Molecular Cloning and Expression of RSSG58 Gene in Rice Sperm Cells

Myosins, a large family of structurally diverse mechanoenzymes, which, upon interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force, play an important role in male reproductive processes. In this study we report the rice ( Oryza sativa L.) RSSG58 gene, which was cloned from the cDNA library of rice sperm cells by using sperm cell mainly expression subtractive clone as probe. This gene encodes a putative 66.7 W polypeptide, which shows similarity to the myosin heavy chain of Arabidopsis thaliana, and consists of 579 amino acids with an isoelectric point (pI) of 4.885. RSSG58, which is a member of a divergent gene family, generates transcripts of 2 278 bp and 2 437 bp that differ only in their polyadenylation sites. Southern hybridization showed that RSSG58 has only one copy in rice genome and RSSG58 transcripts are most abundant in sperm cells, with two distinct signals. The RT-PCR analysis indicated that the transcriptions of the RSSG58 gene were various in the different development stages and tissues. The greatest accumulation of RSSG58 mRNA was detected in sperm cells, while weaker expression was detected in leaves, microspore mother cells, unicellular microspore pollen stage, two-cell stage pollens, mature pollens and pollinated ovaries. These results suggest that RSSG58 is especially abundantly expressed in rice sperm cells.

Molecular Cloning and Sequence Analysis ofBoPGIP2 Gene from Brassica oleracea L. var. alboglabra

PGIP gene was obtained from Brassica oleracea L. var. alboglabra, named BoPGIP2. The full length of BoPGIP2 gene is 1 102 bp and the exon is 993 bp which encodes a protein of 330 amino acids with a predicted molecular mass of 37.1 kDa, interrupted by one intron of 95 bp in, length. Sequence analysis revealed that it has five potential N-giycosylation sites, two protein kinase C phosphrylation sites, five casin kinase Ⅱ phosphrylation sites and four N-myristoylation sites. The amino acids sequences alignment confirmed that ^145 LRR stucture was highly conserved in all aligned PGIP sequences.

Molecular Characterization, Expression Patterns and Binding Properties of Two Pheromone-Binding Proteins from the Oriental Fruit Moth, Grapholita molesta(Busck)

Insect pheromone-binding proteins （PBPs） play important roles in transporting hydrophobic pheromone components across the sensillum lymph to the surface of olfactory receptors （ORs）. However, the PBPs of the oriental fruit moth, Grapholita molesta, an important destructive pest of stone fruits worldwide, are not well characterized. In this study, two new putative PBP genes, GmolPBP2 and GmolPBP3, were identiifed from G. molesta antennae. The deduced amino-acid sequences of these two putative PBP genes are characteristic of the odorant binding protein family, containing six conserved cysteine residues. The genomic DNA sequence of each gene contained two introns. However, the lengths and positions of the introns differed. RT-PCR analyses revealed that the two GmolPBP genes are only expressed in the antennae of female and male moths. Quantitative real-time PCR indicated that the transcription levels of GmolPBP2 are far greater than those of GmolPBP3 in both female and male antennae. GmolPBP3 showed higher transcription levels in female antennae than in male antennae, while GmolPBP2 showed similar transcription levels in both female and male antennae. The transcript levels of both genes were signiifcantly different in premating and post-coitum individuals, implying that mating affects the process of sex pheromone reception. To better understand the functions, two GmolPBPs were expressed in Escherichia coli, and the ligand binding assays were conducted. Results showed that GmolPBP2 has strong binding afifnities to two sex pheromone components, E8-12：Ac and Z8-12：Ac, as well as weaker binding afifnities to Z8-12：OH and 12：OH. GmolPBP2 also bound some ordinary odor molecules. However, the afifnity of GmolPBP3 to both sex pheromones and ordinary odor molecules was very weak. These results show that GmolPBP2 plays the main role in pheromone discrimination and recognition in the oriental fruit moth.

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.

Infectivity Analysis of Cloned Genomic DNA of P1 Agent in vitro

The ultrastructure of porcine kidney(PK)-15 cells was examined after lipofectamine-aided transfection of the molecular clone of the P1 agent.PK-15 cells transfected with the tandem dimer of the P1molecular DNA clone had numbers of intracytoplasmic inclusions,and a few cells had intranuclear inclusions.Intracytoplasmic inclusions were round to oval and 0.1-0.3μm in diameter,and intranuclear inclusions,which were more electron dense,were of two general types:the first were round and small(0.1μm approximately)and the second were hexagonal and larger(0.4-0.8μm in diameter).Cells transfected with the tandem dimer of the P1 molecular DNA clone tested positive for P1 DNA at passage 5.This is the first report that the P1 molecular clone has infectivity in vitro and it will provide fundamental materials for further study of the biological characterization of P1.

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 and characterization of human age-related NADH oxidase (arNOX) proteins as members of the TM9 superfamily of transmembrane proteins

Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is present in blood and other body fluids. The activity was purified to apparent homogeneity from human urine and resolved by 2-D gel electrophoresis into a series of 24 to 32 kDa components of low isoelectric point. The purified proteins were resistant both to N-terminal sequencing and trypsin cleavage. Cleavage with pepsin revealed peptides corresponding to the TM9 family of transmembrane proteins. Peptide antisera raised to all five members of the human TM9 family sequentially blocked the arNOX activity of human saliva and sera. The soluble truncated N-terminus of the human homolog TM9SF4 was expressed in bacteria. The recombinant protein was characterized biochemically and exhibited ar-NOX activity. The findings identify five arNOX isoforms each of which correspond to one of the five known TM9 family members. The exfoliated soluble arNOX forms are derived from the 24 to 32 kDa N-termini exposed to the cell’s exterior at the cell surface. Each of the shed forms contain putative functional motifs characteristic of ECTO-NOX (ENOX) proteins despite only minimal sequence identity. Our findings identify arNOX as having functional characteristics of ENOX proteins and the TM9 superfamily of proteins as the genetic origins of the five known arNOX isoforms present in human sera, plasma and other body fluids1.

Molecular Cloning and Phylogenetic Analysis of a Chitin Deacetylase Isolated from the Epidermis of the Red Snow Crab <i>Chionoecetes japonicas</i>

Chitin deacetylase (CDA;EC 3. 5. 1. 41) catalyzes the deacetylation of chitin. In this study, we successfully cloned and sequenced a chitin deacetylase gene from the red snow crab Chionoecetes japonicas. By using reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends, we obtained a 2141-bp amplicon containing a chitin deacetylase gene (CjCDA) from the epidermis of C. japonicas. The amplicon contains a 1575-bp open reading frame that is predicted to encode a 525-amino acid protein. The structure predicted from the deduced amino acid sequence included an N-terminal signal peptide, chitin-binding domain (CBD), low-density lipoprotein receptor class A domain (LDL-A), and catalytic domain. Comparative analysis of the deduced amino acid sequence of CjCDA revealed the highest homology (74%) to gastrolith protein 59 of Cherax quadricarinatus. We used RT-PCR to evaluate the expression of CjCDA in various tissues of C. japonicas, and we observed that CjCDA was expressed only in the epidermis. A phylogenetic analysis, using the amino acid sequences of CjCDA and other known chitin deacetylases, showed that CjCDA belonged to a group of crustacean chitin deacetylases. To our knowledge, this is the first study reporting the cDNA cloning of a chitin deacetylase from a crab.

Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.

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, 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.

MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING PARTIAL PUTATIVE MOLT-INHIBITING HORMONE FROM PENAEUS CHINENSIS

Total RNA was extracted from eyestalks of shrimp Penaeus chinensis . Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase polymerase chain reaction (RT PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt inhibiting hormone from shrimp Penaeus japonicu s. A specific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 base pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt inhibiting hormones of P. japonicus . The cDNA could be a partial gene of molt inhibiting hormones from P. chinensis . This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis .

Molecular cloning and characterization of a lipid transfer protein gene(PsLTP1) from Pinus sylvestris(L.)

Plant nonspecific lipid transfer proteins (nsLTPs) are widely distributed through plant kingdom and are characterized by the presence of a central hydrophobic cavity, suitable for binding various hydrophobic molecules. Despite extensive research on nsLTP in different plant species, mostly angiosperm, and the great diversity of physiological processes in which they seem to be involved, their exact functions still remain unclear. Also, very limited experimental data are available on nsLTP in gymnosperm. In this study, we report for the first time on the molecular cloning of nsLTP, from Pinus sylvestris L.(PsLTP1, GenBank accession JN980402.1) and the expression pattern of PsLTP1 during ontogenesis and in response to environmental stress conditions. Total RNA from roots of 7-day old pine seedlings was used to isolate the cDNA clone, corresponding to Scots pine lipid transfer protein. The open reading frame of PsLTP1 consists of 372 bp encoding a protein of 123 amino acids. Amino acid sequence alignment revealed that mature PsLTP1 shares high level of similarity with nsLTP from other conifers and with well-studied nsLTPs from angiosperms. The PsLTP1 contains a 27-amino-acid N-terminal signal sequence and presents all the features of a plant nsLTP. Amino acid comparison analysis and 3D structure prediction showed that PsLTP1 is a type 1 nsLTP. The results of the expression analysis of Scots pine PsLTP1 gene revealed that its transcripts accumulate in actively growing tissues. Furthermore, transcription of PsLTP1 was upregulated in response to cold and salt treatments, and downregulated during acidic, osmotic and water stresses.

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 of IGFBP-1 Gene and Developmental Expression of Its mRNA in Different Tissues of Nanjiang Mongolian Gazelles

The objective of the present study was to investigate the developmental expression patterns of Insulin-like growth factor-binding protein 1 (IGFBP-1) gene in different tissues of postnatal Nanjiang Mongolian Gazelles. Samples of heart, liver, spleen, lung, longissimus dorsi, semimembranosus, m. triceps brachii and biceps muscle of thigh were collected from a total of 36 Nanjiang Mongolian Gazelles at the age of 0, 15, 30, 60, 90 and 120 days after birth (3 males and 3 females at each age). The CDS was sequenced and ontogeny of mRNA levels of IGFBP-1 were measured by real-time fluorescence quantitative RT-PCR. The size of IGFBP-1 ORF was 792 bp encoding 263 amino acid residues, and displayed higher nucleotide/amino acid sequence identities with other ruminants compared to non-ruminants. The levels of IGFBP-1 mRNA in liver were highest (P<0.01), levels were medium in lung, spleen and heart, and the lowest in the muscles; there were no significant differences among the muscles (P>0.05). Three expression patterns of IGFBP-1 mRNA during postnatal growth from birth to day 60 were found: consistently decreasing (liver), fluctuating as increasing then decreasing (heart) or as decreasing then increasing then decreasing (spleen, lung and muscles). The results indicate that the IGFBP-1 gene is highly conserved among species, and liver has the highest expression. It was concluded that IGFBP-1 plays important roles in early postnatal growth and is expressed in a developmental-tissue-dependent manner.

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