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.

The structural analysis of protein sequences based on the quasi-amino acids code

Proteomics is the study of proteins and their interactions in a cell. With the successful completion of the Human Cenome Project, it comes the postgenome era when the proteomics technology is emerging. This paper studies protein molecule from the algebraic point of view. The algebraic system （∑, ＋, ＊） is introduced, where ∑ is the set of 64 codons. According to the characteristics of （∑, ＋, ＊）, a novel quasi-amino acids code classification method is introduced and the corresponding algebraic operation table over the set ZU of the 16 kinds of quasi-amino acids is established. The internal relation is revealed about quasi-amino acids. The results show that there exist some very close correlations between the properties of the quasi-amino acids and the codon. All these correlation relationships may play an important part in establishing the logic relationship between codons and the quasi-amino acids during the course of life origination. According to Ma F et al （2003 J. Anhui Agricultural University 30 439）, the corresponding relation and the excellent properties about amino acids code are very difficult to observe. The present paper shows that （ZU, ＋,×） is a field. Furthermore, the operational results display that the eodon tga has different property from other stop codons. In fact, in the mitochondrion from human and ox genomic codon, tga is just tryptophane, is not the stop codon like in other genetic code, it is the case of the Chen W C et al （2002 Acta Biophysiea Siniea 18（1） 87）. The present theory avoids some inexplicable events of the 20 kinds of amino acids code, in other words it solves the problem of ＇the 64 codon assignments of mRNA to amino acids is probably completely wrong＇ proposed by Yang （2006 Progress in Modern Biomedicine 6 3）.

A reduced computational load protein coding predictor using equivalent amino acid sequence of DNA string with period-3 based time and frequency domain analysis

Development of efficient gene prediction algorithms is one of the fundamental efforts in gene prediction study in the area of genomics. In genomic signal processing the basic step of the identification of protein coding regions in DNA sequences is based on the period-3 property exhibited by nucleotides in exons. Several approaches based on signal processing tools and numerical representations have been applied to solve this problem, trying to achieve more accurate predictions. This paper presents a new indicator sequence based on amino acid sequence, called as aminoacid indicator sequence, derived from DNA string that uses the existing signal processing based time-domain and frequency domain methods to predict these regions within the billions long DNA sequence of eukaryotic cells which reduces the computational load by one-third. It is known that each triplet of bases, called as codon, instructs the cell machinery to synthesize an amino acid. The codon sequence therefore uniquely identifies an amino acid sequence which defines a protein. Thus the protein coding region is attributed by the codons in amino acid sequence. This property is used for detection of period-3 regions using amino acid sequence. Physico-chemical properties of amino acids are used for numerical representation. Various accuracy measures such as exonic peaks, discriminating factor, sensitivity, specificity, miss rate, wrong rate and approximate correlation are used to demonstrate the efficacy of the proposed predictor. The proposed method is validated on various organisms using the standard data-set HMR195, Burset and Guigo and KEGG. The simulation result shows that the proposed method is an effective approach for protein coding prediction.

Metagenomic analysis revealing the metabolic role of microbial communities in the free amino acid biosynthesis of Monascus rice vinegar during fermentation

Free amino acid(FAA)is the important component of vinegar that infl uences quality perception and consumer acceptance.FAA is one of the major metabolites produced by microorganisms;however,the microbial metabolic network on FAA biosynthesis remains unclear.Through metagenomic analysis,this work aimed to elucidate the roles of microbes in FAA biosynthesis during Monascus rice vinegar fermentation.Taxonomic profiles from functional analyses showed 14 dominant genera with high contributions to the metabolism pathways.The metabolic network for FAA biosynthesis was then constructed,and the microbial distribution in different metabolic pathways was illuminated.The results revealed that 5 functional genera were closely involved in FAA biosynthesis.This study illuminated the metabolic roles of microorganisms in FAA biosynthesis and provided crucial insights into the functional attributes of microbiota in vinegar fermentation.

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.

Sequence Analysis of Polymorphic Fragments in the Third Intron of Porcine H-FABP Gene

[ Objective] The aim of this study was to provide a theoretical basis for exploring the major genes affecting intramuscular fat （IMF） deposition. [Method] Taking 383 pigs from five breeds including Mashen Pig, Large White Pig, Landrace, Duroc and Shanxi White Pig as the experimental animals, polymorphisms of partial fragments in the third intron of porcine H-FABP gene were detected by PCR-SSCP method, and then the polymorphic fragments were sequenced. [ Result] Two alleles, designated as A and B, were found at the locus 346 in the third intron of porcine H-FABP gene, and the mutation was caused by a A→G substitution. [ Conclusion] A polymorphic locus was discovered in the third intron of porcine H-FABP gene in this experiment, laying a foundation for the further study on the relationship between H-FABP gene and IMF content.

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.

Sequence Analysis of HA Genes from Three H9N2 Subtype Avian Influenza Viruses

[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% （ Dk/HK/Y439/97 ） -98.0% （ Ck/GX17/00 ）, 85.1% （Dk/HK/Y439/97） - 99.1% （ Ck/GXl 7/00）, 90.7% （ Ck/BJ/3/01 ） - 99.1% （Ck/GX17/00） with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L （ Leu）, suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin.

Determination and Analysis of Mitochondrial ND2 Gene Sequence of Anas platyrhynchos

[Objective] The study was to analyze the phylogenesis of Anas platyrhynchos. [Method] Complete sequence of mitochondrial ND2 gene of 4 Anas platyrhynchos was determined by direct DNA sequencing based on PCR products. Combined with ND2 gene sequences of the Anas Linnaeus accessed in GenBank, phylogenetic tree was constructed by Neighbor-joining and maximum parsimony methods. [Result] The ND2 gene sequences of 4 Anas platyrhynchos were identical(1 041 bp in length; the nucleotide contents of A, G, T, and C were 28.91%, 13.35%, 20.75% and 36.98% respectively; A+T content approximated to that of C+G). Sequences of ND2 gene of mallard were same as spotbill duck, and had high homology with others. The phylogenetic trees indicated mallard and spotbilled duck were close in genetic relationship, both shared a haplotype; then Philippine duck, green-winged teal and northern pintail fell into branch ''A". [Conclusion] The domestic duck may be domesticated from mallard and spotbilled duck.

Cloning and Sequence Analysis of 16S rRNA and COI Gene in Mitochondrial DNA of Scortum barcoo

[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing were used to study the 16S rRNA and COI gene fragments.[Result] As for 16S rRNA gene fragments,nucleotide sequences of 791 bp were obtained,and the A,T,G and C contents in this fragment were 31.6%,21.4%,20.4% and 26.7%respectively.As for the COI gene fragments,the size was 631 bp and the A,T,G And C contents were 27.7%,23.6%,29.8% and 18.9% respectively.Among these two gene fragments,the content of GC was lower than AT,and AT/GC of these two fragments was 1.13 and 1.05 respectively.[Conclusion] The genetic characteristics of gene fragments of 16S rRNA and COI of S.barcoo suggested that the variation in the same species was relatively low.The sequences of 16S rRNA gene in three samples the same,while the sequences of COI gene was also the same,indicating that these two gene of S.barcoo were conservative.

Isolation of Rice EPSP Synthase cDNA and Its Sequence Analysis and Copy Number Determination

In order to isolate the total cDNA of rice (Oryza sativa L.) epsps gene, RT-PCR was carried out with template of rice first-strand cDNA and primers designed according to rice EPSP synthase genomic sequence obtained in previous study. A 1 585-bp cDNA fragment was amplified and cloned. The 1 585-bp cDNA contains an open reading frame (ORF) comprising of 1 533 nucleotides (nt) which encodes a 511 residue polypepetides, including 67 amino acids chloroplast transit peptide and 444 amino acids EPSP synthase mature peptide. A comparison between the EPSP synthase of different sources indicates that the mature peptide shows more than 51% identity except for the fungi EPSP synthase and the transit peptide shows considerably less sequence conservation. The copy number of rice epsps gene is estimated to be one copy per haploid rice genome using southern blot. RT-PCR indicated that rice epsps gene is expressed in rice leaves, endosperms and roots and has the highest expression level in leaves.

Cloning and Sequence Analysis of Lactate Dehydrogenase C(LDH-C)Gene from Black-lipped Pika in Western Sichuan Plateau

[Objective] The aim was to lay a foundation for research of contraceptive rodenticide with LDH-C4 as target protein. [ Method] EST sequence of LDH-C gene from black-lipped pika was cloned by PCR with degenerate primers; then the full length open reading frame （ORF） and 3＇UTR sequence were cloned by RACE technique. [ Result] The full length cDNA was 1 498 bp containing an ORF of 996 bp and a 3＇UTR of 486 bp. The ORF encoded a polypeptide of 332 amino acids. The alignment of LDH-C gene ORF nucleotide sequences from different species showed that the gene was conserved even between large taxons. The phylogenic tree showed that black-lipped pika LDH-C was closer to prima- tes and artiodactyla than to rodents. [Conclusion] cDNA sequence of LDH-C gene from black-lipped pika was cloned successfully.

Sequence Analysis of ITS Region of rDNA of Alternaria Nees. from Some Areas of China

[Objective] The study aimed to identify Alternaria Nees from some areas of China at molecular level by analyzing the rDNA ITS sequence. [ Method ] The DNA sequences coding for the 5.8S rDNA and the flanking internal transcribed spacers （ ITS1 and ITS2） were amplified by PCR with universal primers ITS4 and ITS5 and subsequently sequenced for 34 Alternaria isolates from different areas of China. [Result] Sequences analysis showed that 5.8S rDNA was 159 bp and no variation in tested 34 isolates. There had variables sites in ITS. The isolates that had same sequences as A. tenuissima or A. alternata all put up eurytopicity to area and host. The variables sites of the isolates showed the diver- sity of Alternaria in the hosts of Oleaceae, Rosaceae and Solanaceae. At the same time that ITS could not clearly separated the isolates was indicated. The results indicated that the phylogenetic relationship were not closely related to the geographical origin and hosts of these isolates. [ Conclusion] The sequence analysis of ITS region could provide theory basis for the identification of Alternaria Nees..

Molecular Cloning, Sequence Analysis and Prokaryotic Expression of Ovine Activin Receptor Type IIB(ActRIIB) Gene

Objective] This study aimed to clone ovine activin receptor type llB （Ac-tRIIB） gene, construct the prokaryotic expression vector and express the target gene in vitro, thus providing basis for further function verification. [Method] The template cDNA which was reversely transcribed from total RNA of sheep liver tissue, was subjected to polymerase chain reaction （PCR） using specific primers of ActRIIB. The ful-length cDNA of ovine ActRIIB was obtained by pMD18-T cloning and sequencing for bioinformatics analysis. Ovine ActRIIB encoding sequence was subcloned into prokaryotic expression vector pET41a with restriction sites BamHl/Notl, and then transformed into BL21 （DE3）. The induced products by lPTG were analyzed with SDS-PAGE and Western Blot. [Result] The amplified ful-length cDNA of ovine Ac-tRllB gene was 1 564 bp in length （Genbank accession number： JX422071.1） with an open reading frame of 1 539 bp, encoding 512 amino acides. Ovine ActRllB shared the highest homology （99.6%） with bovine ActRllB. ActRllB had highly ho-mologous C-terminal domains and belonged to the TGFβ family. After prokaryotic expression, an approximately 92 kD His-tagged ActRllB recombinant protein was obtained, which was consistent with the excepted result. [Conclusion] Cloning and successful expression of ovine ActRIIB laid solid foundation for further investigation of its biological function.

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 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.

Sequence Analysis of Mitochondrial DNA D-loop Region in Xinjiang Goose

[Objective] The sequences of mitochondrial DNA D-loop region of Xinjiang Goose with three different colors of plumage were analyzed in order to study the genetic diversity of Xinjiang Goose, as well as the phylogeny and evolution. [Method] Ten geese were selected randomly from the core populations of grey-, mosaic- and white-plumaged Xinjiang Goose respectively with a total number of thirty as experi- mental materials, of which the blood samples were collected from the largest vein under the wing （brachial vein） for DNA extraction. Sequences of mitochondrial DNA D-loop regions were determined using DNA sequencing technology to analyze the polymorphism. In addition, the genetic distances among different populations were estimated through the comparison with the reference sequences. [Resull] The con- tents of A, G, C and T nucleotides in the D-loop region of Xinjiang Goose were 28.85%, 17.05%, 25.38% and 28.72%, respectively. The average haplotype diversity and nucleotide diversity of Xinjiang Goose were 0.583 and 0.056. Xinjiang Goose and Greylag Goose were clustered into the same group. [Conclusion] The results showed that Xinjiang Geese with three different colors of plumage all descend from Greylag Goose （Anser anser）.

Construction of Midgut Tissue-Specific cDNA Library of Bombyx mandarina M. and Isolation and Sequence Analysis of Serine Protease Gene Fragment

[Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed via cDNA Library Construction Kit (TaKaRa), then the serine protease gene was cloned via sequencing of the yielded cDNA library. [Result] The titer of cDNA library reached 6.2×105 pfu/ml, average insert size was about 1.2 kb. The serine protease gene cDNA fragment was obtained from colony sequencing (Accession No: EU672968). The nucleotide sequence of the cloned 854 bp fragment encodes 284 amino acid residues. Homology analyses showed some homology between putative amino acid sequence of the cloned fragment and amino acid sequences of serine proteases from other ten insects. [Conclusion] The results may avail to reveal the resistance of silkworm and wild silkworm to exotic intrusion.

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 Sheeppox Virus RPO30 Gene

[Objective] The study aimed to clone RPO30 gene from Sheeppox virus （SPPV） and predict the structure and function of the sequence. [Method] RPO30 gene of SPPV was cloned with PCR, linked into pMD18-T simple vector and then transformed into E. coli DH5a. In blue-white screen, the white colonies were selected to prepare plasmids. The positive plasmids were selected by double digestion and PCR, and then sequenced. Finally, the structure and function of the sequence obtained were predicted by bioinformatics methods. [Results] The RPO30 gene was successfully obtained; its ORF was 585 bp, encoding 193 amino acids and containing a recognition site for Hind III. Moreover, the SPPV RPO30 gene shared different homologies with the RPO30 gene sequences of other pox virus strains from GenBank database. Further analysis by biological software showed that in RPO30 protein, amino acids 4-12, 18-26, 50- 61, 68- 92 and 176-190 had a high possibility to form the active center, and acting to these regions was likely to inactivate the enzyme encoded by the sequence, thus to inhibit viral replication efficiently. [Conclusion] This study will lay foundation for further study on the structure and function of RPO30.