肝癌中高表达基因fup1的克隆及其功能

原发性肝细胞癌是我国高发的恶性肿瘤之一，开展肝癌相关基因的研究具有重要的意义。从已经获得的在肝癌和正常肝对照中表达量有明显差异的EST片段入手，克隆了一个功能未报道的而可能与肝癌相关的基因。暂命名为fup1，该基因编码区全长1233bp，其产物的分子量约为46kD，等电点为5.48,可能是一个核蛋白。Northern 迹结果表明该基因在人类除心脏以外的多种正常组织中表达量很小，说明其分布具有一定的组织特异性，将整合有该基因的真核表达载体转染NIH3T3细胞后，MTT检测结果证明该基因的产物可能对细胞的增殖具有促进作用。     

橡胶草TkCPT1基因的生物信息学分析及功能验证

TkCPT1基因是橡胶草(Taraxacum kok-saghyz)合成顺式橡胶过程中的一个重要基因.本实验运用生物信息学手段对橡胶草中顺式异戊烯基转移酶(cis isoprene transferase,CPT)进行一系列特征分析,并利用农杆菌介导的遗传转化法,通过超量表达TkCPT1基因遗传转化烟草,获得转基因烟草...     

水稻蛋白激酶基因Pti1的克隆与分析

植物Pti1基因编码丝氨酸/苏氨酸蛋白激酶,是重要的抗病相关基因。在水稻抗褐飞虱基因Qbp1所在的染色体区段存在一个与番茄Pti1基因高度同源的序列片段。从抗虫水稻B5中分离了Pti1基因的全长cDNA克隆,测定了基因组序列。分析发现,水稻中Pti1基因长度有5644bp,含有7个内含子,编码368个氨基酸的激酶蛋白。其蛋白质的C末端在不同植物之间具有高度的保守性,而N末端的变异则相对较大。对不同水稻材料Pti1基因的序列进行了比较,发现药用野生稻与栽培稻之间存在较大的差异,而栽培稻各品种之间的差异较小。讨论了Pti1基因在抗虫防卫反应中可能的作用。     

十字花科植物SAMDC基因同源序列的克隆与进化分析

腺苷甲硫氨酸脱羧酶(SAMDC)是参与植物多胺合成的一个关键酶。根据GenBank中已报道的SAMDC基因编码序列保守区域设计特异引物, 运用PCR技术分别从十字花科6属14个物种中新克隆分离了SAMDC基因的同源序列。比较分析结果表明: 这些同源序列的相似性达87%以上, 所推导的氨基酸序列相似性达90%以上, 且两者种间差异分别为0.2%~10.1%和0.3%~6.6%, 属间差异除“圆白”萝卜外分别是4.9%~13.6%和3.1%~10.3%; SAMDC基因的核苷酸及其可能编码的氨基酸序列差异属间较种间大, 可用于属间的分类等级研究; 且氨基酸序列间的差异比核苷酸序列间的差异小的多, 因而根据核苷酸序列构建了NJ与ME分子系统树。进化树直观地表明在亲缘进化关系上芸薹属与萝卜属较近, 其他依次为山芥属、蔊菜属、拟南芥属, 与荠菜属最远。研究结果有助于从分子水平阐明十字花科植物间的亲缘进化关系, 可为其种质资源利用提供理论依据。     

小麦BBC1基因cDNA的克隆与分析

从小麦(Triticum aestivum L．)中克隆了一个BBC1基因的cDNA。分析结果表明,该基因编码一亲水多肽,富含丙氨酸、赖氨酸、精氮酸和谷氦酸。该基因的转录受低温调控。在小麦基因组中,BBC1基因以一个小家族的形式存在。     

沙鞭转录因子PvCBF1的基因克隆及功能验证

沙鞭(Psammochloa villosa)是西北地区重要的防风固沙植物,关于其耐旱调控基因克隆与功能验证的研究尚未见报道。基于三代转录组测序技术,本研究克隆出沙鞭中调控耐旱的关键转录因子PvCBF1基因,对其编码产物的理化性质、蛋白质结构特点和进化关系进行了分析,测定了其在沙鞭不同组织及模拟干旱胁迫条件下的表达,并验证了PvCBF1在转基因烟草(Nicotiana tabacum)中的功能。结果表明,PvCBF1基因编码区长度为690 bp,编码229个氨基酸,包括AP2保守结构域,并含有CBF转录因子的两段特征序列PKRPAGRTKFRETRHP和DSAWR;进化分析显示,沙鞭PvCBF1与普通小麦(Triticum aestivum)CBF1转录因子亲缘最近;RT-qPCR结果表明,PvCBF1基因在沙鞭根、茎、叶和种子中均能表达,无组织表达特异性;模拟干旱胁迫0～6 h,PvCBF1基因迅速表达并在6 h时达到峰值;20%PEG-6000模拟干旱胁迫条件下,随模拟干旱胁迫时间的增加,转PvCBF1基因烟草叶片萎蔫程度明显低于野生型烟草,转PvCBF1基因烟草和野生型烟草的超氧...     

十字花科植物CYP86MF基因同源序列的克隆与进化分析

为了从分子水平阐明十字花科植物间的亲缘进化关系,给植物种质资源的创建提供理论依据,试验根据课题组已报道的CYP86MF基因编码的氨基酸保守区域设计特异引物,运用PCR技术分别从十字花科6个属11个物种中分离克隆到了CYP86MF基因的同源序列,经比较分析,结果表明：这些同源序列的相似性达80%以上,所推导的氨基酸序列相似性达70%以上,且两者种间差异分别为1.0% ~ 5.7%和2.6% ~ 7.3%,属间差异分别是5.6% ~ 22.5%和7.3% ~ 31.2%;由氨基酸序列构建的分子系统树可知,在亲缘进化关系上芸薹属与萝卜属较近,其他依次为蔊菜属、拟南芥属、荠菜属,而与诸葛菜属最远。因此,CYP86MF基因的核苷酸及其可能编码的氨基酸序列差异属间较种间大,它可用于属间的分类等级研究,而难用于属以下的分类等级研究。     

厚叶悬蒴苣苔BcWRKY1基因启动子克隆及功能元件分析

为了更好的了解BcWRKY1基因的表达调控方式,我们利用接头介导的PCR(LM-PCR)技术,从厚叶悬蒴苣苔(Boea crassifolia)基因组中克隆了943bp的BcWRKY1基因的启动子序列。序列分析显示,该启动子中存在多种非生物胁迫反应元件,如MYC recognition site、MYB binding site、DRE\CRTCOREAT等。本研究有助于阐明WRKY家族基因在非生物胁迫下的作用机制。     

克隆与克隆动物基因相同吗

美籍华人牛满江教授,著名生物学家,１９１２年生于中国河北省。１９３２年考入北京大学,１９３６年毕业留校任助教,１９３７年在昆明西南联大任教。１９４４年由北大派出去美国斯坦福大学深造,１９４６年获博士学位。自此,先后在斯坦福大学,洛克菲勒医学研究所,后改名为洛克菲勒大学和坦普尔大学从事教学和研究工作。并获得坦普尔大学终身教授,名字被载入美国科学家名人录。从１９５３年起,他潜心于“核糖核酸在发育中独特功能”的研究,是这一研究领域的先行者。他创立了“外基因学说”,开创了人工培育新物种的新思路。获得“利利学术”及“古根海姆”奖。１９７０年被选为台湾中央研究院院士。牛教授身居海外,对祖国科学教育事业极为关注,１９７８年,首先接收中科院派去美国的访问学者,打破中美学术交流的禁令,１９８０年中科院授予他科学顾问,中国各地的大学及科研机构授予他名誉教授、顾问头衔者３０多个。近来,为促进中国生命科学的研究和教育事业的发展,在他主持下在北京成立了“牛满江基金会”,将为培养优秀人才,促进国际学术交流方面作更多的贡献。     

马铃薯Y病毒p1基因的克隆与序列分析

利用依据马铃薯Y病毒(PVY)p1基因序列设计合成的一对引物YP1,YP2,以带毒烟草总RNA为模板,通过RT-PCR方法扩增得到了0．83kb的目的条带,测序结果表明为PVY p1基因。通过对PVY P1蛋白氨基酸序列分析发现PVY不同分离物间P1蛋白氨基酸序列存在明显差异,氨基酸序列同源性在64％～94％间。依据P1蛋白氨基酸序列建立了PVY系统关系树并对PVY进行了类型分析。     

Evolutionary Pattern of the FAE1 Gene in Brassicaceae and Its Correlation with the Erucic Acid Trait

The fatty acid elongase 1 (FAE1) gene catalyzes the initial condensation step in the elongation pathway of VLCFA (very long chain fatty acid) biosynthesis and is thus a key gene in erucic acid biosynthesis. Based on a worldwide collection of 62 accessions representing 14 tribes, 31 genera, 51 species, 4 subspecies and 7 varieties, we conducted a phylogenetic reconstruction and correlation analysis between genetic variations in the FAE1 gene and the erucic acid trait, attempting to gain insight into the evolutionary patterns and the correlations between genetic variations in FAE1 and trait variations. The five clear, deeply diverged clades detected in the phylogenetic reconstruction are largely congruent with a previous multiple gene-derived phylogeny. The Ka/Ks ratio (<1) and overall low level of nucleotide diversity in the FAE1 gene suggest that purifying selection is the major evolutionary force acting on this gene. Sequence variations in FAE1 show a strong correlation with the content of erucic acid in seeds, suggesting a causal link between the two. Furthermore, we detected 16 mutations that were fixed between the low and high phenotypes of the FAE1 gene, which constitute candidate active sites in this gene for altering the content of erucic acid in seeds. Our findings begin to shed light on the evolutionary pattern of this important gene and represent the first step in elucidating how the sequence variations impact the production of erucic acid in plants.     

Cloning and functional characterization of the fatty acid elongase 1 (FAE1) gene from high erucic Crambe abyssinica cv. Prophet

A genomic fatty acid elongation 1 ( FAE1 ) clone was isolated from Crambe abyssinica . The genomic clone corresponds to a 1521-bp open reading frame, which encodes a protein of 507 amino acids. In yeast cells expression of CrFAE led to production of new very long chain monounsaturated fatty acids such as eicosenoic (20 : 1^Δ11) and erucic (22 : 1^Δ13) acids. Seed-specific expression in Arabidopsis thaliana resulted in up to a 12-fold increase in the proportion of erucic acid. On the other hand, in transgenic high-erucic Brassica carinata plants, the proportion of erucic acid was as high as 51.9% in the best transgenic line, a net increase of 40% compared to wild type. These results indicate that the CrFAE gene encodes a condensing enzyme involved in the biosynthesis of very long-chain fatty acids utilizing monounsaturated and saturated acyl substrates, with a strong capability for improving the erucic acid content.     

Orthologs of arabidopsis CLAVATA 1 gene in cultivated Brassicaceae plants

In arabidopsis (Arabidopsis thaliana), the CLAVATA1 (CLV1) gene is involved in maintaining the balance between the stem cells in the central zone of the stem apical meristem and the determined cells at its periphery. However, CLV1 has not been previously characterized in other Brassicaceae. Using the direct amplification of genomic DNA, we obtained a full-length CLV1 ortholog from canola plants (Brassica napus), and also three CLV1 fragments from rape (B. rapa), canola (B. napus), and false flax (Camelina sativa), which corresponded to the transmembrane domain and a part of the kinase domain of the CLAVATA1 protein. The nucleotide and deduced amino acid sequences of the full-size CLV1 ortholog from B. napus were similar by 81 and 87% to the prototype gene from arabidopsis; in the case of shorter gene fragments, the similarity was as high as 91-93 and 98%, respectively. By their primary structure, the CLV1 genes in the Brassicaceae considerably differ from its putative structural homologs beyond this family.     

Engineering and mechanistic studies of the Arabidopsis FAE1 beta-ketoacyl-CoA synthase, FAE1 KCS.

The Arabidopsis FAE1 beta-ketoacyl-CoA synthase (FAE1 KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoAs. Sequence analysis of FAE1 KCS predicted that this condensing enzyme is anchored to a membrane by two adjacent N-terminal membrane-spanning domains. In order to characterize the FAE1 KCS and analyze its mechanism, FAE1 KCS and its mutants were engineered with a His6-tag at their N-terminus, and expressed in Saccharomyces cerevisiae. The membrane-bound enzyme was then solubilized and purified to near homogeneity on a metal affinity column. Wild-type recombinant FAE1 KCS was active with several acyl-CoA substrates, with highest activity towards saturated and monounsaturated C16 and C18. In the absence of an acyl-CoA substrate, FAE1 KCS was unable to carry out decarboxylation of [3-(14)C]malonyl-CoA, indicating that it requires binding of the acyl-CoA for decarboxylation activity. Site-directed mutagenesis was carried out on the FAE1 KCS to assess if this condensing enzyme was mechanistically related to the well characterized soluble condensing enzymes of fatty acid and flavonoid syntheses. A C223A mutant enzyme lacking the acylation site was unable to carry out decarboxylation of malonyl-CoA even when 18:1-CoA was present. Mutational analyses of the conserved Asn424 and His391 residues indicated the importance of these residues for FAE1-KCS activity. The results presented here provide the initial analysis of the reaction mechanism for a membrane-bound condensing enzyme from any source and provide evidence for a mechanism similar to the soluble condensing enzymes.     

拟南芥ats1A基因启动子的克隆和功能分析

通过PCR扩增,从拟南芥中克隆出ats1A基因启动子(包括叶绿体转运肽),将此启动子与GUS基因相连构建植物瞬时表达载体,用基因枪法将之导入烟草进行瞬时表达。GUS基因检测分析表明,ats1A基因启动子能特异的启动GUS基因在烟草叶片中高效表达。     

Cloning of the flap endonuclease-1 gene in Bombyx mori and identification of an antiapoptotic function

The flap endonuclease-1 (FEN-1) gene is involved in DNA replication and repair, and it maintains genomic stability as well as the accuracy of DNA replication under normal growth conditions. However, FEN-1 also plays an important role in apoptosis and cancer development. We cloned the BmFEN-1 gene from Bombyx mori, which was 1343?bp in length and possessed an 1143?bp ORF (123-1266). It consists of seven introns and eight exons that encode a protein with 380 amino acids that has the typical XPG domain. The N-terminal motif is located at amino acids 95-105, and the proliferating cell nuclear antigen interaction motif is located at amino acids 337-344. RNA interference-mediated reduction of BmFEN-1 expression induced cell cycle arrest in S phase in BmE-SWU1?cells. These results suggest that BmFEN-1 can inhibit apoptosis and promote cell proliferation.     

Cloning of fatty acid elongase1 gene and molecular identification of A and C genome in Brassica species

The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at po- sition 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and be- tween the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.     

Cloning of TTG1 gene and PCR identification of genomes A,B and C in Brassica species

Arabidopsis Transparent Testa Glabra 1 (TTG1) genes were cloned from three diploid Brassica species (B. rapa, B. nigra and B. oleracea) and two amphidiploids species (B. juncea and B. carinata) by homology cloning. TTG1 homologues identified in all the accessions of the investigated species had a coding sequence of 1,014 bp. One copy was obtained from each diploid species and two copies from each amphidiploid species. Combined analysis of the TTG1 sequences cloned in this study with those obtained from public databases demonstrated that three, forty-five and seven nucleotides were specific variations in TTG1 genes from genomes A, B and C, respectively. Primers designed with genome-specific nucleotide variations were able to distinguish among TTG1 genes originating from genomes A, B and C in Brassica. Therefore, the TTG1 gene could serve as a candidate marker gene to detect the pollen flow of Brassica and provide an alternative method for the detection of pollen drift and risk assessment of gene flow in Brassica species.