Isolation, cloning and sequencing of AFLP markers related to disease-resistance traits in Fenneropenaeus chinensis

Amplified fragment length polymorphisms (AFLP) technique was used to analyze the fingerprint- ing of four successive generations of Fenneropenaeus chinensis to reveal their disease-resistance traits. Some loci showed quite different genetic frequencies due to artificial selection, which implied that these fragments were putative markers related to the disease-resistance trait. We developed a simple and effective method to fur- ther characterize these AFLP fragments. Specific AFLP bands were cut directly from polyacrylamide gels, re-amplified, cloned and sequenced. Eight putative genetic markers were sequenced and their sizes ranged from 63 to 209 bp. The sequences were submitted to dbGSS (database of Genome Sequence Survey); and the BLAST analysis showed low similarity to the function genes, indicating these markers were tightly linked to a dis- ease-resistance trait but were not functional genes.

CLONING AND SEQUENCING OF THE FERREDOXIN GENE OF BLUE-GREEN ALGA ANABAENA SIAMENSIS

The structure gene for ferredoxin, petFI, from Anabaena siamensis has been ampli-fied by polymerase chain reaction(PCR) and cloned into cloning vector pGEM-3zf(+). The nucleotidesequence of petFI has been determined with silver staining sequencing method. There is 96. 8% homologybetween coding region of petFI from A. siamensis and that of petFI from A. sp. 7120. Amino acid se-quences of seven strains of blue-green algae are compared.

Cloning and Sequencing of Gtx-2 Homeobox Sequence

murine genomic DNA was surveyed by polymerase chain reaction (PCR) to detect homeobox sequence of mammal. The PCR product (413 bp) was cloned and sequenced. The nucleotide sequence and the deduced amino acid sequence of a homeobox, which was isolated in this study, designated Gtx 2 were obtained. The result of sequence analysis showed that there is an intron between the nucleotides at position 138 and 139 in the Gtx 2 homeobox. Southern blot analysis revealed that Gtx 2 is likely to exist as a single copy in the murine genome. Comparison of the encoded polypeptide sequence with other homeodomains reveals that Gtx 2 has 96% and 58% identity to that of Gtx 1 and Tcl 3 , respectively.

Cloning, Sequencing, and Characterization of Porcine Sterol Regulatory Element Binding Proteins-1c Gene

Primers were designed according to the known sequences of Sterol Regulatory Element Binding Proteins-1c (SREBP-1c) genes of human, rat and pig. RT-PCR was then used to amplify porcine SREBP-1c gene with total RNA of spleen tissue. A 760 bp segment of cDNA was cloned and sequenced. Homogeneous comparison showed that the sequence of porcine SREBP-1c had 99.9% and 99.1% homogeneity with the two known partial mRNA sequences of porcine SREBP-1c gene. The complete cDNA was obtained mainly based on the known partial sequences, which has 3 830 bp, encoding 1 151 amino acids with a calculated molecular weight of 121 479 Da. It is the first time that we get complete encode sequence of porcine SREBP-1c gene. The complete cDNA sequence has high homogeneity with SREBP-1c gene of other species. A characteristic structure of HLH (Helix-Loop-Helix) and four transmembrane segments were found in the amino acids. The sequence had been submitted to GenBank (Accession No.NM_-214157).

Cloning and Sequencing of cSZ1 Gene of Eimeria acervulina Changchun Strain

[ Objective] To analyze Eimetria acervulina cSZl gene sequence and thus to find a candidate antigen for vaccine development.[ Method] According to the cSZl gene sequence of Eimeria acervulina published in GenBank, a pair of specific primers was designed. Then, the cSZl gene of Eirneda acervulina Changchun strain was amplified by reverse-transcription polymerase chain reaction （RT-PCR） and sequenced. [ Result] Compared with the published cSZ1 gene sequence, five nucleotide mutation sites appeared in the cSZ1 gene of Eirneda acervulina Changchun strain. They had a nucleotide sequence similarity of 99.47%, and their encoding region had a nucleotide sequence similarity of 99.61%. Among these mutation sites, two appeared in cSZl gene ORF, and A185G mutation led to substitution of valine by isoleucine. [Conclusion] Protein encoded by cSZ1 gene can be used as a candidate antigen for vaccine development.

Amplification,cloning,sequencing and expression of the gene encoding the major surface antigen of Toxoplasma gondii isolated in China

According to the published gene sequence of the major surface antigen (P30) of Toxoplasma gondii, a pair of primers were designed and synthesized. Using polymerase chain reaction (PCR), the coding sequences of P30 gene were amplified from a Chinese strain of T. gondii, The amplified gene fragment and plasmid pB220 were digested with EcoRI and BamHI and then ligated. The inserted gene fragment was sequenced by the chain termination method, the reading reveals that nucleotide sequence determined was the same as the P30 sequecne of RH strain pubilished by Burg (1988), except that one base was changed. The recombinant plasmid containing P30 gene was transformed to E. coli DH5α.After temperature inducing culture, the total cellular proteins were analysed by SDS-PAGE and Western blot. The results show that the p30 gene cloned into the plasmid could express in E. coli, and the expression product had immunogenicity.

Cloning and sequencing of the fourth exon of transforming growth factor α gene

Using normal brain cell geneomic DNA as a template,transforming growth factor(TGFa)-IV exon gene was amplified by polymerase chain reaction(PCR). The sequence of amplified fragment was analysed with a DNA sequencing kit.The results showed that the cloned fragment is proved to be the TGFa-IV exon gene.

MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING PARTIAL PUTATIVE MOLT-INHIBITING HORMONE FROM PENAEUS CHINENSIS

Total RNA was extracted from eyestalks of shrimp Penaeus chinensis . Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase polymerase chain reaction (RT PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt inhibiting hormone from shrimp Penaeus japonicu s. A specific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 base pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt inhibiting hormones of P. japonicus . The cDNA could be a partial gene of molt inhibiting hormones from P. chinensis . This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis .

Cloning and Sequencing of Kappa Light Chain Gene of a Mouse Monoclonal Antibody Directed Against Potato Virus Y

A cDNA library was constructed in λgt11 vectors, complementary to the mRNA isolated from a mouse hybridoma raised against potato virus Y(PVY). Thirty cDNA clones were selected from the cDNA library by in situ immunohybridization with goat anti-mouse kappa-chain-specific antibody conjugated to alkaline phosphatase, from which one clone, k6, having the largest insert was characterized by sequence analysis. The result shows that the immunoglobulin messenger RNA corresponding to k6 is 956 nucleotides in length excluding the poly(A) region, among which 31 bases code for the 5’ non-coding region, 57 for the leader sequence of the protein, 657 for the mature protein and 211 for the 3’ non-coding region. Comparison of deduced amino acid sequences of the protein and other kappa light chains shows that they share a 100% identity in their constant regions(CL) and 93.7% identity in their variable regions(VL). The kappa light chain encoded by k6 is considered to be specific to PVY since only one type of light chain is expressed in the hybridoma.

Cloning, sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.

PCR Amplification，Cloning and Sequencing of RbcL Coding Region in Mesophyll Cell and Bundle Sheath Cell of Sorghum（Sorghum bicolor L．）

The Primacy question addressed in our study is: Is the difterntial expression of rbcL gene in mesophyll cells and in bundle sheath cells related to the sequence of the gene per se?An enzymatic approach was fist established to separate the two groups of cells. Microscopic examination revealed satisfactory separation effect: minimal mutual contamination was found so that no mistake might be introduced into biochemical or molecular biological expeitments using such preparations. CpDNA were isolated from mesophyll cells and from bundle sheath cells and coding region of rbcL gene was obtained from each by PCR ampilfication.Cloning and sequencing were then done on them.Compartive analysis , however, revealed identical sequence, with a length of 1,368 bp, encoding 456 amino acids. Since sequences of the non-coding regions of rbcL gene in masephyll sad bundle sheath have not been obtained, it can not yet be concluded that the differential expression is not related to the sequence itself. Nevertheless,It sesems justifiable to infer that whatever difference there may be between the sequences of rbcL gene in two groups of cells can only be found in the non-coding regions(including promoter and the 3' down stream region).

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 Expression of Conserved Sequences of cry Gene in E.coli Strain Rosetta(DE3)

[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta （DE3）. [Method] Specific primers were designed according to NCBI database information and the conserved sequences of cry gene were amplified by PCR from Bt transgenic cotton. Then recombinant plasmids were constructed and expressed in E. coil strain Rosetta （DE3）. Finally, the effects of different concentrations and inducing time of IPTG on the expression level of protein were investigated. [Result] Two conserved sequences （304 and 853 bp respectively） of cry gene were amplified. The result of SDS-PAGE confirmed that the recombinant plasmids pGEX-4t-I-304 and pGEX-4t-1-853 could express fusion proteins by IPTG induction and the molecular weight of protein products was 39 and 62.4 kDa respectively, which was in accordance with predicted result. The optimal protein ex- pression conditions were confirmed as induction with 0.15 mmol/L IPTG for 7 h. [Conclusion] This study prepared the ground for the further detection of Bt transgenic crops.

Cloning and Sequence Analysis of HN and F Protein Genes from a Strain of Goose Paramyxovirus

[ Objective] The study was to clone HN and F genes from GX1 strain of goose paramyxovirus and analyze their sequences. [ Method] According to the full nucleotide sequence of GPV-SF02 strain of goose paramyxovirus, two pairs of pdmers were designed to amplify the HN and F genes from GX1 strain of goose paramyxovirus isolated from diseased goose in Guangxi Zhuang Autonomous Region; the amplified products were ligated into pMD18-T vector and sequenced. [ Result ] HN and F genes of this strain tested were 1 716 and 1 662 bp in full nucleotide length, respectively; both showed the homologues of about 97.3% with GPV- SF02 strain, of 80.3% -97.5% with strains LaSota, F48E9 and JS, of just 84.8% with Miyadera strain. [ Conclusion] The results show that isolated strain BX1 matches to virulent APMV-1 strain, belonging to genotype Ⅶ of APMV-1 strain.

Cloning and Sequence Analysis of Interleukin 10 (IL-10) Full-length cDNA from Cyprinus carpio L.

[Objective] This study aimed to obtain IL-10 (interleukin 10) full-length cDNA of common carp (Cyprinus carpio L.) and conduct the sequence analysis. [Method] The differentially expressed cDNA fragment was obtained by DD-RTPCR (differential display RT-PCR). The cDNA library of peripheral blood leukocytes which were separated from common carp and stimulated by mitogen was screened with a probe labeled with DIG (digoxigenin). The IL-10 full-length cDNA was cloned from 0.8×104 pfu of recombinant phages, and the sequence analysis and homology comparison were carried out. [Result] Sequence analysis indicated that the IL-10 full-length cDNA of common carp was 1 117 bp long, containing a 55 bp 5’-UTR, a 522 bp 3’-UTR, and a 540 bp open reading frame(ORF) encoding 179 amino acids. In addition, there were three mRNA instability motifs (ATTTA) in the 3’-untranslated region. The deduced protein sequence shared typical sequence features of the IL-10 family. Homology comparison indicated that the obtained sequence shared 89.1% homology with the carp IL-10 gene from GenBank. [Conclusion] This study laid foundation for further study of the expression manner, functional characteristic and regulation mechanism of IL-10 in vivo and the interaction mechanism in the inflammatory 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 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,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 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.