瓠瓜LINE逆转座子RT序列的克隆与特征分析

以瓠瓜[Lagenaria siceraria（Molina）Standl．]品种‘大籽葫芦’（‘Dazihulu’）和‘杂交瓠瓜’（‘Hybridbottlegourd’）为供试材料，对其基因组总DNA中的LINE逆转座子RT序列进行扩增，并对30个克隆产物的核苷酸序列及其编码的氨基酸序列进行比对、同源性分析和系统进化分析。结果表明：瓠瓜品种‘大籽葫芦’与‘杂交瓠瓜’的基因组总DNA均包含长度约580bp的RT序列片段。从2个瓠瓜品种中获得的30条LINE逆转座子RT核苷酸序列（编号为LsRT1至LsRT30）长度为564～599bp，碱基A、T、G和C的数量分别为143—193、157～205、104～139和83～134，AT／GC比为1．29～1．76，表现出高度异质性。缺失突变和终止密码子突变是造成瓠瓜LINE逆转座子RT核苷酸序列长度差异的主要因素。30条瓠瓜LINE逆转座子RT核苷酸序列的相似性为47．1％～99．5％，其编码的氨基酸序列相似性为26．7％～100．0％。根据核苷酸替代值，30条瓠瓜LINE逆转座子RT核苷酸序列可分为4个家族（family），分别包含14、8、1和7条序列。氨基酸序列分析结果显示：瓠瓜LINE逆转座子RT氨基酸序列包含20个保守氨基酸残基和多个半保守氨基酸残基；有14条氨基酸序列具有终止密码子突变。Family1、Family2和Family4是可能具有转座活性的逆转座子家族，分别包含8、3和5条无终止密码子的RT氨基酸序列。根据瓠瓜与其他15种植物的LINE逆转座子RT氨基酸序列构建的系统进化树，瓠瓜与葡萄（Vitis vinifera Linn．）和黄瓜（Cucumis sativus Linn．）等种类的LINE逆转座子RT氨基酸序列有较高同源性。研究结果表明：瓠瓜LINE逆转座子是一类较古老元件，LINE逆转座子可在瓠瓜与其他种类的基因组间横向传递。

Molecular Characterization of a Chinese Soybean Mosaic Virus Isolate by RT_PCR, cDNA Sequence Analysis and Direct Expression of PCR Products in Bacteria

Soybean mosaic virus (SMV) causes one of the most severe viral diseases in soybean ( Glycine max L.) and is known to contain many pathogenically and serologically related isolates. In the present study, the authors have obtained cDNAs to all cistrons of a Chinese SMV isolate, SMV_ZK, by RT_PCR. By analysing the nucleotide and amino acid sequence of the HC_PRO, NIb and CP cistrons, it was found that SMV_ZK was highly homologous to the G2 strain of SMV, thus confirming the existence of G2_like isolates in soybean crop in China. The amplified cDNAs were directly cloned into a bacterial expression vector. With the exception of the P3 cistron, expression of the cDNAs of all other cistrons in bacteria gave rise to polypeptides of expected molecular weight. The expressed viral proteins were subsequently purified by gel elution. The preparation of viral_specific cDNAs and gene products will be useful in future functional study of the SMV genome.

登革2型病毒NS3基因的扩增及序列测定

采用热酚法从登革２ 型病毒４３ 株（Ｄ２４３） 感染的Ｃ６／３６ 细胞中提取了病毒ＲＮＡ，以病毒ＲＮＡ 为模板，进行Ｄ２４３ 株ＮＳ３ 基因ｃＤＮＡ 片段的反转录ＰＣＲ 扩增，片段长度为１１７６ ｂｐ。将扩增的ｃＤＮＡ 片段克隆到Ｔ 载体ｐＢｌｕｅｓｃｒｉｐｔｋｓⅡ（ ＋ ） 中。通过双脱氧法测定了ｃＤＮＡ片段序列，与国际标准株ＮＧＣ株序列一致。

Na^+/K^+-ATPase α-subunit in swimming crab Portunus trituberculatus: molecular cloning, characterization, and expression under low salinity stress

Na^＋/K^＋-ATPases are membrane-associated enzymes responsible for the active transport of Na^＋ and K^＋ ions across cell membranes, generating chemical and electrical gradients. These enzymes＇ α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na^＋/K^＋-ATPase α-subunit eDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction （RT-PCR） and rapid amplification of eDNA end methods. Analysis of the nucleotide sequence revealed that the eDNA had a full-length of 3 833 base pairs （bp）, with an open reading frame of 3 120 bp, 5＇ untranslated region （UTR） of 317 bp, and 3＇ UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na^＋/K^＋-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of arnino acid sequences showed that the P. tritubereulatus α-subunit possessed an overall identity of 75%-99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit＇s transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab＇s Na^＋/K^＋-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress.

Cloning and Sequence Analysis of Three Plant Ran Genes

[Objective]The aim was to study homology between Ran gene in Allium cepa,Allium sativum and Brassica napus and Ran2 gene in Arabidopsis in order to determine whether three kinds of plant material as substitute for Arabidopsis. [Method]By using RT-PCR method,homology gene was cloned from totoal RNA which extracted from splinter cells of Allium cepa,Allium sativum and Brassica napus with Arabidopsis Ran2 primer,then,carrying out sequence and comparative analysis. [Result]The results showed that the open reading frames of Ran genes in Allium cepa,Allium sativum and Brassica napus were 666,663,666 bp,coding 221,220 and 221 amino acids respectively,with the molecular weight of 24.3 kDa. The sequence analysis showed that the amino acid homology of Ran genes between Allium cepa,Allium sativum,Brassica napus and Arabidopsis Ran2 were respectively 99.1 %,100 % （except an Asp D at Allium sativum C terminal）,96.4 %. The phylogenetic tree indicated that Ran genes from Allium cepa and Allium sativum had closer evolutionary relationship with Arabidopsis Ran2. [Conclusion]The research laid a foundation for further study on the biological function of plant Ran gene.

Finding noncoding RNA transcripts from low abundance expressed sequence tags

It has been proved that noncoding RNA （ncRNA） genes are much more numerous than expected. However, it remains a difficult task to identify ncRNAs with either computational algorithms or biological experiments. Recent reports have suggested that ncRNAs may also appear in the expressed sequence tags （EST＇s） database. Nevertheless, intergenic ESTs have received little attention and are poorly annotated owing to their low abundance. Here, we have developed a computational strategy for discovering ncRNA genes from human ESTs. We first collected ESTs that are located in the intergenic regions and do not have detailed annotations. The intergenic regions were divided into non-overlapping 50-nt windows and PhastCons scores obtained from the UCSC database were assigned to these windows. We kept conserved windows that had PhastCons scores of over 0.8 and that had at least three supporting ESTs to act as seeds. Each cluster of ESTs corresponding to the seeds was assembled into a long contig. We used two criteria to screen for ncRNA transcripts from these contigs： the first was that the longest predicted open reading frame was less than 300 nt and the second was that the likely Pol-II promoters exist within 2 000 nt upstream or downstream of the contigs. As a result, 118 novel ncRNA genes were identified from human low abundance ESTs. Of seven randomly selected candidates, six were transcribed in human 2BS cells as shown by RT-PCR. Our work proves that the EST is a ＇hidden treasure＇ for detecting novel ncRNA genes.

Isolation and Complete Genomic Sequence Analysis of a New Sindbis-like Virus

The Sindbis-like virus was first discovered in China in 1986. Its complete genomic sequence consists of more than 11 000 bp encoding more than 3 700 amino acids. It contains a 5＇ non-transcriptional region （5＇-NTR） in a non-structural region, four non-structural proteins （nsP1, nsP2, nsP3, nsP4） regions, capsids in conserved and non-conserved regions and structural El, E2, E3, 6K regions and a 3＇ non-transcriptional region （3＇-NTR）. The Sindbis-IMB was isolated from the blood of a patient suspected to have encephalitis, and was followed by identification and passage. The virus RNA was extracted from virus supernatant in infected cells and the whole genome was divided into 12 fragments; RT-PCR was then performed to amplify the 12 fragments for complete sequencing. The results showed that the whole genomic sequence of Sindbis-IMB consists of 11 717 bp encoding 3 773 amino acids. Homology comparison with other Sindbis-like isolates demonstrated the highest similarity was the YN87448 with a variation of 1% strain isolated in Yunnan Province and the second highest to the SAAR86 strain with a variation of -1.2%. The nucleotide sequence variations were present in non-structural regions, resulting in amino acids K, E, N, R, H, and L in protein sequences in positions 230, 231,443,781, 1 582, and 1746 in the new isolation respectively. Furthermore, three additional amino acids-glutamic acid, serine and alanine-were noted in nsp4 terminus as compared to the YN87448 isolate

Construction of RNAi Vector Which Resist Cucumber Mosaic Virus and Transformation of Tobacco

[Objective] Aimed to construct RNAi vector resistant to cucumber mosaic virus and transferred this vector into tobacco. [Method] RT-PCR method was used to amplify cucumber mosaic virus NS04 and process RNA2 gene sequen of tomato isolates. The analysis results of phylogenetic tree demonstrated that the sequence in RNA2 encoded CMV-2a had 98.0% and 96.5% homology with nucleotide and amino acid of DQ412731 isolate of Zhejiang,China. The replicase fragment in CMV RAN2 gene was taken as target sequence to construct pBi35SCR2 eukaryotic expression vector,then the expression vector was identified. Through agrobacterium-mediated method,the expression vector was transferred into tabacco and PCR method was used to check the transfer. The PCR results demonstrated that the experiment had successfully construct eukaryotic expression vector of pBi35SCR2 and the expression vector was successfully transferred into tabacco. [Conclusion] The obtained transgenic tobacco could be used as challenge test material in following experiment and provided foundation for studying processing tomato resist cucumber mosaic virus.

Cloning and Sequence Analysis of CHS Gene Fragment from Acer truncatum

[Objective] This study aimed to clone and analyze the sequence of CHS gene from Acer truncatum leaves. [Method] Using A. truncatum cultivars No.1-6 as experimental materials, total RNA was extracted from A. truncatum leaves with the modified CTAB method. CHS gene sequences were downloaded from the NCBI and aligned by BLAST. Degenerate primers were designed by DNAMAN and Primer- premier5 to amplify the target band. CHS gene fragment was amplified by RT-PCR and ligated to pMD18-T vector. The identified positive colonies were sequenced. [Result] A 1 365 bp fragment was amplified. Sequence analysis suggested that the obtained fragment encoded 365 amino acids and shared above 90% homology to nucleotide sequence of CHS gene from A. palmatum and A. [Conclusion] In this study, CHS gene was successfully cloned from A. truncatum for the first time, which laid the foundation for efficient utilization of CHS gene.

Identification and characterization of a Dna J gene from red alga Pyropia yezoensis(Bangiales, Rhodophyta)

Members of the Dna J family are proteins that play a pivotal role in various cellular processes, such as protein folding, protein transport and cellular responses to stress. In the present study, we identified and characterized the full-length Dna J c DNA sequence from expressed sequence tags of Pyropia yezoensis( Py Dna J) via rapid identification of c DNA ends. This c DNA encoded a protein of 429 amino acids, which shared high sequence similarity with other identified Dna J proteins, such as a heat shock protein 40/Dna J from Pyropia haitanensis. The relative m RNA expression level of Py Dna J was investigated using real-time PCR to determine its specific expression during the algal life cycle and during desiccation. The relative m RNA expression level in sporophytes was higher than that in gametophytes and significantly increased during the whole desiccation process. These results indicate that Py Dna J is an authentic member of the Dna J family in plants and red algae and might play a pivotal role in mitigating damage to P. yezoensis during desiccation.

Characterization, Expression and Function Analysis of DAX1 Gene of Scallop(Chlamys farreri Jones and Preston 1904) During Its Gametogenesis

DAX1, a member of nuclear receptor superfamily, has a function in the sex determination and gonadal differentiation of several vertebrate species. However, little information about DAX1 of invertebrates is available. Here we cloned a homolog of scallop(Chlamys farreri Jones and Preston 1904) dax1, Cf-dax1, and determined its expression characteristics at m RNA and protein levels. The c DNA sequence of Cf-dax1 was 2093 bp in length, including 1404 bp open reading frame(ORF) encoding 467 amino acids. Unlike those of vertebrates, no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1. Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes. Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues, with the highest expression level found in adductor muscle, moderate level in mantle, gill and testis, and low level in kidney, ovary and hepatopancreas. The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher(P<0.05) in testis than in ovary at the same stage, showing a sex-dimorphic expression pattern. Furthermore, immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary, implying that DAX1 may involve in gametogenesis of bivalves.

SEQUENCE ANALYSIS OF THE NS5 REGION OF GBVC/HGV AND DETECTION OF THE VIRUS BY REVERSE TRANSCRIPTASE PCR

GBV C/HGV is a newly identified virus associated with human hepatitis In this study, the nucleotide sequences of the partial NS5 gene of GBV C/HGV derived from sera of 8 Chinese patients were determined The nucleotide homology among the 8 isolates were 92% on average On the basis of sequence analysis, two sets of oligonucleotide primers derived from highly conserved region of GBV C/HGV NS5 gene were designed to establish both sensitive and specific nested PCR for detection of GBV C/HGV RNA 253 Chinese patients were examined for the virus RNA GBV C/HGV RNA positive rates in patients infected with HBV, HCV and patients with chronic non B,non C hepatitis were 18 4%, 19 8% and 8 9% respectively This result suggested that HBV,HCV and GBV C/HGV shared the same transmission risk factors 8 patients with GBV C/HGV and HCV coinfection were retrospectively observed for the response to interferon Coinfection with GBV/HGV did not negatively influence the responsiveness of HCV, and GBV C/HGV was sensitive to interferon to a certain degree

Cloning and Bioinformatics Analysis of zmERECTA

[Objective] This study was conducted to clone zmERECTA gene according to Arabidopsis ERECTA sequence and predict its characteristics by bioinformatics. [Method] The c DNA of zmERECTA gene was isolated from B73 using RT-PCR, and analyzed by bioinformatics methods. [Result] zmERECTA gene was 2 985 bp in size, which encoded a protein consisting of 944 amino acids, containing leucine-rich repeats, a PKC domain, two transmembrane regions, 14 N-glycosylation potential sites and41 kinase specific phosphorylation sites. The theoretical p I and molecular weight of zmERECTA protein was 6.01 and 10 8495.5respectively. [Conclusion] Cloning and bioinformatics of zmERECTA gene laid a foundation for further research.

CLONING AND COMPARISON OF THE GENES ENCODING PREPROAPAMIN FROM THE VENOM OF 2 HONEYBEE AND 4 WASP SPECIES

Preproapamin genes were amplified by RT-PCR from total RNA from the venom glands of 2 honeybee species, Apis mellifera, A. cerana cerana, and 4 wasp species, Vespa magnifica, V. velutina nigrothorax and Polistes hebraeus, respectively. Their PCR products were ligated into pGEM -T easy vector and the nucleotide sequences analyzed. The six fragments were all 141?bp in length and contained a n ORF coding the precursor of apamin. The apamin precursors of V. magnifica, V. velutina nigrothorax and P. hebraeus had 95% and 93% similarity with that of A. melliera in nucleotide and amino acid sequences, respectively. That of Vespu la maculifrons was identical to that of A. mellifera in nucleotide and amino acid sequences. Apamin precursors of V. magnifica, P. hebraeus and V. velutina nigrothorax also had the same nucleotide sequences. The nucleotide sequences o f preproapamin genes from the Chinese honeybee, A. cerana cerana and 4 wasp sp ecies are described for the first time. A notable discovery was that the wasps species had exactly same apamins as the honeybees despite the fact they belong to different insect families.

Pod1 is involved in the sexual differentiation and gonadal development of the Nile tilapia

Pod1 is a member of the basic helix-loop-helix(bHLH) family of transcription factors that have been implicated in the regulation of sexual differentiation and gonadal development in mammals.However,to date,little is known about the role of Pod1 in nonmammalian vertebrate gonadogenesis.We cloned and characterized the Pod1 gene from tilapia.The tilapia Pod1 gene contains an open reading frame(ORF) of 525 nucleotides which potentially codes for a protein with 174 amino acids.Sequence alignment revealed that the deduced tilapia protein sequence shared high homology(79.5% to 90.5%) with the Pod1 sequences of other vertebrates.The tissue distribution of Pod1 revealed by RT-PCR showed that it had varied expression patterns in adult tilapia.In situ hybridization was performed to examine the temporal and spatial expression patterns of Pod1 during tilapia sexual differentiation and gonadal development.In the undifferentiated gonad,Pod1 was expressed in the somatic cells of both sexes.Subsequently,Pod1 expression in tilapia persisted in differentiated juvenile and adult ovary and testis.Our data indicate for the first time that Pod1 is not only necessary for the onset of sexual differentiation,but also plays an important role in gonadal development in the teleost.