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

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

Cloning and Sequence Analysis of Beta-actin Gene Full Length cDNA from Trachidermus fasciatus

[Objective] The purpose of this experiment was to reveal the sequence and characteristics of Trachidermus fasciatus beta-actin gene.[Method] Using total RNA of muscle in T.fasciatus as template,three cDNA fragments of beta-actin gene in T.fasciatus were amplified by RT-PCR,5＇-RACE and 3＇-RACE.[Result] A full-length of 1905 bp beta-actin cDNA sequence in T.fasciatus,which includes a 1128 bp length open reading frame encoding a 375-amino acid peptide,was obtained.Sequence alignment of nucleotide and amino acid sequence revealed that T.fasciatus beta-actin shared high homology with that of Epinephelus coioides,Rachycentron canadum,Chrysophrys auratus and of relatively low homology with mammalian and bird.The phylogenetic analysis showed that T.fasciatus beta-actin had closest relationship with Epinephelus coioides.And RT-PCR analysis suggested that the beta-actin gene expressed in four tissues,i.e.,muscle,liver,intestine and brain.[Conclusion] The full length sequence of beta-actin gene with high conservation in T.fasciatus was obtained for the first time.

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,Identification and Differential Expression Analysis of Full-length cDNA of Carp Interleukin-1β

[Objective] The research aimed to carry out the cloning,identification and differential expression analysis of carp interleukin-1β （IL-1β） cDNA. [Method] By using DD-RTPCR method,the differential expression cDNA fragments were gained. The cDNA library of carp peripheral blood leucocytes which was stimulated by the mitogen was screened,and the full length cDNA of carp IL-1β was cloned. Moreover,the sequence analysis and differential expression analysis were carried out. [Result] The positive clone which had a whole ORF that encoded 276 amino acids was obtained. The cluster analysis showed that the amino acid sequence of carp IL-1β and Japanese carp closely gathered as a branch,and the homoeology of amino acid sequence reached 95%. The clustering order was the carassius,zebra fish,pig,cattle,horse,human and mouse in turn. The differential expression analysis showed that the expression of IL-1β in the leucocytes significantly increased in the prior period （4 h） after the mitogen stimulated. But as the time went by （12 and 24 h）,it didn＇t increase in the same period. The total trend of expression amount presented the peak type. [Conclusion] The research laid the foundation for further studying the expression manner,function characteristic,regulation mechanism of IL-1β in vivo and its action mechanisms in the inflammatory reaction,emergency reaction and immune response.

玉米大斑病菌cDNA文库的构建及转录因子StMR1互作蛋白的筛选

【目的】筛选玉米大斑病菌(Setosphaeria turcica)转录因子的互作蛋白,解析黑色素调控转录因子StMR1调控玉米大斑病菌致病性的分子机制。为解析玉米大斑病菌侵染过程中转录因子的调控网络,阐明病菌的致病机理提供参考。【方法】收集玉米大斑病菌菌丝和孢子不同萌发阶段作为试验材料,采用Gateway方法构建玉米大斑病菌cDNA文库,使用同源重组的方法构建转录因子StMR1的诱饵载体,采用酵母双杂交技术筛选其互作蛋白并进行一对一验证。【结果】构建的玉米大斑病菌文库插入的平均片段长度大于1000 bp,初级文库及次级文库的库容量为1.2×107和1.04×107CFU,重组率为100%,可以用于酵母双杂交筛选。成功构建可以用于筛库的诱饵载体pGBKT7-StMR1,经初筛与复筛得到3个互作蛋白,一对一验证短链脱氢酶、糖基转移酶、富含亮氨酸重复序列蛋白均与转录因子StMR1存在互作。【结论】成功构建了丰富度高且质量好的玉米大斑病菌cDNA文库并筛选到了与转录因子StMR1互作的蛋白。

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 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.

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.

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.

矢车菊不同颜色花瓣酵母cDNA文库的构建

【目的】矢车菊花瓣的蓝色呈色和品种间花色变异的分子调控机制尚不明晰。本研究采用Gateway技术构建了矢车菊6个不同花色品种花瓣的酵母cDNA文库,以期进一步通过酵母单杂交或双杂交技术筛选参与调控花瓣呈色的关键互作蛋白。【方法】本研究以白色、粉色、红色、蓝色、紫色和墨色矢车菊花瓣为材料,提取总RNA后分离和纯化mRNA,合成双链cDNA后依次进行BP重组反应和LR重组反应,分别获得初级和次级文库。最后将次级文库质粒转化酵母Y187,获得矢车菊不同颜色花瓣的酵母cDNA文库。【结果】质量鉴定结果显示:初级文库的库容量为1.3×10^(7) CFU,重组率为100%,且插入片段长度均在1000 bp以上;次级文库的库容量为1.6×10^(7) CFU,重组率为100%,且插入片段长度均在1000 bp以上。酵母文库的滴度为3.5×10^(7) CFU/mL,随机挑选的24个单克隆经PCR检测后均扩增出明亮条带,重组率为100%,插入片段长度均大于1000 bp。【结论】本研究构建的矢车菊不同颜色花瓣酵母cDNA文库的质量较高,能满足酵母文库筛选的试验要求,为后续探究矢车菊花瓣的蓝色呈色和品种间花色变异的分子调控机制提供了材料基础。

Cloning of A Full-Length cDNA Encoding 4-Coumarate:CoA Ligase of Amorpha fruticosa by PCR-Based Methods

An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE) PCR. We designed 5′RACE primers based on 4CLA1 fragment which obtained from degenerate PCR. Inverse PCR and nested PCR enabled cloning of the remainder fragments of the gene included 5′ and 3′ end sequence. The ORF encodes a polypeptide of 540 amino acids. The predicted amino acid sequence exhibits significant homology with those of other cloned 4CL genes, contain domains typical of predicted 4CL proteins, in particular a postulated AMP binding site, catalytic domain, and conserved Cys residues.

橡胶树棒孢霉落叶病菌酵母单杂交cDNA文库及CcCas5启动子诱饵载体的构建

为了筛选出与CcCas5启动子结合的转录因子,本研究构建了橡胶树棒孢霉落叶病菌的酵母单杂交cDNA表达文库和CcCas5启动子诱饵载体,并对3-氨基-1,2,4-三唑(3-amino-1,2,4-triazole,3-AT)的最佳抑制浓度进行了筛选。结果显示,构建的酵母单杂交cDNA表达文库总容量为3.49×10^(6)cfu,插入片段主要分布于750~2000 bp之间,重组率为95.8%;克隆了CcCas 5基因2600 bp的启动子序列,预测获得真菌主要的10大类转录因子的结合位点165个;构建了分别携带1500 bp和1000 bp启动子序列的酵母单杂交启动子诱饵载体,10 mmol/L的3-AT能抑制pHis2.1-pCcCas5-1000和pHis2.1-pCcCas5-1500的背景表达。试验结果为通过酵母单杂交筛选与CcCas5启动子结合的转录因子和进一步研究转录因子对CcCas5表达的调控作用奠定基础。

灰霉菌胁迫下番茄叶片cDNA文库构建及SlbZIP1互作蛋白筛选鉴定

为筛选番茄在灰霉菌侵染下与bZIP转录因子SlbZIP1(Solyc01g008730.2.1)互作的蛋白,以感病番茄Moneymaker为材料,提取接种灰霉菌胁迫处理后0、24、48、72、96 h的番茄叶片的总RNA,通过三框法构建cDNA酵母文库,计算文库库容量、重组效率。同时,以SlbZIP1为诱饵,利用构建的文库探索其在灰霉菌侵染过程中的互作蛋白,并对其中可能参与病原菌防御反应的潜在蛋白在酵母中进行互作验证。结果表明,酵母文库库容达到1.5×10^(6)CFU,重组效率为98%。酵母双杂交共计筛选到14个与SlbZIP1互作的潜在蛋白。进一步互作验证表明,SlbZIP1与谷氧还蛋白SlGRXC9、SlGRXC6、bZIP转录因子SlTGA2.2及钙调蛋白SlCaM1均存在互作。上述结果可为进一步阐明SlbZIP1对腐生型真菌病害的抗病分子机制奠定基础。

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.

Accelerating Oxygen Electrocatalysis Kinetics on Metal-Organic Frameworks via Bond Length Optimization

Metal-organic frameworks(MOFs)have been developed as an ideal platform for exploration of the relationship between intrinsic structure and catalytic activity,but the limited catalytic activity and stability has hampered their practical use in water splitting.Herein,we develop a bond length adjustment strategy for optimizing naphthalene-based MOFs that synthesized by acid etching Co-naphthalenedicarboxylic acid-based MOFs(donated as AE-CoNDA)to serve as efficient catalyst for water splitting.AE-CoNDA exhibits a low overpotential of 260 mV to reach 10 mA cm^(−2)and a small Tafel slope of 62 mV dec^(−1)with excellent stability over 100 h.After integrated AE-CoNDA onto BiVO_(4),photocurrent density of 4.3 mA cm^(−2)is achieved at 1.23 V.Experimental investigations demonstrate that the stretched Co-O bond length was found to optimize the orbitals hybridization of Co 3d and O 2p,which accounts for the fast kinetics and high activity.Theoretical calculations reveal that the stretched Co-O bond length strengthens the adsorption of oxygen-contained intermediates at the Co active sites for highly efficient water splitting.

Construction of a Full-Length cDNA Library of Gossypium hirsutum L. and Identification of Two MADS-Box Genes

A full-length normalized cDNA library for the flower development stages of short-season cotton （Gossypium hirsutum L.） （CCRI36） was constructed. A total of 3 421 clones were randomly selected for sequencing, with a total of 3 175 effective sequences obtained after removal of empty-carriers and low-quality sequences. Clustering the 3 175 high-quality expressed sequence tags （ESTs） resulted in a set of 2 906 non-redundant sequences comprised of 233 contigs and 2 673 singletons. Comparative analyses indicated that 913 （43.6%） of the unigenes had homologues with function-known genes or functionassumed genes in the National Center for Biotechnology Information. In addition, 763 （36.4%） of the unigenes were functionally classified using Gene Ontology hierarchy. Through EST alignment and the screening method, the full-length cDNA of two MADS-box genes viz., GhMADSll and GhMADS12 were acquired. These genes may play a role in flower development. Phylogenetie analysis indicated that GhMADS11 and GhMADS12 had high homology and close evolutionary relationship with AGL2/SEP-type and PI-type genes, respectively. The expression of both GhMADSll and GhMADS12, genes was high in reproductive organs. In floral organs, GhMADSll expression was high in petals （whor12） and ovules, while GhMADS12 expression was high in petals （whor12） and stamens （whor13）. Results show that the EST strategy based on a normalized cDNA library is an effective method for gene identification. The study provides more insights for future molecular research on the regulation mechanism of cotton flower development.

A NEW METHOD TO CONSTRUCT A FULL-LENGTH cDNA LIBRARY OF HUMAN NORMAL BLADDER TISSUE

Objective Using template switch mechanism at the 5’ end of mRNA technique (SMART) to construct a full length cDNA library of human normal bladder tissue. Methods The novel procedures used the template switching activity of powerscript reverse transcriptase to synthesize and anchor first strand cDNA in one step. Following reverse transcription, 5 cycles of PCR were performed using a modified oligo(dT) primer and an anchor primer to enrich the full length cDNA population with 1.0 g human normal bladder poly(A) + RNA, then double strand cDNA was synthesized. After digestion with sfiI and size fractionation by CHROMA SPIN 400 columns, double strand cDNA was ligated into λ TripIEx 2 vector and was packaged. We determined the titer of the primary library and the percentage of recombinant clones and finally amplified the library. Results The titer of the cDNA library constructed was 2.1×10 6 pfu·mL -1 , and the amplified cDNA library was 6×10 11 pfu·mL -1 , the percentage of recombination clones was 99%. Conclusion Using SMART technique helps us to construct full length cDNA library with high efficiency and high capacity which lays solid foundation for screening target genes of bladder diseases with probes and antibodies.

Construction of a Normalized Full-Length cDNA Library of Cephalopod Amphioctopus fangsiao and Development of Microsatellite Markers

Amphioctopus fangsiao is one of the most economically important species and has been considered to be a candidate for aquaculture. In order to facilitate its fine-scale genetic analyses, we constructed a normalized full-length library successfully and developed a set of microsatellite markers in this study. The normalized full-length library had a storage capacity of 6.9×105 independent clones. The recombination efficiency was 95% and the average size of inserted fragments was longer than 1000 bp. A total of 3440 high quality ESTs were obtained, which were assembled into 1803 unigenes. Of these unigenes, 450(25%) were assigned into 33 Gene Ontology terms, 576(31.9%) into 153 Kyoto Encyclopedia of Genes and Genomes pathways, and 275(15.3%) into 22 Clusters of Orthologous Groups. Seventy-six polymorphic microsatellite markers were identified. The number of alleles per locus ranged from 4 to 17, and the observed and expected heterozygosities varied between 0.167 and 0.967 and between 0.326 and 0.944, respectively. Twelve loci were significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and no linkage disequilibrium was found between different loci. This study provided not only a useful resource for the isolation of the functional genes, but also a set of informative microsatellites for the assessment of population structure and conservation genetics of A. fangsiao.

麦根腐平脐蠕孢cDNA文库的构建及BsTup1互作蛋白筛选

【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为诱饵来筛选酵母双杂交文库,确定与BsTup1相互作用的蛋白。【结果】1)利用SMART(switching mechanism at 5′end of the RNA transcript)技术首次成功构建了麦根腐平脐蠕孢(B.sorokini-ana)分生孢子和菌丝体的混合cDNA文库。文库鉴定结果表明,构建的cDNA文库库容为4.8×10^(7) cfu·mL^(-1),文库插入片段重组率达100%且平均大小为1000 bp。2)构建了pGBKT7-BsTup1诱饵载体,无自激活活性。3)使用诱饵蛋白载体pGBKT7-BsTup1对麦根腐平脐蠕孢(B.sorokiniana)酵母双杂交cDNA文库进行筛选,经测序、序列比对和酵母回转验证,获得38个与BsTup1相互作用的候选蛋白。【结论】成功构建了麦根腐平脐蠕孢(B.sorokiniana)的cDNA文库,并鉴定出38个与BsTup1相互作用的候选蛋白。

Chord length sampling correction analysis for dispersion fuel in Monte Carlo simulation

Dispersion fuels,knowned for their excellent safety performance,are widely used in advanced reactors,such as hightemperature gas-cooled reactors.Compared with deterministic methods,the Monte Carlo method has more advantages in the geometric modeling of stochastic media.The explicit modeling method has high computational accuracy and high computational cost.The chord length sampling(CLS)method can improve computational efficiency by sampling the chord length during neutron transport using the matrix chord length?s probability density function.This study shows that the excluded-volume effect in realistic stochastic media can introduce certain deviations into the CLS.A chord length correction approach is proposed to obtain the chord length correction factor by developing the Particle code based on equivalent transmission probability.Through numerical analysis against reference solutions from explicit modeling in the RMC code,it was demonstrated that CLS with the proposed correction method provides good accuracy for addressing the excludedvolume effect in realistic infinite stochastic media.