桃ACC合酶基因的分离及其差异表达

利用5′/3′RACE PCR技术,从桃(Prunus persica (L.) Batsch)果实中克隆了植物乙烯生物合成的关键酶--ACC合酶的全长cDNA pacs,对pacs基因进行全序列测定表明,该基因全长1 848个碱基,编码区为1 449个碱基,5′端有177个碱基的非编码区序列,3′端有219个碱基的非编码区序列(不包括终止密码子TAA).pacs基因编码区共编码483个氨基酸,蛋白质大小为54 kD,等电点为6.43.pacs与番茄(S19677)、梅(AB031026)、番木瓜(U68216)、苹果(AB034993)等其他植物ACC合酶cDNA氨基酸序列同源性分别为65%、70%、75%、90%,并存在与这些ACC合酶氨基酸的活性位点保守序列SLSKDMGFPGFR.RT-PCR结合杂交分析表明,pacs和我们以前克隆的桃ACC合酶cDNA pacs12(AF467782)在叶片和花中基因表达模式基本一致,伤处理和IAA均能诱导叶片pacs 和pacs12基因的表达,但pacs在伤处理叶片的表达水平比pacs12高;pacs 和pacs12基因在果实表达有所不同,pacs在绿熟和成熟果实中均有表达,而pacs12在绿熟果实中基本检测不到,在成熟果实中才有表达,两者在果实中的表达水平比伤处理和IAA处理叶片和花中要低.     

香蕉果实特异性ACC合酶的cDNA克隆及序列分析

根据ACC合酶高度保守区氨基酸序列设计两种兼并引物。通过RTPCR,克隆了香蕉果肉ACC合酶1693bp的cDNA片段。再根据其序列测定结果进行5′RACE(RapidamplificationofcDNAends)。最终确定香蕉果肉中ACC合酶的mRNA全长为1752个核苷酸。其中5′非翻译区74个核苷酸,编码区1461个核苷酸,3′非翻译区217个核苷酸,编码产物为486个氨基酸。通过Northern杂交分析,证明此ACC合酶基因的表达具有果实特异性     

一个新高产青蒿倍半萜合酶基因的克隆、表达和分析

用RACE方法从青蒿（Artemisia annua L.)高产株系001中克隆了一个新的1886bp的全长倍半萜合酶cDNA。克隆的倍半萜合酶氨基酸序列与烟草马兜铃烯合酶,莨菪岩兰螺旋二烯合酶,棉花杜松烯合酶的一致性分别为39％,38％和41％;与青蒿柏木脑合酶,紫穗槐二烯合酶和一个推测的倍半萜合酶克隆cASC125的一致性为50％,48％和59％。cDNA编码区序列被克隆进原核表达载体pET-30a,并在大肠杆菌（Escherichia coli)BL21(DE3)中诱导表达,但过量表达的蛋白主要是以不溶性蛋白形式存在。Northern blotting分析表明此基因在茎,叶,花中表达,在根中没有表达。     

长春花钙调素基因克隆及其假基因的识别

以长春花[Catharanthus roseus(L.)G.Don]叶片cDNA和基因组DNA为模板,利用PCR技术扩增得到了长春花钙调素基因447 bp的全长编码cDNA序列和2个大小不同的DNA片段.序列分析表明,DNA长片段全长1 551 bp,由2个外显子和1个内含子构成,为长春花钙调素基因编码区DNA片段;DNA小片段全长447 bp,与447 bp的长春花钙调素基因cDNA核苷酸一致性高达87%,有56个碱基的差异,其中位于226 bp处的碱基A突变为T,即由AAG突变为终止密码子TAG使翻译提前终止.推测此447 bp的DNA小片段可能为长春花钙调素基因的假基因,命名为CCaMP1.     

白皮黄瓜鲨烯合酶基因cDNA克隆及生物信息学分析

为认识葫芦科植物中葫芦素生物合成途径所需鲨烯合酶基因结构特征,克隆白皮黄瓜鲨烯合酶(squalene synthase,SS)基因c DNA并进行生物信息学分析。根据葫芦科植物绞股蓝、罗汉果、红花栝楼等的鲨烯合酶基因cDNA序列,设计白皮黄瓜鲨烯合酶引物,分别采用3'RACE和5'RACE技术扩增鲨烯合酶基因3'端和5'端。获得白皮黄瓜鲨烯合酶基因的两个cDNA克隆,命名为CsSS1和CsSS2,其cDNA序列全长分别为1 627 bp和1 534 bp,都编码417个氨基酸残基,分子质量为47.6 k D。成功克隆得到白皮黄瓜鲨烯合酶基因全长cDNA序列并对其进行序列分析,后续可用于葫芦素生物合成途径所需鲨烯合酶基因正选择位点功能分析。     

砀山酥梨不同程度缺铁叶片生长素抑制蛋白基因表达分析

以‘砀山酥梨’为材料,采用酶联免疫分析法(ELISA),测定叶片中内源IAA的含量.依据已构建的缺铁叶片SSH文库中生长素抑制蛋白(ARP)基因片段的序列信息,应用RACE技术克隆其cDNA全长,通过实时荧光定量(qRT-PCR)技术,分析ARP基因的相对表达量.结果表明:(1)ARP基因cDNA全长为707 bp,其中开放阅读框为351 bp,编码116个氨基酸,推测的蛋白质分子量为12.82 kD;该蛋白可能定位于微体,属于非分泌型、非跨膜蛋白类,并具有ARP基因家族的保守结构域.(2)在不同程度缺铁叶片中ARP基因的表达量存在差异,随着缺铁程度的增加,表达量显著升高,同时叶片中内源IAA含量逐渐降低.据此推测,ARP基因可能负反馈调节缺铁黄化叶片中IAA的水平,从而调控叶片的生长发育.     

草菇漆酶基因vv-lac1和vv-lac6的克隆及异源表达

【目的】克隆草菇漆酶基因vv-lac1和vv-lac6的全长cDNA,证实其编码的蛋白具有漆酶活性,最终建立草菇漆酶基因的异源表达及纯化体系。【方法】利用RACE技术克隆全长cDNA序列,并利用生物信息学技术进行序列分析,在此基础上,去除全长cDNA的编码信号肽的序列并在3'端添加His-tag碱基序列,把修饰后的cDNA片段克隆到表达载体pPIC9K上,并转化到毕赤酵母GS115中进行表达,对重组蛋白利用Ni柱进行分离纯化并以ABTS底物法检测重组蛋白的漆酶活性。【结果】vv-lac1和vv-lac6的全长cDNA的长度分别为1599 bp和1554 bp,且分别含有19和15个外显子;其编码的蛋白的理论分子量分别是57.3 kDa和56.3 kDa,理论等电点分别为4.73和5.62,且都属于分泌型的胞外蛋白;表达出的重组蛋白RBvvlac1和RBvvlac6,其分子量大小约为70 kDa,说明存在翻译后修饰;含有150 mmol/L咪唑的缓冲液对RBvvlac1和RBvvlac6发酵液进行洗脱所得到的蛋白溶液具有最高漆酶活性(333.17 U/L和227.63 U/L)。【结论】草菇漆酶基因vv-lac1和vv-lac6能够编码有活性的漆酶蛋白,本研究建立的异源表达及纯化体系适用于草菇或其它漆酶基因的异源表达与纯化。     

苎麻纤维素合酶基因BnCesA1全长cDNA的克隆与表达分析

苎麻是中国的传统纤维作物,能够生产最长的自然纤维。本研究旨在克隆苎麻纤维素合酶基因BnCesA1全长编码序列,对其表达模式进行分析。以已知的苎麻纤维素合酶基因序列(DQ077190)为基础,设计5’RACE引物,以湘苎三号为材料,得到了BnCesA1的5’端,拼接后得到了BnCesA1的全长序列,并从湘苎三号的cDNA中成功克隆到包括BnCesA1全部编码序列的cDNA序列。扩增得到的BnCesA1基因cDNA为3253bp,编码区3246bp,编码含1082个氨基酸的多肽。通过对这个基因进行核酸序列和蛋白结构域分析表明,BnCesA1和毛果杨、欧美山杨、巨桉、大叶相思等其他物种的纤维素合酶基因都有很高的同源性,根据得到的BnCesA1的5’端设计特异性表达检测引物,分析其在湘苎三号苎麻品种各组织中的表达情况,结果显示BnCesA1在所检测的各组织中均有表达,表达量为茎皮>叶>顶芽>根。本研究首次克隆到苎麻中编码全长蛋白的纤维素合酶基因,并且苎麻BnCesA1在茎皮中高表达提示该基因可能在苎麻韧皮纤维合成中有重要作用。     

草莓ξ-胡萝卜素脱氢酶基因ZDS的克隆及特征分析

用RT-PCR和RACE技术从草莓(Fragaria ananassa Duchesne)果实中克隆到ξ-胡萝卜素脱氧酶基因ZDS,命名为FaZDS,其cDNA全长2148 bp,具有1个1710 bp的完整开放阅读框(ORF),编码569个氨基酸.序列分析表明,FaZDS编码的氨基酸序列与其它植物的ZDS蛋白有很高的相似性.系统进化树分析表明,草莓与苹果的ZDS蛋白亲缘关系较近.原核表达结果表明FaZDS基因在大肠杆菌中能高效表达.用半定量RT-PCR技术进行组织表达模式分析表明,FaZDS基因在草每的花、叶片和果实中均有表达,表达量为花＞红果＞粉红果＞白果＞青果＞叶.     

漳州水仙ZDS 基因cDNA克隆及其表达分析

采用RACE技术从漳州水仙‘金盏银台’Narcissus tazetta var. chinensis花器官总RNA中克隆ZDS的cDNA序列,用相关软件进行序列分析,并利用实时荧光定量技术检测ZDS基因在各水仙品种不同花器官的表达。序列分析表明,ZDS基因cDNA全长2189 bp,其中包含69 bp 5'非编码区,395 bp 3'非编码区,1725 bp编码区（编码574个氨基酸,分子量约63.6 kDa）,命名为Ntzds （GenBank登录号: EU573238）,其编码的氨基酸序列（ACB87206.1）与喇叭水仙（CAA12062.1）、葡萄（XP_002277348.21）和温州蜜柑（ABC33728.1）ZDS基因编码产物的相似性分别为97%、85%和83%。实时荧光定量PCR分析表明,Ntzds基因在漳州水仙‘金盏银台’、‘Minnow’、‘Fortissimo’中均有表达,且同一品种中副冠的表达量高于花瓣;随着花色的加深,其转录水平也逐渐趋高。     

LINE-1编码蛋白L1-ORF1的原核表达纯化和多克隆抗体制备

目的: 制备具有肿瘤组织特异性表达的L1-ORF1蛋白多克隆抗体并进行初步应用研究。方法:采取基因工程表达方法制备L1-ORF1蛋白,免疫家兔制备多克隆抗体,间接ELISA检测抗体效价,Western blot和细胞免疫荧光方法检测抗体特异性,免疫检测验证其识别肿瘤细胞内L1-ORF1蛋白的特异性。结果:制备的抗L1-ORF1蛋白多克隆抗体具有很高的敏感性与特异性,免疫学检测表明该抗体不仅能检测出正常细胞中瞬时表达的L1-ORF1蛋白,而且可检测出肿瘤细胞中天然表达的L1-ORF1蛋白。结论:制备的多克隆抗体具有较高的敏感性与特异性,为以后该抗体的进一步应用奠定了基础。     

DEC1 and MIC-1

Comment on: Qian Y, et al. Proc Natl Acad Sci USA 2012; 109:11300-5.     

Role of the ASK1-SEK1-JNK1-HIPK1 signal in Daxx trafficking and ASK1 oligomerization

Overexpression of JNK binding domain inhibited glucose deprivation-induced JNK1 activation, relocalization of Daxx from the nucleus to the cytoplasm, and apoptosis signal-regulating kinase 1 (ASK1) oligomerization in human prostate adenocarcinoma DU-145 cells. However, SB203580, a p38 inhibitor, did not prevent relocalization of Daxx and oligomerization of ASK1 during glucose deprivation. Studies from in vivo labeling and immune complex kinase assay demonstrated that phosphorylation of Daxx occurred during glucose deprivation, and its phosphorylation was mediated through the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. Data from immunofluorescence staining and protein interaction assay suggest that phosphorylated Daxx may be translocated to the cytoplasm, bind to ASK1, and subsequently lead to ASK1 oligomerization. Mutation of Daxx Ser667 to Ala results in suppression of Daxx relocalization during glucose deprivation, suggesting that Ser667 residue plays an important role in the relocalization of Daxx. Unlike wild-type Daxx, a Daxx deletion mutant (amino acids 501-625) mainly localized to the cytoplasm, where it associated with ASK1, activated JNK1, and induced ASK1 oligomerization without glucose deprivation. Taken together, these results show that glucose deprivation activates the ASK1-SEK1-JNK1-HIPK1 pathway, and the activated HIPK1 is probably involved in the relocalization of Daxx from the nucleus to the cytoplasm. The relocalized Daxx may play an important role in glucose deprivation-induced ASK1 oligomerization.     

Generation of 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-) mouse line

Human/rodent CYP1A1 and CYP1A2 orthologs are well known to exhibit species-specific differences in substrate preferences and rates of metabolism. This lab previously characterized a BAC-transgenic mouse carrying the human CYP1A1_CYP1A2 locus; in this line, human dioxin-inducible CYP1A1 and basal vs dioxin-inducible CYP1A2 have been shown to be expressed normally (with regard to mRNAs, proteins and three enzyme activities) in every one of nine mouse tissues studied. The mouse Cyp1a1 and Cyp1a2 genes are oriented head-to-head and share a bidirectional promoter region of 13,954 bp. Using Cre recombinase and loxP sites inserted 3' of the stop codons of both genes, we show here a successful interchromosomal excision of 26,173 bp that ablated both genes on the same allele. The Cyp1a1/1a2(-) double-knockout allele was bred with the "humanized" line; the final product is the hCYP1A1_1A2_Cyp1a1/1a2(-/-) line on a theoretically >99.8% C57BL/6J genetic background-having both human genes replacing the mouse orthologs. This line will be valuable for human risk assessment studies involving any environmental toxicant or drug that is a substrate for CYP1A1 or CYP1A2.     

Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected LC50s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Isolation and Characterization of Ubiquitin-activating Enzyme E1-domain Containing 1, UBE1DC1

Ubiquitin and other ubiquitin-like proteins play important roles in post-translational modification. They are phylogenetically well-conserved in eukaryotes. Activated by other proteins, ubiquitin and ubiquitin-like proteins can covalently modify target proteins. The enzymes responsible for the activation of this modification have been known to include UBA1, SAE2, UBA3, SAE1 and ULA1. Here we report a new ubiquitin activating enzyme like cDNA, named ubiquitin activating enzyme E1-domain containing 1 (UBE1DC1), whose cDNA is 2654 base pairs in length and contains an open reading frame encoding 404 amino acids. The UBE1DC1 gene consists of 12 exons and is located at human chromosome 3q22. The result of RT-PCR showed that UBE1DC1 is expressed in most of human tissues. These two authors contributed equally to this paper. The nucleotide sequence reported in this paper has been submitted to GenBank under accession number AY253672.     

Regulation of PCTAIRE1 protein stability by AKT1, LKB1 and BRCA1

PCTAIRE1, also known as CDK16, is a cyclin-dependent kinase that is regulated by cyclin Y. It is a member of the serine-threonine family of kinases and its functions have primarily been implicated in cellular processes like vesicular transport, neuronal growth and development, myogenesis, spermatogenesis and cell proliferation. However, as extensive studies on PCTAIRE1 have not yet been conducted, the signaling pathways for this kinase involved in governing many cellular processes are yet to be elucidated in detail. Here, we report the association of PCTAIRE1 with important cellular proteins involved in major cell signaling pathways, especially cell proliferation. In particular, here we show that PCTAIRE1 interacts with AKT1, a key player of the PI3K signaling pathway that is responsible for promoting cell survival and proliferation. Our studies show that PCTAIRE1 is a substrate of AKT1 that gets stabilized by it. Further, we show that PCTAIRE1 also interacts with and is degraded by LKB1, a kinase that is known to suppress cellular proliferation and also regulate cellular energy metabolism. Moreover, our results show that PCTAIRE1 is also degraded by BRCA1, a well-known tumor suppressor. Together, our studies highlight the regulation of PCTAIRE1 by key players of the major cell signaling pathways involved in regulating cell proliferation, and therefore, provide crucial links that could be explored further to elucidate the mechanistic role of PCTAIRE1 in cell proliferation and tumorigenesis.     

Antagonism between Cry1Ac1 and Cyt1A1 Toxins of Bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC₅₀s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC₅₀s were obtained. All of these LC₅₀s were significantly higher than the expected LC₅₀s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC₅₀ of Cry1Ac1 was 2.46 ng/cm² of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC₅₀s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm² of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm² of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.