32~44℃高温胁迫下随温度的升高而显著增高, 证实Cphsp90是诱导型热激基因, 且mRNA相对表达量与胁迫程度正相关。Cphsp90基因的表达还具有组织特异性, 35℃处理幼虫的表皮中Cphsp90相对表达量显著高于血淋巴、 脂肪体和中肠, 应激响应最为活跃。与未经温热预处理的昆虫相比, 35℃温热预处理3 h后的5龄幼虫在40, 45和50℃更高的温度胁迫下, Cphsp90 mRNA达到最高表达量所需要的胁迫温度有所提升, 由未经预热处理的40℃处理10 min提高到45℃处理10 min, 这与温热预处理会增强5龄幼虫耐热性的现象相符, 表明Cphsp90基因的响应表达在苹果蠹蛾耐热性及其可塑性过程中发挥重要的作用。     

Five small heat shock protein genes from Chilo suppressalis: characteristics of gene, genomic organization, structural analysis, and transcription profiles

Small heat shock proteins (sHSPs) are the most diverse but also the most poorly known family of molecular chaperones, and they play essential roles in various biological processes. The striped stem borer, Chilo suppressalis (Insecta: Lepidoptera: Pyralidae), is one of the most serious pests of rice, causing extensive damage and yield loss. In this study, we isolated and characterized five members of the sHSPs family—Cshsp19.8, Cshsp21.4, Cshsp21.5, Cshsp21.7a, and Cshsp21.7b—from C. suppressalis. The cDNAs of these genes encoded proteins of 177, 187, 191, 191, and 191 amino acids with isoelectric points of 7.0, 5.6, 6.1, 6.3, and 6.3, respectively. While Cshsp19.8, Cshsp21.5, and Cshsp21.7b had no introns, Cshsp21.4 and Cshsp21.7a contained one and two introns, respectively. Structural analysis indicated that all five Cshsps possessed conserved arginine and a V/IXI/V motif, which is related to hydrophobic characteristics of sHSPs. The five heat shock proteins can be classified into two main groups: an orthologous type (Cshsp21.4 and Cshsp21.7a) and a species-specific type (Cshsp19.8, Cshsp21.5, and Cshsp21.7b). Real-time quantitative PCR analyses revealed that Cshsp19.8, Cshsp21.5, Cshsp21.7a, and Cshsp21.7b all exhibited their highest expression levels within Malpighian tubules or the hindgut, while such levels were found in the head for Cshsp21.4. The expression of Csshsps at different developmental stages revealed that the mRNA levels of Cshsp19.8, Cshsp21.4, Cshsp21.5, and Cshsp21.7b peaked in adults, whereas the highest level of Cshsp21.7a was observed in first instar larvae. Cshsp19.8 and Cshsp21.7b were both upregulated dramatically by heat and cold, and Cshsp21.5 could be induced by cold stress. Neither Cshsp21.4 nor Cshsp21.7a responded to heat or cold. These results demonstrated that different Csshsps play distinctive roles in the regulation of the physiological activities in C. suppressalis.     

Molecular cloning and expression analysis of ultraspiracle (USP) from the rice stem borer Chilo suppressalis

cDNA for ultraspiracle (USP) from the lepidopteran rice stem borer Chilo suppressalis was cloned using PCR techniques. The deduced amino acid sequence of C. suppressalis USP (CsUSP) was very similar to those of other lepidopteran USPs, especially to the Manduca sexta USP-2 isoform. Northern hybridization analysis detected a 6.5-kb message in the epidermis, fat body, and midgut of wandering larvae. CsUSP mRNA expression in the epidermis varied little during the last larval instar. Gel mobility shift assays showed that in vitro translated C. suppressalis ecdysone receptor (CsEcR) and CsUSP proteins bound to the Pal1 or Drosophila melanogaster hsp27 ecdysone response element as a heterodimer. In a ligand-receptor binding assay, [(3)H]ponasterone A ([(3)H]PoA) did not bind to individual CsEcR or CsUSP protein, but bound strongly to the CsEcR/CsUSP complex. [(3)H]PoA binding to CsEcR/CsUSP complex was competed by 20-hydroxyecdysone and a non-steroidal ecdysteroid agonist, RH-5992, but not by cholesterol, indicating that compounds with molting hormone activity against C. suppressalis can bind specifically to the CsEcR/CsUSP complex.     

Molecular cloning,expression analysis and functional confirmation of two ecdysone receptor isoforms from the rice stem borer Chilo suppressalis

PCR techniques were used to clone and identify cDNAs for ecdysone receptor A and B1 (EcR-A and EcR-B1) isoforms from the rice stem borer, Chilo suppressalis. They differ only in the N-terminal A/B regions and show high sequence identities to other insects' EcRs. At the wandering stage, EcR-B1 mRNA was expressed more abundantly in the midgut than in the epidermis and fat body, whereas expression levels of EcR-A mRNA were similar in the three tissues. In the epidermis of the last instar larvae, the maximal mRNA expression of both EcR-A and EcR-B1 was observed from the wandering to prepupal stages prior to the peak of ecdysteroid titer in the hemolymph. In gel mobility shift assays, in vitro translated C. suppressalis EcR-B1 (CsEcR-B1) and Bombyx mori ultraspiracle (BmUSP) proteins bound to the Pal 1 and Drosophila melanogaster hsp27 ecdysone response element as a heterodimer. These results indicate that the cDNAs isolated here encode functional ecdysone receptors.     

Molecular cloning of a steroid-regulated 108K heat shock protein gene from hen oviduct.

The natural gene for a steroid inducible 108K heat shock protein has been isolated from a lambda genomic library prepared from hen oviduct tissue. Genomic DNA blots indicate that it exists as a single copy gene in the chick oviduct haploid genome. The 9.9 kilobase gene codes for a messenger RNA of 2733bp (21) and is split into 18 exons as established by sequence comparison of cDNA and genomic clones. The 3' end of the gene contains a repetitive element which shares homology with the CR1 family of repeats. The first exon contains both the untranslated leader and coding regions of the gene. The promoter region is rich in G + C residues (70%) and the dinucleotide CG. This 5' flanking segment contains bases similar both in sequence and location to the Goldberg-Hogness TATA homology and consensus sequence CCAAT. A consensus sequence located upstream of steroid hormone responsive chicken genes is found at -267 and on a reverse orientation at -593. The structure of this gene is of interest since the presence of introns in heat shock genes is rare in any species examined to date. Furthermore, this gene lacks the previously described heat shock promoter consensus sequence (C-GAA-TTC-G) present in other species.     

Molecular cloning, mRNA expression, and characterization of heat shock protein 10 gene from humphead snapper Lutjanus sanguineus

Heat shock protein 10 (HSP10) gene of humphead snapper (Lutjanus sanguineus), designated as ByHSP10, was cloned by rapid amplification of cDNA ends (RACE) techniques with the primers designed from the known EST sequence identified from the subtracted cDNA library of the head kidney of humphead snapper. Sequence analysis showed the full length cDNA of ByHSP10 was 529 bp, containing a 5′ terminal untranslated region (UTR) of 51 bp, a 3′ terminal UTR of 181 bp, and an open reading frame (ORF) of 297 bp encoding a polypeptide of 99 amino acids. Based on the deduced amino acid sequence, the theoretical molecular mass of ByHSP10 was calculated to be 10.92 kDa with an isoelectric point of 9.46. Moreover, chaperonins hsp10/cpn10 signature was found in the amino acids sequence of ByHSP10 by PredictProtein. BLAST analysis revealed that the amino acids of ByHSP10 had the highest homology of 88% compared with other HSP10s. Fluorescent real-time quantitative RT-PCR was used to examine the expression of ByHSP10 gene in eight kinds of tissues of humphead snapper after the challenge with Vibrio harveyi. There was a clear time-dependent expression pattern of ByHSP10 in head kidney, spleen and thymus after bacteria challenge. The expression of mRNA reached the maximum level at the time point of 9 h, 6 h and 24 h, respectively and then returned to control level in 36 h. The up-regulated mRNA expression of ByHSP10 in humphead snapper after bacteria challenge indicated that the HSP10 gene was inducible and might be involved in immune response. A phylogenetic tree was constructed based on the ORF nucleotide sequences of HSP10 for 30 species. The relatonships among them were generally in agreement with the traditional taxonomy which suggested that HSP10 genes could aid in the system classification research.     

二化螟piggyBac类转座子的克隆与分析

piggyBac转座子是DNA型转座子, 广泛分布于生物体内。基于piggyBac转座子超家族成员IFP2开发的转基因工具载体是目前转基因研究中使用最广泛的载体之一, 因此piggyBac转座子的研究受到广泛的关注和重视。本文是对二化螟Chilo suppressalis内源性piggyBac类转座子（piggyBac-like element, CsuPLE）的首次报道。克隆的CsuPLE（GenBank登录号： JX392388）全长2 537 bp, 包含一个长1 914 bp的完整开放阅读框（open reading frame, ORF）, 编码含637个氨基酸残基的转座酶, 转座酶中含有piggyBac家族保守的“DDD-domain”。CsuPLE全长序列具有完全对称的13 bp反向末端重复序列（inverted terminal repeats, ITRs）以及非完全对称的21 bp内部重复序列（internal repeats, IRs）, 在二化螟基因组上插入在特征性的“TTAA”靶位点重复（target site duplication, TSD）处。在我国地理跨度很大的不同二化螟种群中均存在结构完整的CsuPLE序列。本研究结果为深入研究piggyBac转座子的结构与功能的关系提供了新的素材, 也为评价利用转座子载体系统在二化螟体内进行转基因操作的可行性和安全性提供了重要的理论基础。