Molecular cloning and expression analysis of Crustin-like gene from Chinese shrimp Fenneropenaeus chinensis

A new member of antimicrobial protein genes of the Crustin family was cloned from haemocytes of the Chinese shrimp Fennero- penaeus chinensis by 3 ′and 5′ RACE. The full-length cDNA of Crustin-like gene contains a 390 bp open reading frame, encoding 130 amino acids. The deduced peptide contains a putative signal peptide of 17 amino acids and mature peptide of 113 amino acids. The molecular mass of the deduced mature peptide is 12. 3 ku. It is highly cationic with a theoretical isoelectric point of 8.5. The deduced amino acids sequence of this Crustin showed high homology with those of Penaeus （ Litopenaeus ） setferus. Northern blotting showed that the cloned Crustin gene was mainly expressed in haemocytes, gill, intestine, and RNA in situ hybridization indicated that the Crustin gene was constitutively expressed exclusively in haemocytes of these tissues. Capillary elec- trophoresis RT-PCR analysis showed that Crustin was up-regulated dramatically from 12 to 48 h after a brief decrease of mRNA during first 6 h in response to microbe infection. The level of Crustin mRNA began to restore at 72 h post-challenge. This indicated that Crustin gene might play an important role when shrimps are infected by bacterial pathogen.

Molecular cloning of human heat shock protein 27 and study of its protective effects on oxidative damage in rat cardiomyocte H9c2

Objective： To clone human cardiac heat shock protein 27 （HSP27） gene and to determine the effects of HSP27 on the oxidative stress in rat cardiomyocyte cell line H9c2. Methods： Full length of HSP27 cDNA which got by RT-PCR was constructed into pCDNA3.1^＋ . The recombinant was transfected into rat cardiomyocyte cell line H9c2 and the stable trahsfection cell line was selected by G418. Then we observe the effects of HSP27 over-expression on LDH release and apoptosis induced H2O2 in H9c2. Results： ①pCDNA3.1^＋/HSP27 provided a sound expression of HSP27 in both 293T and H9c2. ②LDH releasing induced by 0, 100,250,500, 1000 μmol/L H2O2 in HSP27 over-expression group and wild type group were 0.396±0.017 vs. 0.390±0.01）9 （p 〉0.05）, 0.437±0. 014 vs. 0.416±0.015 （P〈0.05）, 0.471±0.018 vs. 0.417±0.009 （P 〈0.001）, 0.505±0.030 vs. 0.657± 0.022（P 〈0.001）, 0.547 ±0.027 and 0.661 ± 0.011（ P 〈 0. 001 ）, respectively. ③Apoptosis induced by 150 μmol/L H2O2 in HSP27 over-expression group and wild type group were （10.693± 1.122）% vs. （4.027 ± 1.628）%（ P 〈0.01）. Conclusion： We cloned and constructed human cardiac HSP27 gene successfully, and over-expression of human HSP27 could inhibit oxidative damage significantly in H9c2.

Molecular Cloning and Characterization of Three Novel Genes Related to Fatty Acid Degradation and Their Responses to Abiotic Stresses in Gossypium hirsutum L.

Fatty acid metabolism is responsible not only for oilseed metabolism but also for plant responses to abiotic stresses. In this study, three novel genes related to fatty acid degradation designated GhACX, Gh4CL, and GhMFP, respectively, were isolated from Gossypium hirsutum acc. TM-1. The phylogenetic analysis revealed that amino acid sequences of GhACXand GhMFP have the highest homology with those from Vitis vinifera, and Gh4CL has a closer genetic relationship with that from Camellia sinensis. Tissue- and organ-specific analysis showed that the three genes expressed widely in all the tested tissues, including ovules and fiber at different developing stages, with expressed preferentially in some organs. Among them, GhACX showed the most abundant transcripts in seeds at 25 d post anthesis （DPA）, however, GhMFP and Gh4CL have the strongest expression level in ovules on the day of anthesis. Based on real-time quantitative RT-PCR, the three genes were differentially regulated when induced under wounding, methyl jasmonate （MeJA）, cold, and abscisic acid （ABA） treatments. The characterization and expression pattern of three novel fatty acid degradation related genes will aid both to understand the roles of fatty acid degradation related genes as precursor in stress stimuli and to elucidate the physiological function in cotton oilseed metabolism.

Molecular Cloning and Sequence Analysis of IGF-Ⅰfrom Triangular Bream (Megalobrama terminalis)

The insulin-like growth factor Ⅰ(IGF-Ⅰ) gene of triangular bream (Megalobrama terminalis)(GenBank No.AY247412)(Tb)was cloned for the first time from liver by RT-PCR. Thenucleotide sequence analysis showed the Tb IGF-ⅠcDNA consisted of 486 nucleotides andencoded 117 amino acids including B, C, A, D and E five domains. Analysis of E-domainindicated that cloned Tb IGF-Ⅰbelonged to IGF-ⅠEa-2 subtype. Identity analysis showedthe IGF-Ⅰnucleotide sequence shared 99.8% homology with bluntnose bream, 88.8% withgrass carp, 85.8% with common carp; the pre-IGF-Ⅰamine acid sequence shared 99.4% withbluntnose bream, 88.8% with grass carp, 85.4% homology with common carp. In the CyprinusCarpio, the higher homology of nucleotide sequence and amino acid sequence in IGF-Ⅰshowed that the closer relationship the fishes have. These results could provide basicdata for the research on Tb germplasm and the development and utilization of biologicalfeed additives.

Molecular Cloning and Nucleotide Sequence of the Attenuated Hog Cholera VirusLapinized Chinese Strain

The genomic sequence of the attenuated hog cholera virus Lapinized Chinese strain (HCLV) was determined from overlapping cDNA clones. The viral RNA of HCLV stain comprised 12 310 nucleotide (nt) including 374 nt and 239 nt at the 5′ and 3′-noncoding region, respectively. The complete genome sequence contained one large open reading frame which encoded an amino acid sequence of 3 898 residues with a calculated molecular weight of 437×103. Although there were mostly only small differences between the sequence of the HCLV strain and the published sequences of strains ALD, GPE?, Alfort and Brescia, there was one notable insertion of 12 nucleotides, TTTTCTTTTTTC in the 3′ non-coding region of HCLV strain.

Molecular cloning, characterization, and antioxidant function of catalase in Lymantria dispar asiatic (Lepidoptera: Lymantriidae) under avermectin stress

The critical antioxidant catalase(CAT)breaks down hydrogen peroxide induced by environmental stresses.Here we cloned full length catalase cDNA from Lymantria dispar asiatic(LdCAT).Bioinformatic analyses showed that open reading frames of LdCAT contains 1524 bp,encoding 507 amino acids with molecular weight of 126.99 kDa,theoretical pI of 5.00,aliphatic index of 29.92,grand average of hydropathicity of 0.764,and instability index(II)of 46.56.Protein BLAST and multiple sequence alignment indicated that LdCAT had high identity with CAT from other insects,especially lepidopterans.In a phylogenetic analysis,LdCAT was most similar to CAT from Spodoptera litura and S.exigua.Quantitative realtime polymerase chain reaction showed that LdCAT transcripts in all instar larvae and the five tissues tested,verifying the ubiquity of LdCAT in L.disapr.Moreover,LdCAT of third instar larvae was significantly upregulated after they fed on avermectin at sublethal and LC10 doses.The highest relative transcript levels were found 2 h after an avermectin spray at LC90,and in the cuticula,rather than heads,fat bodies,malpighian tubes,and midguts after a spray avermectin at a sublethal concentration.The expression level of LdCAT under pesticide stresses here suggested that CAT is an important antioxidant enzyme of L.disapr defensing against pesticide stress and may be a good target for controlling this pest.

Porcine LEM domain-containing 3: Molecular cloning, functional characterization, and polymorphism associated with ear size

Ear size exhibits remarkable diversity in pig breeds. LEM domain-containing 3 （LEMD3） on chromosome 5 is considered as an important candidate for porcine ear size. This is the first study on cloning and characterization of LEMD3 cDNA. The complete cDNA contains 4 843 bp, including a 2 736-bp open reading frame （ORF）, a 37-bp 5＂-untranslated region （UTR） and a 2070-bp 3＂-UTR. The complete LEMD3 gene is 126241-bp and contains 13 exons and 12 introns. The ORF encodes a deduced LEMD3 protein of 911 amino acids, which shares 82-94% nucleic acid and 51-96% amino acid identity with other species. A phylogenetic tree constructed based on the amino acid sequences revealed that the porcine LEMD3 protein was closely related with cattle LEMD3. Resequencing of the ORF and promoter of LEMD3 from Minzhu pig and Large White revealed three single nucleotide polymorphisms （SNPs）： L964C〉A in the complete coding region, L4625A〉G in the 3＂ UTR, and L-394T〉C in the promoter region. Genome-wide association study （GWAS） revealed that all of SNPs were shown significant association with ear size in Large WhitexMinzhu pig intercross population. With conditional GWAS, -Iogl0（P-value） decreased by more than 80% when each of three SNPs was included as a fixed effect. These results suggested direct involvement of LEMD3 or close linkage to the causative mutation for ear size. The findings of this study might form the basis for understanding the genetic mechanism of ear size variation in pigs and provide potential molecular markers for screening ear size diversity in pig breeds.

Molecular Cloning and Characterization of the Actin-depolymerizing Factor Gene in Gossypium barbadense

Sea Island cotton(Gossypium barbadense L.) has been highly valued in Verticillium wilt resistance and many fiber qualities including fiber length,strength,and fineness.To identify whether

Molecular Cloning and Characterization of Genes Involved in Cotton(Gossypium barbadense L.) Response to Verticillium dahliae

Verticillium dahliae Kleb.is a necrotrophic plant pathogen which causes serious soil borne vascular disease in cotton.The molecular basis the defense response of cotton to this pathogen is

Molecular Cloning,Expression,and Characterization of an Adenylyl Cyclase-associated Protein from Gossypium arboreum Fuzzless Mutant

CAP,an adenylyl cyclase-associated protein,is predicted to be involved in cytoskeletal organization and signal transduction.Recently,we found that CAP may play an important role in fuzz-like fiber cell initiation in cotton.For the further research,we isolated two CAP homologues from wild

Molecular Cloning and Expression Analysis of a Cys2/His2 Type Zinc Finger Protein Gene in Upland Cotton

The zinc finger proteins belong to the largest family of transcription factors.But there is little research of Cys2/His2 type zinc finger proteins in cotton,and there is no submission of correlating

Molecular cloning,expression pattern and phylogenetic analysis of Guanine nucleotide exchange factor Vav2 in lamprey,Lampetra japonica

The Guanine nucleotide exchange factor Vav2（Vav2） is a member of the Vav family that serves as an important regulators for the Rho family of Ras-related GTPases. In the current study, an ortholog（Lj-Vav2） of Vav2 was identified in the lamprey（Lampetra japonica）. To elucidate the phylogenetic relationship of Vav2, the metazoan genome databases were analyzed to mine the ortholog of Vav. It was found that Vav2 genes were only existed in vertebrates and Lj-Vav2 was the original one found in agnathans. The evolutionary dynamics of conserved motifs of Vav2 were explored using combined amino acid sequence as markers, and it is revealed that the Calponin homology（CH） domain, Dbl-homologous（DH） domain, Pleckstrin homology（PH） domain, Cysteine-rich（C1）domains, Src homology 3（SH3） domains and Src homology 2（SH2） domain were conserved throughout the Vav2 gene family in vertebrates during gene evolution. Relative quantitative real-time PCR analysis showed that the LjVav2 was distributed in the heart, kidney, supraneural myeloid body, liver, gill and lymphocyte-like cells. The LjVav2 was found to be expressed in these tissues, and the level of which was upregulated in lymphocyte-like cells after the animal was stimulated with LPS. These results indicated that the Lj-Vav2 might be involved in the immune response of lymphocyte-like cells in lamprey. Meanwhile, our findings provided a foundation for further investigation of the function of Lj-Vav2 in the primary vertebrate.

Molecular cloning and functional analyses of low-temperature induced genes from Ammopiptanthus mongolicus

Studies on the cold-responsive genes and cold signaling of woody species drop far behind in comparison to herbaceous plants.Due to similar lignified structure,perennial characteristic,and enhanced tolerance,it seems much easier to find strongly antifreeze genes and obtain effective results in transgenic woody plants.In this study,Ammopiptanthus mongolicus,an evergreen,broadleaf and cold-resist leguminous shrub growing in the desert of Inner Mongolia,was used as a material for low-temperature induced gene isolation.Through differential expression analysis induced by low-temperature,thirteen up-regulated cDNAs were identified.One of them,AmEBP1,(accession number:DQ519359)confers enhanced cold-tolerance to both transgenic E.coli and transgenic Arabidopsis.Results suggest that AmEBP1 can stimulate the synthesis of ribosome and the dephosphyration of the α-subunit of initiation factor 2(eIF2α),and subsequently promote the translation process.By which the transgenic plants obtained increased cold-resistant ability.

Molecular Cloning of a Chitinase Gene from the Ovotestis of Kuroda’s Sea Hare Aplysia kurodai

In this study, we report that we successfully cloned and sequenced a chitinase gene from the ovotestis of Kuroda’s sea hare Aplysia kurodai. By using reverse transcription-polymerase chain reaction (RT-PCR) and a system for the 5’ and 3’ rapid amplification of cDNA ends, we obtained a 1352 bp chitinase gene (AkChi) from the ovotestis of A. kurodai. AkChi contains a 1263 bp open reading frame that encodes 421 amino acids. The domain structure predicted from the deduced amino acid sequence was an N-terminal signal peptide and a catalytic domain of glycoside hydrolase (GH) family 18 chitinase. A comparative analysis of the deduced amino acid sequences of AkChi with those of the acidic mammalian chitinase of the California sea hare Aplysia californica revealed the highest homology at 83%. The purified chitinase from the ovotestis was digested by trypsin, and 119 residues of digested peptides were consistent with the deduced amino acid sequence of AkChi. We used RT-PCR to evaluate the expression of AkChi in various tissues of A. kurodai, and we observed that AkChi was expressed only in the ovotestis. A phylogenetic tree analysis, performed using the amino acid sequences of AkChi and known GH family 18 chitinases, showed that AkChi was separated from the molluscan chitinases with a chitin binding domain. To our knowledge, this is the first study demonstrating the cDNA cloning of an ovotestis chitinase from a sea hare.

Characterization and molecular cloning of a serine hydroxymethyltransferase 1(OsSHM_1) in rice

Serine hydroxymethyltransferase (SHMT) is important for one carbon metabolism and photorespiration in higher plants for its participation in plant growth and development, and resistance to biotic and abiotic stresses. A rice serine hydroxymethyltransferase gene, OsSHM1, an ortholog of Arabidopsis SHM1, was isolated using map-based cloning. The osshm1 mutant had chlorotic lesions and a considerably smaller, lethal phenotype under natural ambient CO2 concentrations, but could be restored to wild type with normal growth under elevated CO2 levels (0.5% CO2), showing a typical photorespiratory phenotype. The data from antioxidant enzymes activity measurement suggested that osshm1 was subjected to significant oxidative stress. Also, OsSHM1 was expressed in all organs tested (root, culm, leaf, and young panicle) but predominantly in leaves. OsSHM1 protein is localized to the mitochondria. Our study suggested that molecular function of the OsSHM1 gene is conserved in rice and Arabidopsis.

Molecular cloning, sequencing and expression of obese gene in the Chinese

Objective To construct the human obese(ob) cDNA clone in the Chinese, and analyze the expression of the ob gene in adipose tissue of obese, non obese subjects and nooinsulin dependent diabetes mellitus (NIDDM) Chinese patients Methods A ob cDNA clone was isolated by reverse transcription polymerase chain reaction (RT PCR) Four groups of Chinese subjects participated in the study: 1) 12 obese subjects [body mass index (BMI): 28 5±2 3? kg/m 2]; 2) 11 non obese subjects (BMI: 21 0±1 5? kg/m 2); 3) 8 obese NIDDM patients (BMI: 27 0±1 4?kg/m 2); 4) 11 non obese NIDDM patients (BMI: 21 2±1 4?kg/m 2) The expression of ob gene mRNA in abdominal subcutaneous adipose tissue was examined using RNA dot blot hybridization with a digoxigenin labeled human ob cDNA probe The hybridized signals were quantitated by densitometry Results A full human ob cDNA fragment which included a glutamine codon at +49 was obtained A base substitution (A to G) in the coding region at position 287 was found, resulting in a glutamine being replaced by an arginine Expression of the ob gene was significantly higher in Chinese obese subjects compared to non obese ones ( P <0 05), and positively correlated with the BMI No significant difference in the amount of ob mRNA was detected between non diabetic and diabetic groups at the same BMI level Conclusions We constructed a full length human ob cDNA clone. The expression of the ob gene was significantly higher in Chinese obese subjects than in non obese ones. The metabolic and hormonal changes associated with NIDDM are not the main factors regulating the expression of the ob gene.

Molecular cloning and characterization of three phenylalanine ammonia-lyase genes from Schisandra chinensis

Phenylalanine ammonia-lyase(PAL),which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid,is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes.Schisandra chinensis,a woody vine plant belonging to the family of Magnoliaceae,is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity.However,the functional role of PAL in the biosynthesis of lignan is relatively limited,compared with those in lignin and flavonoids biosynthesis.Therefore,it is essential to clone and characterize the PAL genes from this valuable medicinal plant.In this study,molecular cloning and characterization of three PAL genes(ScPAL1−3)from S.chinensis was carried out.ScPALs were cloned using RACE PCR.The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis.In order to determine their catalytic activity,recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli(BL21-DE3),followed by Ni-affinity purification.The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds.The optimal temperature,pH value and effects of different metal ions were determined.Vmax,Kcat and Km values were determined under the optimal conditions.The expression of three ScPALs in different tissues was also determined.Our work provided essential information for the function of ScPALs.

MOLECULAR CLONING OF A cDNA ENCODING FILARIAL COMMON ANTIGEN

It is estimated that more than 100 million peo-ple are infected with filaria and that 10 times as many are at risk of infection.Epidemiological

Molecular Cloning and Expression Analysis of a Hexokinase Gene,MdHXK1 in Apple

A hexokinase gene named MdHXK1(MDP0000309677) was cloned from ‘Gala' apple(Malus × domestica Borkh.). Sequence analysis showed that the MdHXK1 gene was 1 497 bp long and encoded 499 amino acids. The predicted molecular mass of this protein was 54.05 kD, and the pI was 5.76. A phylogenetic tree indicated apple MdHXK1 exhibited the highest sequence similarity to Pyrus bretschneideri PbHXK1. Analysis of the functional domain showed that the MdHXK1 protein included two conserved kinase domains. The prediction of subcellular localization suggested that the MdHXK1 protein was mainly localized in the cytoplasm. There was an indication that MdHXK1 existed as one copy in the apple genome by Southern blotting. Silico analysis suggested that the promoter sequence contained several typical cis-acting elements, including defense, sugar signaling and phytohormone responsive elements. Quantitative real-time PCR analysis demonstrated that the MdHXK1 gene was mainly expressed in stem and flower tissues. During the development of apple fruits, the expression of the MdHXK1 gene initially increased and then decreased. The changes on Glc phosphorylation relative activity and glucose concentration showed the same trend. In addition, the expression of this gene was induced by salt stress, low temperature, and abscisic acid(ABA). Finally, we obtained and purified the fused MdHXK1 protein by recombinant prokaryotic expression. Studies have demonstrated that MdHXK1 may participate in sugar metabolism in apple fruits. Enzyme encoded by MdHXK1 is a key factor in the mediation of sugar accumulation. Recently,researchers on hexokinase at home and abroad mainly focused on model plants, such as Arabidopsis, tobacco and rice, but orchard fruit like apple were underresearched. Our research established the foundation for the further study of the functions of MdHXK1.

Purification, Biological Activities, and Molecular Cloning of a Novel Mannose-Binding Lectin from Bulbs of Zephyranthes candida Herb （Amaryllidaceae）

A novel mannose-bindlng aggiutinln was purified from bulbs of Zephyranthes candida Herb by extraction, precipitation with 80% （NH4）2SO4, and ion-exchange chromatography on DEAE-Sepharose followed by gel flitration on Sephscryl S-100. The purified Z. candida agglutlnln （ZCA） migrated as a single band of 12 kDa on sodium dodecyi suifate-poiyecryiamide gel electrophoresis under reducing and non-reducing conditions. The apparent molecular mass of the iectln, as datermlned by gel filtration chromatography, was 48 kDa. The results Indicated that ZCA was composed of four Identical subunlts of 12 kDa each （homotetramerlc nature）. The ZCA agglutlhated rabbit erythrocytes, Escherichla coil and Saccharomyces cerevislae ceils at concentrations of 0.95, 1.90, and 31.30 μg/mL, respectively. Bloassays Indicated that ZCA has a significant effect on wheat aphid survival. Mortality after 7 d was 〉 90% at 0.26%. A degenerate primer was designed In accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by 3＇- and 5＇-rapid amplification of cDNA ends. The full-length cDNA had 661 bp and the sequence encoded an open reading frame of 168 amino acids. The mature protein of ZCA Includes 109 amino acid residues and the molecular weight of the protein was 12.1 kDa. The result show that the zca gene encodes a protein precursor with a signal peptlde, a mature protein, and a C-terminal cleavage amino acids sequence. Molecular modeling of ZCA Indicated that Its three-dimensional atructure strongly resembies that of the snowdrop aggiutinin. Blocks＇ analysis revealed that the deduced amino acid sequence of ZCA has three functional domains specific for agglutination and three carbohydrate binding boxes （QDNY）.