Full-length transcriptome sequence and SSR marker development for genetic diversity research in yellowfin seabream Acanthopagrus latus

Yellowfin seabream Acanthopagrus latus is an important economic fish in Chinese coastal areas.Given its narrow distribution and overfishing,the genetic diversity of yellowfin seabream has been restricted for artificial breeding and reproduction.We performed full-length transcriptome sequencing and assembly of the genome of yellowfin seabream.A total of 68086 unigenes were obtained,with an N50 of 3391 bp on average length of 2933 bp.A total number of 50593 expressed sequence tags linked to simple sequence repeats(EST-SSR)were identified,among them dinucleotide repeats(40.6%)and AC/GT motifs(38.5%)were the most frequent.Of the 190 EST-SSRs for which PCR primer pairs were designed,150 primer pairs successfully amplified target loci and 15 SSRs showed high polymorphism.The alleles per locus ranged 6-50 on average of 25.3.The expected and observed heterozygosity varied from 0.632 to 0.969 and from 0.519 to 0.953,respectively.The polymorphic index content(PIC)values of each locus ranged 0.587-0.966 on average of 0.851.Among six yellowfin seabream population samples preliminarily tested for genetic diversity and differentiation,the Fangchenggang(FCG)population in Guangxi Province had the highest mean observed heterozygosity(H_(o))value(0.786),whereas the Zhangzhou(ZZ)population in Fujian Province had the lowest(0.678).The pairwise fixation index(Fst)values indicated significant population differentiation among six yellowfin seabream populations.This study provided evidence for the usefulness of the transcriptomic resource information and EST-SSR markers for natural resource conservation,population genetics,and breeding studies of yellowfin seabream in South China.

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.

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.

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.

Molecular Cloning and Sequence Analyses of cDNAs Encoding Seven C-type Lectin-like Protein Subunits from Daboia russellii siamensis

Total mRNA was extracted from a venom gland of snake Daboia russellii siamensis following the manufacturer＇s protocol of the PolyATtract System 1000 kit purchased from Promega Biotech. cDNAs encoding C-type lectins were amplified by RT-PCR and subcloned into a pMD18-T vector. Fourteen positive clones were selected for nucleotide sequencing and seven cDNAs encoding various snake venom C-type lectin-like protein precursors, designated as DRS-L1, DRS-L2, DRS-L3, DRS-L4 DRS-L5, DRS-L6 and DRS-L7, were obtained. Amino acid sequences of these proteins were deduced and each contains a carbohydrate recognition domain. Of all the deduced protein sequences, only DRS-L1 seemed to represent a closer sequence similarity to α subunits of other known snake venom C-type lectin-like proteins using the BLAST program. Homology comparison combined with analysis of cysteine position indicate that DRS-L1 and DRS-L2 are probably the light chain LC2 and LC1 of factor X activator from Daboia russellii siamensis venom, respectively. DRS-L3 and DRS-L4 might be the β subunits of higher molecular weight C-type lectin-like proteins while DRS-L5 and DRS-L6 might be β subunits of lower molecular weight C-type lectin-like proteins. DRS-L7 might be the β subunit of a platelet membrane glycoprotein Ib-binding protein similar to echicetin.

Cloning and Sequencing of a Full-Length cDNA Encoding the RuBPCase Small Subunit (RbcS) in Tea (Camellia sinensis)

This study was aimed to isolate ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit （RbcS） from tea plant [Camellia sinensis （L.） O. Kuntze]. In the study of transcriptional profiling of gene expression from tea flower bud development stage by cDNA-AFLP （cDNA amplified fragment length polymorphism）, we have isolated some transcript-derived fragments （TDFs） occurring in both the young and mature flower bud. One of them showed a high degree of similarity to RbcS. Based on the fragment, the full length of RbcS with 769-bp （EF011075） cDNA was obtained via rapid amplification of cDNA ends （RACE）. It contained an open reading frame of 176 amino acids consisting of a chloroplast transit peptide with 52 amino acids and a mature protein of 124 amino acids. The amino acids sequence presented a high identity to those of other plant RbcS genes. It also contains three conserved domains and a protein kinase C phosphorylation site, one tyrosine kinase phosphorylation site and two N-myristoylation sites. Analysis by RT-PCR showed that the expression of RbcS in tea from high to low was leaf, young stem, young flower bud and mature flower bud, respectively. The isolation of the tea Rubisco small subunit gene establishes a good foundation for further study on the photosynthesis of tea plant.

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.

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

Generation and characterization of expressed sequence tags(ESTs) from coralloid root cDNA library of Cycas debaoensis

A normalized full-length cDNA library was constructed from the coralloid roots of Cycas debaoensis by the DSN （duplex-specific nuclease） normalization method combined with the SMART （Switching Mechanism At 5＇ end of the RNA Transcript） technique. The titer of the original cDNA library was about 1.5 × 10^6 cfu·mL^-1 and the average insertion size was about 1 kb with a high recombination rate （97%）. The 5011 high-quality expressed sequence tags （ESTs） were obtained from 5393 randomly picked cDNA clones. Clustering and assembly of ESTs resulted in 2984 unique sequences, consisting of 618 contigs and 2366 singlets. EST sequence annotation revealed that 2333 and 1901 unigenes were functionally anno- tated in the NCBI non-redundant database and Swiss-Prot protein database, respectively. Functional analysis demonstrated that 1495 （50.1%） unigenes were associated with 4082 Gene Ontology （GO） terms. A total of 847 unigenes were grouped into 22 Cluster of Orthologous Groups （COG） functional categories. Based on the EST dataset, 22 ESTs that encoded putative receptor-like protein kinase （RLK） genes were screened. Furthermore, a total of 94 simple sequence repeats （SSRs） were discovered, of which 20 loci were successfully amplified in C debaoensis. This study is the first EST analysis for the coralloid roots of C debaoensis and provides a valuable genomic resource for novel gene discovery, gene expression and comparative genomics, conservation and management studies as well as applications in C debaoensis and related cycad species.

Cloning and Sequence Analysis of the Full-Length Genome of Japanese encephalitis virus Strain SXBJ07 Isolated from Swine

A virus strain, showing cytopathic effect in BHK-21 cell, was isolated from swine brains in Shaanxi Province, China, in 2007. The isolate was confirmed as Japanese encephalitis virus （JEV） by immunofluorescence assay （IFA） and reverse transcription polymerase chain reaction （RT-PCR）, and named SXBJ07. The complete nueleotide and deduced amino acid sequences of the JEV strain SXBJ07 were determined. Its single open reading frame has a total of 3 432 amino acid residues. An extensive E gene based phylogenetic analysis was performed, the result showed that SXBJ07 strain belongs to genotype I. Comparison of the SXBJ07 genomic sequence with those of the 24 fully sequenced JEV strains in published databases showed nucleotide homology ranging from 99.0 to 83.7%; amino acid homology ranged from 99.8 to 94.8%. Compared SXBJ07 with SA14-14-2 strain, the current live vaccine strain in China, the homology of amino acid in envelope gene was 97.0%; and there were amino acid substitutions in 13 sites of the active domains of E protein （E1-E411）.

Construction and Quality Analysis of Full-length cDNA Library of Phyllostachys heterocycla Germinating Seeds

[Objective] This study aimed to construct the full-length cDNA library for ger- minating seeds of Phyllostachys heterocycla [Method] Germinating seeds of P. hetero- cycla were used as experimental materials to construct the full-length cDNA library by using Oligo-capping method. [Result] The constructed library has a total capacity of 6.5×10^6 recombinant clones, and a low proportion of clones without inserted frag- ments; the size of inserted fragments ranges between 0.3-5.0 kb, with strict classifi- cation and ideal consistency. Furthermore, the proportion of clones harboring long in- serted fragments （1.0-5.0 kb） is as high as 30%, achieving the standard for high- quality full-length cDNA library. [Conclusion] The full-length cDNA library of germinat- ing seeds of P. heterocycla was successfully constructed, which laid important foun- dation for the functional genomics research of bamboo plants.

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.

Construction of a Normalized Full-Length cDNA Library of Sesame Developing Seed by DSN and SMART^(TM)

Sesame （Sesamue indicum L.） is one of the most important oilseed crops with high oil yield. Here, we described a simple and efficient method for constructing a normalized cDNA library from a high oil content cultivar of sesame Zhongzhi 14, during its oil accumulation stages. It combined switching mechanism at 5？end of RNA transcript （SMART） technique and duplex-specific nuclease （DSN） normalization methods. Double-stranded cDNAs were synthesized from mRNAs, processed by normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into Escherichia coli DH10B by electroporation. The capacity of the library was 1.0？06 clones in this library. Gel electrophoresis results indicated the fragments ranged from 700 to 2 000 bp, with the average size of 1 800 bp. Random picking clones showed that the recombination rate was 100%. The results showed that the cDNA library constructed successfully was a full-length library with high quality, and could be used to screen the genes related to development of oil synthesis.

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.

Complementary DNA sequencing (cDNA): an eff ective approach for assessing the diversity and distribution of marine benthic ciliates along hydrographic gradients

The Yellow Sea Cold Water Mass(YSCWM)is a distinct hydrographic phenomenon of the Yellow Sea,and the distribution pattern of meio-and macrobenthos diff ers inside and outside of the YSCWM.However,such a pattern has never been observed in the microbenthic ciliate communities.Therefore,we hypothesized that benthic ciliates followed a similar distribution pattern as meio-and macrobenthos,but this pattern has not been uncovered by morphological methods.We evaluated the diversity and distribution of benthic ciliates at fi ve stations along hydrographic gradients across the YSCWM and adjacent shallow water by using morphology and DNA and complementary DNA(cDNA)high-throughput sequencing of the V4 region of 18S rRNA gene.Results showed that the diversity of benthic ciliates detected by DNA(303 OTUs),and the cDNA(611 OTUs)sequencing was much higher than that detected by the morphological method(79 species).Morphological method detected roughly diff erent ciliate communities inside and outside of the YSCWM,but without statistical signifi cance.No clear pattern was obtained by DNA sequencing.In contrast,cDNA sequencing revealed a distinct distribution pattern of benthic ciliate communities like meioand macrobenthos,which coincided well with the results of the environmental parameter analysis.More than half of the total sequences detected by DNA sequencing belonged to planktonic ciliates,most(if not all)of which were recovered from historic DNA originating through the sedimentation of pelagic forms because none of them were observed morphologically.The irrelevant historic DNA greatly infl uenced the recovery of rare species and thus limited the understanding of the benthic ciliate diversity and distribution.Our research indicates that the methods used have signifi cant eff ects on the investigation of benthic ciliate communities and highlights that cDNA sequencing has great advantages in estimating the diversity and distribution of benthic ciliates,as well as the potential for benthic environmental assessments.

Construction of a Full-Length cDNA Library of Solen grandis Dunker and Identification of Defense- and Immune-Related Genes

The basic genetic characteristics, important functional genes, and entire transcriptome of Solen grandis Dunker were investigated by constructing a full-length cDNA library with the ‘switching mechanism at the 5'-end of the RNA transcript'(SMART) technique. Total RNA was isolated from the immune-relevant tissues, gills and hemocytes, using the Trizol reagent, and cDNA fragments were digested with Sfi I before being ligated to the pBluescript II SK* vector. The cDNA library had a titer of 1048 cfu μL-1 and a storage capacity of 1.05×106 cfu. Approximately 98% of the clones in the library were recombinants, and the fragment lengths of insert cDNA ranged from 0.8 kb to 3.0 kb. A total of 2038 expressed sequence tags were successfully sequenced and clustered into 965 unigenes. BLASTN analysis showed that 240 sequences were highly similar to the known genes(E-value < 1e-5; percent identity >80%), accounting for 25% of the total unigenes. According to the Gene Ontology, these unigenes were related to several biological processes, including cell structure, signal transport, protein synthesis, transcription, energy metabolism, and immunity. Fifteen of the identified sequences were related to defense and immunity. The full-length cDNA sequence of HSC70 was obtained. The cDNA library of S. grandis provided a useful resource for future researches of functional genomics related to stress tolerance, immunity, and other physiological activities.

Analysis of Expressed Sequence Tags from Liver Tissue in Swine

In order to study the expression of function gene and its effect on metabolic control and other physiological function in liver, 438 expressed sequence tags (ESTs) were determined, which were from a cD-NA library of porcine liver tissue. The results showed that the nucleotide sequences of 186 ESTs have already presented in GenBank database, and 37 ESTs could be found the homology with human and other species, while the others were not identified. 45 full length insertion of the clones randomly isolated from cDNA library were also completely sequenced with different size, and the results showed that 19 of them were function-known genes, 11 had no open reading frame ( ORF )at all and 15 had ORF but the function were not elucidated yet.

无蹼壁虎EMC3基因cDNA全长克隆与进化分析

为了探究无蹼壁虎(Gekko swinhonis)EMC3基因(命名为GsEMC3)的序列特征,了解GsEMC3蛋白的结构和功能,探讨GsEMC3蛋白与其他物种EMC3蛋白的进化关系,采用SMART RACE技术克隆获得了GsEMC3的全长cDNA序列,通过信息学分析阐明了GsEMC3基因的序列特征以及GsEMC3蛋白的结构和功能,系统发育和选择压力分析研究了GsEMC3的进化特征.GsEMC3基因的cDNA全长为1023 bp,包含1个786 bp的开放阅读框,基因编码1个含261个氨基酸的多肽.GsEMC3蛋白的相对分子质量约为30.074×10^(3),理论等电点为5.57.生物信息学分析表明,无蹼壁虎GsEMC3为疏水性非分泌型蛋白,有2个跨膜区,可能是一个动态定位的蛋白,无信号肽,直接在细胞内发挥作用.其分子量与已知的其他物种的EMC3蛋白相近.GsEMC3蛋白含有1个DUF106结构域,二级结构包含12个α-螺旋、1个β-折叠、13个无规则卷曲.系统发育分析表明,无蹼壁虎GsEMC3蛋白序列与美国短吻鳄(Alligator mississippiensis)、安乐蜥(Anolis carolinensis)、绿海龟(Chelonia mydas)、科莫多巨蜥(Varanus komodoensis)等爬行动物的EMC3蛋白序列高度同源.选择压力分析表明,无蹼壁虎的GsEMC3蛋白受到纯化选择,其功能可能高度保守.

Cloning and Sequence Analysis of Ribosomal Protein L21 Gene from the Ailuropoda melanoleuca

Ribosonal protein L21 which is a component of the 60s large ribosomal subunit plays an important role in ribosome. To explore the structure characteristic of ribosomal protein L21(rpL21) gene of the Ailuropoda melanoleuca and investigate its homologies with other already reported sequenses' including Rattus norvegicus, Mus musculus, Mus musculus, etc. The cDNA of rpL21 was cloned from the Ailuropoda melanoleuca by RT-PCR. The sequence data were analyzed by Genscan software. Blast 2.1 was used to study the homology of the obtained rpL21 sequence with the gene sequence of other species; Open reading frame (ORF) of the DNA sequence was searched using ORF finder software; Protein structure of the rpL21 sequence cloned was deduced using Predict Protein software. The results indicated that the length of cDNA fragment cloned was 504bp; containing an open reading frame of 483bp. Deduced protein was composed of 160 amino acids with an estimated molecular weight of 18.59kD and pI of 11.10. The length of the genomic sequence is 2254bp, containing 5 exons and 4 introns. Alignment analysis indicates that rpL21 is highly similarity with the reported species both at the level of DNA and protein. Topology prediction shows that 6 different patterns were found in the rpL21 protein of the Ailuropoda melanoleuca. The rpL21 gene of the Ailuropoda melanoleuca was studied in this paper, which will enrich and improve the mammals' rpL21 gene database.

Comprehensive analysis of the full-length transcripts and alternative splicing involved in clubroot resistance in Chinese cabbage

Chinese cabbage is an economically important Brassica vegetable worldwide, and clubroot, which is caused by the soilborne protist plant pathogen Plasmodiophora brassicae is regarded as a destructive disease to Brassica crops. Previous studies on the gene transcripts related to Chinese cabbage resistance to clubroot mainly employed RNA-seq technology,although it cannot provide accurate transcript assembly and structural information. In this study, PacBio RS II SMRT sequencing was used to generate full-length transcriptomes of mixed roots at 0, 2, 5, 8, 13, and 22 days after P. brassicae infection in the clubroot-resistant line DH40R. Overall, 39 376 high-quality isoforms and 26 270 open reading frames(ORFs) were identified from the SMRT sequencing data. Additionally, 426 annotated long noncoding RNAs(lncRNAs),56 transcription factor(TF) families, 1 883 genes with poly(A) sites and 1 691 alternative splicing(AS) events were identified. Furthermore, 1 201 of the genes had at least one AS event in DH40R. A comparison with RNA-seq data revealed six differentially expressed AS genes(one for disease resistance and five for defensive response) that are potentially involved in P. brassicae resistance. The results of this study provide valuable resources for basic research on clubroot resistance in Chinese cabbage.