Papers by Keyword: Molecular Cloning

Abstract: The protease gene Arenicola cristata was cloned, sequenced, and expressed in E.coli and the activity of the recombinant protein was investigated. The full-length cDNA of 880 bp consisted of an ORF of 813bp encoding 270 amino acids. This protease contained highly conserved GDSGGP sequence and revealed high homology with trypsin-like proteases of serine family. The recombinant protein for the active form of the protease was purified by affinity chromatography. The activity analysis of the recombinant protein suggested that it was probably a plasminogen activator.

Abstract: To study the relation between α-actinin1 gene and reproduction traits, 442 different varieties cattle were used as experimental populations. PCR-RFLP and DNA sequencing was used to detect the SNP and genotype analysis in these populations. RT-PCR technique was used to isolate cDNA of bovine α-actinin1gene. The association analysis of this ApaⅠ locus polymorphism with reproduction traits were analyzed by using GLM procedure. The result showed that the mutation of G/A,using ApaⅠ endonuclease, was detected in 10th intron of at the α-actinin1 gene locus. The mutation caused ApaⅠpolymorphism in group, there were α-actinin1-G and α-actinin1-A genotypes.The statistical analysis of α-actinin1 163-SNP with reproduction traits showed that there was no associated with the number of calving.The sequence of cDNA of bovine α-actinin1 was 2 911bp. It contained an open reading frame of 2 679 bases (from 53bp to 2 731bp) which encoded an 892 amino acid.

Abstract: Phosphofructokinase (PFK), which catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6- bisphosphate, a key regulatory step in the glycolytic pathway. The former studies indicated the PFK could enhance glycolysis. The full-length cDNA encoding PFK was obtained from oleaginous microalgae Dunaliella parva, which include 1572 bp open reading frame (ORF), 254 bp 5′-untranslated sequence and 323 bp 3′-untranslated sequence. Dunaliella parva PFK showed the highest sequence similarity with the PFK from Chlamydomonas reinhardtii and Volvox carteri. The Dunaliella parva PFK also showed wide similarity with other species.

Abstract: The vast majority of photosynthetic organisms utilize monovinyl chlorophyll for their photosynthetic reactions. For the biosynthesis of monovinyl chlorophyll, the reduction of the 8-vinyl group which is located on the B-ring of the macrocycle is essential. 3,8-Divinyl protochlorophyllide a 8-vinyl reductase (DVR) catalyzes the reduction of 8-vinyl group on the tetrapyrrole to an ethyl group, which is necessary for monovinyl chlorophyll (Chl) synthesis. The former studies indicated the DVR could enhance photosynthesis. The full-length cDNA encoding DVR was obtained from oleaginous microalgae Dunaliella parva, which include 1326 bp open reading frame (ORF), 22 bp 5′-untranslated sequence and 383 bp 3′-untranslated sequence. Dunaliella parva DVR showed the highest sequence similarity with the DVR from Chlamydomonas reinhardtii and Volvox carteri. The Dunaliella parva DVR also showed wide similarity with other species.

Abstract: Microalgae could modulate their photosynthesis to acclimate to CO2-limiting stress through inducing a carbon-concentrating mechanism (CCM) which included carbonic anhydrases and inorganic carbon (Ci) transporters. However, Ci-specific transporters have not been well described in algae so far. The former studies indicated that low CO2-inducible protein (Lci) played a significant role in the CCM, could increase Ci uptake and intracellular Ci accumulation under low CO2 conditions. The full-length cDNA encoding Lci was obtained from oleaginous microalgae Dunaliella parva, which include 1302 bp open reading frame (ORF), 255 bp 5′-untranslated sequence and 447 bp 3′-untranslated sequence. Similarity analysis revealed that the highest identity was found among Dunaliella parva, Chlamydomonas reinhardtii and Volvox carteri. The Dunaliella parva Lci also showed wide similarity with other species.

Abstract: Malic enzymes are a class of oxidative decarboxylases which catalyze the oxidative decarboxylation of malate to pyruvate and carbon dioxide. the former studies on lipid pathways and genetic engineering test for enhanced lipid synthesis suggests that ME are the most promising targets gene for enhanced lipid synthesis. The full-length cDNA encoding NADP malic enzyme was obtained from oleaginous microalgae Dunaliella parva, which include 1293 bp open reading frame (ORF) and 26 bp 3′-untranslated sequence. NCBI-CD search revealed that there are two mainly domains predicted in the Dunaliella parva ME protein. In addition, a 724 bp promoter was obtained. The potential regulatory elements associated with hormone and light responses were also found in the ME promoter region. Similarity analysis revealed that the highest identity was found between Dunaliella parva and Chlamydomonas reinhardtii. The Dunaliella parva ME also showed wide similarity with other species.

Abstract: Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of AMP, phosphoenolpyruvate (PEP) and pyrophosphate (PPi) to ATP, pyruvate and inorganic phosphate (Pi). It is a key enzyme in gluconeogensis and photosynthesis that is responsible for reversing the reaction performed by pyruvate kinase in Embden-Meyerhof-Parnas glycolysis. A cDNA clone for the Dunaliella parva PPDK was isolated by sequencing. Then the 3'-RACE and 5'-cDNA amplification were conducted based on the obtained sequence. The molecular characterization of the PPDK gene was described.The Dunaliella parva PPDK gene cDNA sequence was 3249 bp, which contained 2595 bp coding region and 654 bp 3'-untranslated regions. The deduced amino acid sequence of Dunaliella parva PPDK showed significant homology to the known PPDK from Volvox carteri and Chlamydomonas reinhardtii. This study provided foundation for further research on the function analysis and overexpression of PPDK genes. To our knowledge this is the first reported.

Abstract: The glycoprotein H(gH) gene homologue of pseudorabies virus wild strain SL(PRV-SL) was cloned by degenerate polymerase chain reaction (PCR) from PRV infected vero cells. Bioinformatics analysis was performed to predict the characteristics of gH, the results indicated that gH gene encoded a polypeptide, molecular mass of 71.95kda and comprising 687 amino acids. The protein had one signal peptide between 1 and 24AA, one transmembrane region 645-667AA, 27 antigenic determinants and the hydrophobicity between -2.117 and 3.178. The prediction of secondary structure showed that gH had consisted highly of alpha helix(Hh) and random coil(Cc). The phylogenetic tree showed that PRV-SL was similar to others of the Alphaherpesvirinae, and that gH had higher conservative in Alphaherpesvirinae. Cloning and analysis of gH gene laid foundation to further research and exploitation.

Abstract: A 561-bp complete open reading frame of the duck enteritis virus(DEV) UL55 gene (GenBank accession No EU071034) was isolated in our laboratory and amplified by common PCR using a pair of specific primers .PCR product containing this ORF was cloned into the vector of PMD18-T. Four evolutionary analysis approaches were used to construct phylogenetic trees of DEV in relation to herpesviruses based on the nucleotide sequences. Bootstrap was used as statistic method to testify the reliablity of the constructed phylogenetic tree. Phylogenetic trees indicated that DEV and other herpesviruses generated from one ancestor and DEV were grouped into the subfamily Alphaherpesvirinae. In our result, DEV showed a close relationship with the genus Mardivirus, but formed a single branch. Partial genomic organization and phylogenetic analysis in the present study provides evidence that DEV was a member of the subfamily Alphaherpesvirinae and should be assigned as an individual genus or group.