流感病毒ns1基因的克隆及其原核表达载体的构建

为了解H5N1亚型流感病毒株的ns1基因特性及其规模制备NS1蛋白,首先将病毒在鸡胚中传代,从收获的尿囊液中提取RNA,采用RT-PCR技术扩增流感病毒全长ns基因。测序显示H5N1亚型流感病毒NS1cDNA全长678bp,编码225个氨基酸。BLAST分析表明,Qa/ST/852/01(H5N1)病毒株ns1基因与近年来从华南地区分离的禽H5N1毒株的ns1基因有很高的同源性。之后采用PCR方法扩增ns1基因的cDNA片段,将其克隆到pGEX-4T-3载体中,与谷胱甘肽巯基转移酶(GST)基因融合,构建重组质粒pGEX-4T-3/NS1cDNA,转化大肠杆菌BL21(DE3)并进行诱导表达。SDS-PAGE和凝胶扫描分析,GST-NS1融合蛋白在大肠杆菌中获得了高效表达,并且以可溶形式存在,重组融合蛋白的表达量占菌体总蛋白的28.5%,表达产物经亲和层析纯化后蛋白质纯度达96%以上。经免疫印记证实重组融合蛋白可以被GST特异性抗体所识别。该表达载体的构建为获得大量NS1蛋白进行功能研究及抗体制备提供了基础。

Cloning and Sequencing the Full-length cDNA of Annexin from Strawberry Fruit

采用RACE技术从草莓 (FragariaananassaDuch .)成熟果实中分离克隆了膜联蛋白 (annexin)基因的cDNA 5′_端未知序列 ,并通过测序确定了翻译的起始密码位点、终止密码位点及完整的读码框 ,从而首次获得了草莓果实膜联蛋白基因的cDNA全序列 ,命名为annfaf(annexinofFragariaananassafruit)基因。

Cloning and Analysis of NBS-LRR Type Resistance Gene Analogues in Sweet Potato (Ipomoea batatas)

The degenerate primers were designed based on the conserved NBS-LRR motifs among the known disease-resistance genes. A fragment of about 500 bp was amplified from genomic DNA of sweet potato using the specifically designed degenerate primers. After cloning and sequencing, 20 NBS-LRR type of disease-resistance gene analogue （RGAs） in sweet potato were observed. The deduced amino acid sequence of DNA fragment contains the conserved motifs of NBS-LRR type RGAs, such as P-loop, Kinase-2α, Kinase-3α and GLPL domain. The 20 RGAs could be sorted into two subclasses, namely TIR- NBS-LRR type and non-TIR-NBS-LRR type. Compared with the known resistance genes including N, L6 and M, the percentages of homologous amino acid sequence in 10 TIR-NBS-LRR range between 21% -44%. While other 10 non-TIR-NBS-LRR assume 15% -46% homology with the known resistance genes （Prf, RPM1, RPS2, etc. ）. Consequently the RGAs may further be used as molecular marker for screening the candidate disease-resistance genes in sweet potato.

Cloning and Sequence Analysis of Actin Gene from Rehmannia glutinosa

[ Objective ] The aim of this study is to clone and analyze the actin gene from Rehmannia glutinosa. [ Method ] Degenerate primers were designed according to the conserved regions of actin sequences of Rehmannia glutinosa and its similar species, RT-PCR was next conducted to amplify the actin gene from Rehmannia glutinosa. [ Result] The amplified fragment is 724 bp and correspondingly 240 amino acids. The BLAST results indicate that the homology between the amplified fragment and other higher plants for aetin gene sequences and amino acid are more than 80% and 90%, respectively, suggesting that the amplified fragment is the actin gene of Rehmannia glutinosa. [ Conclusion] Phylogenetic analysis shows that the actin gene of Rehmannia glutinosa has an intimate genetic relationship with actin7 gene of Nicotiana tabacum.

Molecular Cloning and Sequence Analysis of Class Ⅱ Chitinase Gene in Leymus chinensis

[ Objective] The aim of this study was to clone Class Ⅱ chitinase gene in Leymus chinensis grown in saline land in Heilongjiang Province and analyze its sequence, which provided a foundation for further study on the biological function and application of chitinasa gene. [ Method] cDNA library of Leymus chinensis leaves were constructed, and its DNA sequence was determined or analyzed, while the homology of chitinasa gene and amino acid sequence was compared with that in GenBank. [ Result] One full length cDNA fragment with length of 996 bp was cloned from cDNA library of Leymus chinensis leaves. The length of ORF was 768 bp encoding 225 amino acids （GenBank accession number： EU344908）. The encoding products lacked CBD and C-terminal extension region from the view of structure, but had structural characters of Class Ⅱ chitinase gene, which indicated that amino acid sequence had high homology compared with Class Ⅱ chitinase gene of rye and wheat. The constructed recombinant vector pQE-LcChi2 could express a protein of 27 kD through induction, which was consistent with the deduced encoding product of pQE-LcChi2 gene. [ Conclusion] LcChl2 gene is an expression gene, which can express in E. coll.

Cloning and Sequencing of Two Acetylcholinesterase cDNA Fragments from Cotton Aphid,Aphis gossypii Glover

Two acetylcholinesterase (AChE) genes cDNA fragments,Ag.acel and Ag.ace2,have been cloned from cotton aphid,Aphis gossypii Glover using degenerate primers with RT-PCR technique.Ag.acel gene cDNA fragment is of 282?bp encoding 94 amino acids,and Ag.ace2 gene cDNA fragment is of 264?bp encoding 88 amino acids.Both two putative AChE genes cDNA fragments share numerous similarities with those cloned from other insects.This is the first report of two AChE cDNA fragment sequences in the insect species,which provided the direct evidence of multiple AChE existence in insects.

Cloning, Characterization and Chromosome Localization of Two Powdery Mildew Resistance-Related Gene Sequences from Wheat

Reverse_transcription Polymerase Chain Reaction (RT_PCR) was performed using cDNAs as templates from wheat_ Haynaldia villosa 6VS/6AL translocation line and 'Yangmai 5' induced with fungus Erysiphe graminis , and degenerate primers designed based on the conserved amino acid sequences of known plant disease_resistance genes. The cDNA sequences encoding cyclophilin_like and H +_ATPase_like genes were first isolated and characterized in wheat. The putative amino acid sequences of the two clones showed that they were highly homologous to those of cyclophilin proteins and H +_ATPases isolated from other plants. Thus they were designated as Ta_Cyp and Ta_MAH . The obvious expression differences could be observed between wheat_ H. villosa 6VS/6AL translocation line and susceptible wheat cultivar 'Yangmai 5', implying that the two genes may be related with the resistance of wheat_ H. villosa 6VS/6AL translocation line to disease. Southern blot indicated that the wheat genome contained 2-3 copies of Ta_Cyp gene and one copy of the Ta_MAH gene. Chinese Spring nulli_tetrasomic line analysis located the Ta_Cyp homologous genes on wheat chromosome 6A, 6B and 6D. Southern blot using Ta_Cyp clone as a probe showed that the polymorphic bands existed among the H. villosa , amphiploid of Triticum durum _ H. villosa , wheat_ H. villosa 6VS/6AL translocation line and 'Yangmai 5', suggesting that Ta_Cyp homologies exist in wheat genome as well as on the short arm of chromosome 6V in H. villosa .

Cloning and Sequence Analysis of Interferon γ-2β Full-length cDNA in Cyprinus carpio L.

[Objective] The research aimed to carry out the cloning, identification and sequence analysis of full-length cDNA of carp interferon γ-2β （IFNγ-2β）. [Method] The cDNA library of peripheral blood leucocytes which were separated from carp and stimulated with mitogen was screened by a probe labeled with DIG. The IFNγ- 2β EST sequence was picked out from the constructed cDNA library of peripheral blood leucocyte, and the full length of carp interferon γ-2β was cloned. In addition, the sequence analysis was carried out. [Result] Three positive clones were obtained. Sequence analysis indicated that the sequence had a 119 bp 5’-UTR and a 218 bp 3’-UTR, and the open reading frame （ORF）of this gene was 537 bp which putatively coded 178 amino acids and there were several instable motifs for mRNA （ATTTA） in the 3’-untranslated region. Its homology with IFN from GenBank was up to 97% . Analysis on protein sequence and structure showed that the predicted protein sequence was identified as an IFN family signature. [Conclusion] The research laid the foundation for further studying the expression manner, function characteristic and regulation mechanism of IFNγ-2β in vivo and the action mechanism in the inflammatory reaction, emergency reaction and immune response.

Cloning and Sequence Analysis of ATPase Beta Subunit Gene from Eleutherococcus senticosus

[Objective] This study aimed to clone and analyze the cDNA of ATPase β subunit gene from Eleutherococcus senticosus.[Method] A pair of homologous primers was designed according to the chloroplast ATPase β subunit gene sequences of the known species;then the gene cDNA of E.senticosus were amplified by RT-PCR and compared with that of the known species;its structure was predicted finally.[Result] 1 099 bp of ATPase beta subunit cDNA of E.senticosus which encodes 366 amino acids was amplified by RT-PCR.Sequence comparison and structure prediction showed that amino acids encoded by the ATPase beta subunit gene of E.senticosus shared the highest homology,up to 96.41% with that of Oryza sativa.In the secondary structure,the protein contained 171 alpha helixes accounting for 46.72%,53 extended strands accounting for 14.48%,27 beta sheets accounting for 7.38% and 115 random coils which took up 31.42%.The amino acids 262-271 were the symbolic site of ATPase β subunit.The whole peptide chain had no obvious hydrophobic region and was primarily confirmed as a hydrophilic protein.[Conclusion] The cNDA of ATPase β subunit gene cloned from E.senticosus in this study will provide reference for learning the effect of energy metabolism on secondary metabolism,structure and function of ATPase in E.senticosus.

Cloning and Sequence Analysis of ras Promoter from Auricuralia auricular Strain YBS-3

[Objective] The ras promoters were cloned from genomic DNA of Auricuralia auricular so as to provide promoters for breeding better species by genetic technology. [Method] PCR was used to clone promoters with the genomic DNA of A. auricular strain YBS-3 as template,and then the sequences of four ras promoters were obtained. The analysis of promoter sequences was made by three promoter analysis software：Promoter prediction,Place and TFSEARCH ver.1.3. [Result] Four fragments contained core elements of promoter including TATA-box and CAAT-box,and many other important cis-elements such as GATA BOX,GCGC BOX,CCAAT BOX1,etc. At the same time,there was at least one transcriptional start site in these sequences. And several transcription factor binding sites were detected in these sequences. [Conclusion] Four promoter fragments all had promoter function theoretically.

Cloning and Expression of Gonadotrophin Releasing Hormone Receptor in Apis cerana cerana

[Objective] This study was to clone the GnRH （gonadotropin-releasing hormone）, and to investigate its expression in Apis cerana cerana. [Method] The cDNA sequence of GnRHR gene was amplified from Apis cerana cerana by using RT-PCR techniques. It was conducted with bioinformatics analysis and the in situ hybridization histochemistry of its expression products was studied. [Result] The sequence analy- sis showed that the full cDNA sequence was 1 050 bp with the open reading frame of 1 050 bp, and it encoded 349 amino acid residues. The deduced amino sequence included 7 transmembrane regions, and the predicted molecular mass and isoelectric point were 40.6 kD and 9.54, respectively. The cluster analysis showed that the GnRHR from ＇,4. cerana cerana had close relationship to the GnRHR II from other insects. In situ hybridization showed that Bee-GnRHR staining was specifically localized to the brain, intestine, fat body and testis. [Conclusion] The results indicated that the GnRHR provided molecular bond for the reproduction and metabolism for insects, and suggested a functional role for bee-GnRHR signaling in the coupling of reproduction activities and environment conditions.

Cloning,Sequence Analysis and Amino Acid Structure Prediction of TLR9 Gene from Wild Ovis ammon in Xinjiang

[Objective] The study aimed at cloning and identifying the toll receptor gene 9（TLR9） of wild Ovis ammon in Xinjiang,and predicting its structure and function.[Method] The TLR9 complete sequence of wild Ovis ammon was cloned from its peripheral blood by PCR technology.Then the PCR products were purified by agarose gel electrophoresis and then sequenced.Finally,the structure and function of TLR9 sequence were predicted by molecular biological software.[Result] The complete sequence of TLR9 gene was 3 192 bp in length,encoding 1 064 amino acids with a signal peptide composed of 30 amino acids;the leucine percentage reached as high as 18.5%.The TLR9 amino acid possibly contained three hydrophobic regions,at amino acids 455-475,740-760 and 780-800.The 3-D structure of TLR9 was constructed by the extracellular LRR domain and intracellular TIL domain（Toll/IL IR）.[Conclusion] The characteristics of TLR9 provided theoretical basis for further study on the TLR9 gene of wild Ovis ammon in Xinjiang.

Cloning and Sequence Analysis of Adiponectin Receptor 1 and Receptor 2 cDNA from Guangxi Bama Mini-pig

[ Objective] To clone and analyze the sequence of Adiponectin receptor 1 （ AdipoR1 ） and receptor 2 （AdipoR2） cDNA of Guangxi Bama mini-pig. [Method] The Adiponectin receptors cDNAs were amplified by RT-PCR using skeletal muscle total RNA as template and then ligated into pMD18-T vector after purification. The recombinant pMD18-T vector was transformed into the E. coil DH5α for identification and sequencing. And the results were compared with the cDNA sequence from other species. [Result] The fragments, 1 128 bp and 1 161 bp in size, were amplified by RT-PCR and respectively consistent with the coding sequence of AdipoR1 gene and AdipoR2 gene. The homology analysis showed that the sequences of AdipoR1 gene and AdipoR2 gene were respectively 99.8% and 99.7% homologous to the sequence of domestic pig reported in GenBank with one base and three base missense mutations correspondingly. [ Conclusion] The AdipoR1 gene and AdipoR2. gene were successfully amplified from Guangxi Bama mini-pig, laying the foundation for the further study of the biological function of AdipoR genes and the design of novel drugs with AdipoR as target.

Molecular Cloning and Sequence Analysis ofBoPGIP2 Gene from Brassica oleracea L. var. alboglabra

PGIP gene was obtained from Brassica oleracea L. var. alboglabra, named BoPGIP2. The full length of BoPGIP2 gene is 1 102 bp and the exon is 993 bp which encodes a protein of 330 amino acids with a predicted molecular mass of 37.1 kDa, interrupted by one intron of 95 bp in, length. Sequence analysis revealed that it has five potential N-giycosylation sites, two protein kinase C phosphrylation sites, five casin kinase Ⅱ phosphrylation sites and four N-myristoylation sites. The amino acids sequences alignment confirmed that ^145 LRR stucture was highly conserved in all aligned PGIP sequences.

Cloning and Sequence Analysis of 5′ Flanking Region and Exon of Inhibin α Precursor Gene in Goats

[Objective] The aim of this study was to reveal the relationship between inihibin （INH） α precursor gene and seasonal reproduction of goats, and investigate the evolutionary conservation of INHα precursor gene. [ Method] Cloning and sequence analysis of 5＇ flanking region and exon of inihibinα （INHE） precursor gene in twenty ewes between non-seasonal estrous breed （Haimen goats） and seasonal estrous breed （Anhui white goats） was analyzed in this study. [ Result] Compared with Anhui white goats, INHα precursor gene in Haimen goats had three SNP but no amino acid change, while its nucleotide homology was 99.7% and amino acid homology was 100%. The nucleotide homology of INHα precursor gene in goat, cattle, pig, person, chicken, horse, rat and dog ranged from 12.7% to 96.5%. [ Conclusion] INHα precursor gene tends to be highly conserved in species, and any change of nucleotide and amino acid maybe directly influence the function of the whole gene coding and regulation.

Cloning and Analysis of 5’ Flanking Region of Sporamin A Gene from Sweet Potato

[ Objective] The aim of the study is to clone the 5＇ flanking region of Sporamin A gene from sweet potato. [ Method ] The sweet potato ＂Xushu 18＂ was used to amplify the 5＇ flanking region of Sporamin A gene using specifically designed primers and then target fragment was analyzed by PLACE and PlantCare online. [ Result ] Besides the conserved elements including TATA-box and CAAT-box, the cis-acting elements including sucrose responsive element CMSRE1, SP8 acting site and some other regulatory sequence such as MYB binding site were also found in Spo A promoter sequence. The results suggest that the promoter has sucrose responsive function. [ Conclusion ] The study provided reference to reveal the regulation law of Spo A, and to develop high-level expression vectors promoted by Spo A promoter.

Cloning and Sequence Analysis of CmNAC Gene from Cucurbita moschata

[Objective] The aim was to clone the CmNAC gene from Cucurbita moschata and analyze the sequence characteristics. [Method] A pair of degenerate primers was designed based on the conserved sequences of NAC gene from Brassica napus, Lycopersicon esculentum and Capsicum annuum. NAC transcription factor gene was amplified by RT-PCR from Cucurbita moschata leaves and cloned into pMD-19T vector; then the recombinant clones were sequenced. Finally, the sequences of nucleic acid and amino acid were analyzed using BLAST and DNAMAN software. [Result] The NAC transcription factor gene cloned from C. moschata included 442 bp encoding 147 amino acids, named CmNAC. The NAC gene fragment contained a conserved region like other plant NAC genes and belonged to the NAC family ATAF1/2 subfamily. [Conclusion] The stress resistance related gene NAC cloned from C. moschata is a foundation for further study on the biological function of the gene and plant genetic engineering.

Cloning and Sequence Analysis on cDNA of Growth Hormone Releasing Hormone Receptor Gene from Wuzhishan Miniature Pig

[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of Wuzhishan miniature pig as the template, three pairs of primers were designed by the reported cDNA sequence of porcine GHRH, and cDNA was also amplified by RT-PCR. After being recovered and purified, PCR products were ligated to pMD18-T and then transformed into Escherichia coli DH5a. The transformation products were analyzed by PCR and double enzyme digestion to screen positive clones, and the positive clones were sequenced after identification in LB liquid medium. [ Result] cDNA of Wuzhishan miniature pig GHRH receptor gene was obtained successfully, and its length was 1 577 bp coding 423 amino acids. BLAST analysis showed that there were only 23 nuoleotides in difference between this fragment and pomine GHRH receptor gene, and its homology was 98%. However, both GHRH receptor genes were constituted by 423 amino acids with the sequence homology of 96%. [ Conclusion] This study provides theoretical basis for further studies on the dwarf mechanism of Wuzhishan miniature pig.

Cloning and Sequence Analysis of PBF Encoding Genes from Hexaploid Common Wheat Chinese Spring (Triticum aestivum L.)

[Objective] This study aimed to investigate the polymorphism of PBF en- coding genes from common wheat Chinese Spring （Triticum aestivum L.）. [Method] Using common wheat Chinese Spring as the experimental material, gene-specific primers were designed and applied to amplify the genomic DNA of Chinese Spring. PCR products were isolated, purified and ligated into the cloning vector. Positive clones were randomly selected for sequencing. A series of softwares including DNAMAN, Signalp, PSIPRED, Nuc_PLoc and MEGA were employed for sequence assembly and alignment, signal peptide prediction, primary and secondary structure prediction, as well as analyses of subcellular location and phylogenetic relationships between the PBF family members in Poaceae. [Result] Twenty-five target sequences were obtained from the genome of hexaploid common wheat Chinese Spring, which were classified into three clusters based on the sequence similarity. SNPs exist at two loci of the subunit, resulting in the change of encoded amino acid residues and affecting the secondary structure of final product encoded. [Conclusion] PBF encoding sequences are extremely conservative in Chinese Spring with certain variations. This study provides theoretical reference to evaluate the expression efficiency of wheat storage proteins.

CLONING AND ANALYSIS OF THE GENOMIC DNA SEQUENCE OF AUGMENTER OF LIVERR EGENERATION FROM RAT

Objective.To search for genomic DNA sequence of the augmenter of liver regeneration(ALR)of rat.Methods.Polymerase chain reaction(PCR)with specific primers was used to amp lify the sequence from the rat genome.Results.A piece of genomic DNA sequence and a p iece of pseudogene of rat ALR were identified.The lengths of the gene and pseudogene are 1508bp and 442bp,respectively.The ALR gene of rat includes 3exons and 2introns.The 442bp DNA sequence may represent a pseudogene or a ALR-related peptide.Predicted amino acid sequence analysis showed that there were 14different amino acid residues between the gene and pseu do-gene.ALR-related peptide is 84amin o acid residues in length and relates closely to ALR protein.Conclusion.There might be a multigene family of A LR in rat.