Molecular Cloning and cDNA Sequence Analysis of Two New Lepidopteran OR83b Orthologue Chemoreceptors

The aim of this study was to isolate the full-length cDNA sequences of an unusually highly conserved olfactory receptor （orthologue to the Drosophila melanogaster DOR83b） from the antennae of Spodoptera exigua and S. litura by using reverse transcription-polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends （RACE） methods. Bioinformatics methods were used to further analyze the cDNA sequences and putative amino acid sequences. The two full-length cDNA sequences from olfactory receptor （OR） of male S. exigua and S. litura were named as SexiOR2 and SlitOR2, respectively. SexiOR2 and SlitOR2 consisted of nucleotide sequence of 1 906 and 2 483 bp, respectively, and both with deduced amino acid sequences of 473 residues. The sequence analysis indicated that the deduced amino acid sequences of the cDNA shared the high homologies with OR83b orthologue chemoreceptor sequences from previously reported moths, implying that the cDNA sequences were of OR83b orthologue chemoreceptor genes.

cDNA Cloning and Sequence Analysis of Rice Sbe1 and Sbe3 Genes

Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes Sbe1 and Sbc3 encoding SBE Ⅰ and SBE Ⅲ (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA library derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned Sbe1 and Sbe3 cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of Sbe3 was the same as that of sbc3 (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference, which resulted in changes of two deduced amino acids between the cloned Sbel cDNA and the reported sbe1 (Genbank Accession No. D11082). The cloned Sbe1 and Sbe3 cDNAs make it possible to improve rice starch quality through genetic engineering

Molecular Cloning and Sequence Analysis of the Toc33 cDNA in Brassica napus Line Cr3529

Two primers, designed according to the Arabidoposis thaliana Toc33 sequence, were used to amplify the full coding region of the Toc33 cDNAs in leaves of a chlorophyll-reduced (Cr) mutant Cr3529 and its wild type 3529, Brassica napus, by RT-PCR technique. The RT-PCR results showed that the fragment homologous to Toc33 was expressed in Cr3529 as well as 3529 seedlings. PCR fragments were inserted into the pMD18-T vector and transferred into E. coli, then two cDNA clones, BnToc33-c and BnToc33, were obtained. Sequence analysis showed that the two sequences were 894 bp and the nucleotide and the deduced amino acid sequences were highly homologous to those of A. thaliana. There were three diverged nucleotides between the Cr3529 and the 3529 Toc33 cDNAs, i.e., GGT/AGT, TTG/TTT, AGG/AGT, all of which belonged to missense mutation. The amino acid replacement ((Leu/Phe) caused by TTG/TTT mutation located in the membrane anchor domain may result in chlorophyll-reduced character in Cr3529.

Cloning and Sequence Analysis of cDNA Encoding MRJP3 of Apis cerana cerana

By screening the worker （Apis cerana cerana） heads cDNA library using a fragment of the mrjp3 gene of Apis cerana as probe, 120 positive clones were obtained. The clone containing A. cerana cerana MRJP3 （AccMRJP3） cDNA was selected. Based on the sequencing of the inserts of the positive clone, a sequence of AccMRJP3 cDNA which is 1 887 bp long including a poly （A） tail was obtained. The AccMRJP3 cDNA encompassed an open-reading frame （ORF） with 1 779 bp encoding 593 amino acids. The un-translated regions （UTR） of the 5′ end and 3′end are 46 bp and 160 bp in length, respectively. Similar to AmMRJP3 and AdMRJP3, the putative AccMRJP3 also has a repetitive region. The comparison of the repetitive region of AccMRJP3, AmMRJP3 and AdMRJP3 shows some differences between them.

Molecular Cloning and Sequence Analysis of Chitin Synthase cDNA from Mamestra brassicae (L.) (Lepidoptera: Noctuidae) Cuticle

Chitin is the most widespread amino polysaccharide in nature. Chitin synthase （CHS） plays an important role in chitin formation in the cuticle and the peritrophic membrane （PM） lining the midgut. Total RNA was isolated from the cuticle of Mamestra brassicae （L.） fourth instar larva, cDNA sequence was cloned by RT-PCR and Rapid Amplification of cDNA Ends （RACE）. cDNA 5 220 bp in length, contained an open reading frame of 4 704 bp coding for a polypeptide of 1 567 amino acid residues with a predicted molecular weight of 178.3 ku and its pI was 6.42. The deduced amino acid sequence from Mi brassicae （L.） shared the high level of identity with chitin synthase sequences from other insects, especially lepidopteran insects, cDNA sequence has been deposited with GenBank under accession No. GQ281761

Cloning and Expression Analysis of Downy Mildew Resistance-Related cDNA Sequences in Melon

Melon downy mildew caused by Pseudoperonospora cubensis leads to significant losses in melon yields worldwide. Reverse-transcription Polymerase Chain Reaction （RT-PCR） was performed using cDNAs as templates from melon-Huangdanzi induced with fungus Pseudoperonospora cubensis, and degenerate primers designed based on the conserved amino acid sequences of known plant disease-resistance genes. A polymorphic cDNA fragment which we named rap-19 was cloned and sequenced. The Open Reading Frame （ORF） of this product comprised of 510 base pairs which encodes DNA or RNA-binding protein with 170 amino acids. The putative amino acid sequence of mp-19 appeared highly homologous with those of NBS-type resistant-genes isolated from other plants. Southern blot indicated that the melon genome contained more than 3 copies of rap-19. The obvious expression differences detected by semi-quantitative RT- PCR could be observed between resistant-line Huangdanzi and susceptible-line Jiashi after Pseudoperonospora cubensis infection, which implied that mp-19 gene may be related to the resistance of downy mildew in melon.

Generation and Analysis of Expressed Sequence Tags(ESTs) from Muscle Full-Length cDNA Library of Wujin Pig

Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identified in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1 076 bp, and the cDNA fullness ratio was 86.2%. A total of 1 058 unique sequences with 342 contigs （32.3%） and 716 singleton （67.7%） expressed sequence tags （EST） were obtained by clustering and assembling. Meanwhile, 826 （78.1%） ESTs were categorized as known genes, and 232 （21.9%） ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index of Sus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank （accession numbers： EU650784-EU650788, GE843306, GH228978-GH229100）. The abundantly expressed genes in porcine muscle tissue were related to muscle fiber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.

白菜种子cDNA酵母文库的构建及BrTTG1互作蛋白的筛选及分析

【目的】通过构建白菜种子的cDNA文库,筛选WDR 40蛋白TRANSPARENT TESTA GLABRA 1(TTG1)的互作蛋白,探究TTG1参与MBW三元复合体调控种皮原花青素形成的分子机制。【方法】以棕籽白菜自交系‘B147’的种子为材料,提取总RNA并建立cDNA文库,通过gateway技术构建诱饵载体pGBKT7-TTG1并进行酵母双杂交筛库。【结果】酵母文库库容为1.2×10^(7)CFU,文库滴度是5.0×10^(7)CFU/mL,插入片段平均长度大于1000 bp,诱饵载体在酵母中无自激活活性。通过构建的诱饵载体pGBKT7-TTG1与构建的cDNA文库杂交,共获得了38个阳性互作蛋白,功能预测显示其中一个蛋白注释为MYB转录因子,注释为MYB73,序列分析结果显示该基因含有R2R3-MYB型抑制子保守基序C1和C2,推测该基因为白菜中参与种皮颜色形成的R2R3-MYB型抑制子,暗示着白菜中可能存在不同MYB转录因子参与的调控网络,影响着原花青素的形成。【结论】本研究构建了白菜种子组织的酵母双杂交cDNA文库,获得了38个TTG1阳性互作蛋白,首次挖掘到了可能影响白菜种皮颜色原花青素形成的R2R3-MYB型抑制子MYB73,为后期探究白菜种皮原花青素的调控网络奠定良好的基础。

Cloning and Sequence Analysis of Expansin Genes from Litchi chinensis Fruit

Using PCR degenerate primers, designed with reference to the sequences of the conserved amino acids of known expansins, to amplify cDNA fragments in litchi fruit by RT-PCR, two different cDNA fragments , named as Lc-Exp1 and Lc-Exp2 , were cloned. Lc-Exp1 and Lc-Exp2 was respectively composed of 531 bp encoding 177 amino acids and 537 bp encoding 179 amino acids. Eight cysteine residues and three tryp-tophan residues, which is supposed to be the characteristics of expansins, are conserved in both Lc-Exp1 and c-Exp2. In addition, the homology between the two expansins is 71. 6% at nucleotide acid sequences and 76.3% at amino acid sequences. The homology of Lc-Exp1 with Fa-Exp2 or Pp-Exp1 was 92.7% or 92.1%, but that of Lc-Exp2 with Fa-Exp2 or Pp-Exp1 was only 77. 4% or 76.3% at amino acid sequences.

Construction of cDNA Library from Intestine, Mesentery and Coelomocyte of Apostichopus japonicus Selenka Infected with Vibrio sp. and a Preliminary Analysis of Immunity-Related Genes

The aquaculture of sea cucumber Apostichopus japonicus (Echinodermata, Holothuroidea) has grown rapidly during recent years and has become an important sector of the marine industry in Northern China. However, with the rapid growth of the industry and the use of non-standard culture techniques, epidemic diseases of A. japonicus now pose increasing problems to the industry. To screen the genes with stress response to bacterial infection in sea cucumber at a genome wide level, we constructed a cDNA library from A. japonicus Selenka (Aspidochirotida: Stichopodidae) after infecting them with Vibrio sp. for 48 h. Total RNA was extracted from the intestine, mesentery and coelomocyte of infected sea cucumber using Trizol and mRNA was isolated by Oligotex mRNA Kits. The ligated cDNAs were transformed into DH5α, and a library of 3.24×105 clones (3.24×105 cfu mL-1) was obtained with the sizes of inserted fragments ranging from 0.8 to 2.5 kb. Sequencing the cDNA clones resulted in a total of 1106 ESTs that passed the quality control. BlastX and BlastN searches have identified 168 (31.5%) ESTs sharing significant homology with known sequences in NCBI protein or nucleotide databases. Among a panel of 25 putative immunity-related genes, serum lectin isoform, complement component 3, complement component 3-like genes were further studied by real-time PCR and they all increased more than 5 fold in response to Vibrio sp. challenge. Our library provides a valuable molecular tool for future study of invertebrate immunity against bacterial infection and our gene expression data indicates the importance of the immune system in the evolution and development of sea cucumber.

Cloning and Sequence Analysis of Prophenoloxidase from Haemocytes of the Red Swamp Crayfish,Procambarus clarkii

The full-length cDNA sequence of prophenoloxidase was obtained through RACE technology. The complete cDNA sequence is 3 721-bp long, containing an open reading frame （ORF） of 1 881 bp, a 154-bp 5′-untranslated region, and a 1 686- bp 3′-untranslated region with three potential functional poly（A） signals （AATAAA）. The molecular mass of the deduced amino acid sequence （627 aa） was 72.3 kDa with an estimatedpI of 5.88. It contained putative copper-binding sites （copper A： 131, 135, 167 and copper B： 301,305, 341）, and a tentative complement-like motif （GCGWPDHL）. Eight potential N-linked glycosylation sites were predicted to be present in P. clarkii prophenoloxidase. Similar to those in other arthropod prophenoloxidases reported so far, no signal peptide was detected in the crayfish prophenoloxidase. The phylogenetic trees confirmed that P. clarkii prophenoloxidase was most closely related to that of freshwater crayfish P. leniusculus and more closely related to other crustacean prophenoloxidases from shrimp, prawn, and lobster than to the insect prophenoloxidases. Besides, two putative introns were found in this sequence of genomic DNA.

A new Multilocus Sequence Analysis Scheme for Mycobacterium tuberculosis

Objective Tuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis （M. tuberculosis） multilocus sequence analysis （MLSA） was established for the phylogenetic and epidemiology analysis. Methods To establish the scheme of M. tuberculosis MLSA, the genome of H37Rv, CCDC5079 and CCDC5180 were compared, and some variable genes were chosen to be the MLSA typing scheme. 44 M. tuberculosis clinical isolates were typed by MLSA, IS6110-RFLP, and soligotyping, to evaluate the MLSA methods. Results After comparison of the genome, seven high discrimination gene loci （recX, rpsL, rmlC, rpmG1, mprA, gcvH, ideR） were chosen to be the MLSA typing scheme finally. 11 variable SNP sites of those seven genes were found among the 44 M. tuberculosis isolate strains and 11 sequence types （STs） were identified. Based on the Hunter-Gaston Index （HGI）, MLSA typing was not as good for discrimination at the strain level as IS6110-RFLP, but the HGI was much better than that of spoligotyping. In addition, the MEGA analysis result of MLSA data was similar to spoligotyping/PGG lineage, showing a strong phylogenetic signal in the modern strains of M. tuberculosis. The MLSA data analysis by eBURST revealed that 4 sequence types （ST） came into a main cluster, showing the major clonal complexes in those 44 strains. Conclusion MLSA genotyping not only can be used for molecular typing, but also is an ideal method for the phylogenetic analysis for M. tuberculosis.

The EST Analysis of A Suppressive Subtraction cDNA Library of Chinese Wild Vitis pseudoreticulata Inoculated with Uncinula necator

Uncinula necator is a worldwide serious fungus disease causing annual heavy lost on grapevine production. In order to get more informations on defense related EST sequences and help breeding program, we constructed and characterized a suppression subtractive hybridization cDNA library with artificially inoculated leaves and control Chinese wild Vitis pseudoreticulata clone Baihe-35-1, which is highly resistant to powdery mildew. In the library, the length of 58 EST fragments known as putative functions varied from 130 to 800 bp, and 60% of the ESTs exhibited high similarity to known sequences in database of GenBank with BLASTX analysis. These genes were involved in stress/defense response, detoxification, signal transduction, disease defense, and etc., and 14 ESTs remained unknown or hypothetical proteins, which may be new genes. The experiment provided an important basis for studying the disease-resistance mechanism and obtaining the genes for the aim of improving grapevine powdery mildew resistance.

Isolation and Sequence Analysis of HMW Glutenin Subunit 1Dy10.1 Ecoding Gene from Xinjiang Wheat (Triticum petropavlovskyi Udacz. et Migusch)

A novel HMW glutenin subunit gene 1Dy10. 1 was isolated and characterized from Xinjiang wheat （Triticum petropavlovskyi. Udacz. et Migusch） accession Daomai 2. The complete open reading frame （ORF） of 1Dy10. 1 was 1 965 bp, encoding 655 amino acids. The numbers and distribution of cysteines in 1Dy 10.1 were similar to those of 1Dy10 and other y-type subunits. In the N-terminal of 1Dy 10.1, an amino acid was changed from L （leucine） to P （proline） at position 55. The repetitive domain of 1Dy10.1 differed from those of known HMW subunits by substitutions, insertions or/and deletions involving single or more amino acid residues. In the repetitive domain of subunit 1Dyl 0.1, the deletion of tripeptide GQQ in the consensus unit PGQGQQ resulted in the appearance of the motif PGQ that have not been observed in other known y-type HMW subunits. In comparison with the subunit 1Dy 12, a deletion of dipeptide GQ, which occurred in subunit IDy10, was also observed in subunit 1Dy10.1. The cloned IDylO. 1 gene had been successfully expressed in Escherichia coli, and the expressed protein had the identical mobility with the endogenous subunit IDy10.1 from seed.

Vitis amuerensis CBF3 Gene Isolation,Sequence Analysis and Expression

The full length cDNA sequence of CBF3 （CRT/DRE-binding factor） was cloned from Vitis amurensis by reverse transcription polymerase chain reaction （RT-PCR） using the primers designed based on CBF genes available in GenBank. Sequence analysis showed that the gene had 854 bp long and its coding sequence contained 720 bp, encoding a protein with 239 amino acids and an AP2 structural domain. The molecular mass of CBF3 was predicted to be 25.9 kDa and its theoretical isoelectric point was 7.02 and aliphatic index was 59.29. The average hydropathicity of the protein was -0.551. The tertiary structures of CBF3 were also analyzed. The prokaryotic expression vector pGEX-4T-CBF3 containing CBF3 gene was constructed and CBF3 fusion protein （52 kDa） was produced in Escherichia coli after induction with 1 mmol L-1 IPTG. Further studies are needed to evaluate its potential application for improving plant resistance to cold and other stress condition such as drought and salinity.

Cloning and Sequence Analysis of a Novel Cold-Adapted Lipase Gene from Strain lip35 (Pseudomonas sp.)

A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 （Pseudomonas sp.） is described, whereby a lipase gene （lip） was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.

Cloning and sequence analysis of the partial sequence of the rbcL from Bryopsis hypnoides

The partial sequence of the rbcL from Bryopsis hypnoides, including the sequences of the upstream, extron and partial intron, was amplified by PCR and their sequences were determined. With Spinacia oleracea as the outgroup, neighbor-joining method and maximum parsimony method were used respectively to build phylogenetic trees according to the rbcL exon sequence among 13 species that were the typical species of six phyla. Two kinds of trees showed clearly that there were two groups among those species, the green lineage and the non-green lineage. And the relationships of algae in the green lineage were similar in the two trees but those in the non-green lineage were not consistent. Analysis of codon preference indicated that the codon preference of the rbcL exon of Bryopsis hypnoides distinctly differed from that of the relevant sequence of photosynthetic bacteria.

Characteristics of nucleotides and amino acids in cDNA sequence of xyloglucan endotransglycosylase cloned from Anthocephalus chinensis

Xyloglucan endotransglycosylase is an identified relaxation factor with functions of easing and extending of plant cell walls.Its activities are directly related to plant growth and elongation of organisms.Anthocephalus chinensis is a tall and fast growing evergreen tree species.We cloned the full cDNA sequence of AcEXT genes which is abundantly expressed in the cambium of A.chinensis.The sequence analysis of nucleotides and amino acids revealed the presence of a 1396 bp full cDNA sequence,including a 960 bp complete open reading frame（ORF） encoding a 320 amino acid protein.The deduced amino acid sequence of AcXET was homologous to the other known XET proteins and contained the conserved EIDFE catalytic site which was specific to all the XETs.Our data should serve as a foundation for further insight into AcXET gene molecular mechanisms during wood formation and cell wall engineering of woody plants.

IDENTIFICATION AND SEQUENCE OF A cDNA CLONE CORRESPONDING TO A GENE INVOLVED IN DEVELOPMENT OF UNDARIA PINNATIFIDA

During the induction of gamete-producing gametangia, induced gametophytes werecollected at 4 days intervals (0,4,8, 12 d) and total RNAs were isolated by CsCl gradient ultracentrifu-gation. Some stage-specific expressed mRNAs were identified by differential display of mRNAs from dif-ferent developing stages of the gametophytes. The cDNA of one specific mRNA was verified, cloned andsequenced. This gene was specifically expressed during 4 days of induction, and had partial homologoussequence with tobacco IAA-binding protein gene. It suggests that this cDNA may represent a gene whichis related to the LAA regulating function during the development of the gametophytes.

Aequorea taiwanensis n. sp. (Hydrozoa,Leptomedusae) and mtCOI sequence analysis for the genus Aequorea

Nine species of Prionospio complex are recorded from China＇s waters,including one new species and six newly recorded species.Prionospio（Prionospio） pacifica sp.nov.,is characterized by having first and forth pairs of branchiae pinnate,second and third pairs of apinnate,ventral crest on Setiger 9 and dorsal crests on Setigers 10-25.Apoprionospio kirrae（Wilson,1990）,Prionospio（Aquilaspio） convexa Imajima,1990,Prionospio（Minuspio） multibranchiata Berkeley,1927,Prionospio（Prionospio） bocki Sderstrm,1920,Prionospio（Prionospio） dubia Maciolek,1985 and Prionospio（Prionospio） paradisea Imajima,1990 are recorded for the first time from China＇s waters.