Rapid Amplification Of cDNA Ends Method Research Articles

Molecular Characterization of a Vector-Based Candidate Antigen Using the 3'-RACE and Genome Walking Methods and In Silico Analysis of the Correspondent Protein for Vaccine Design and Development.

The main objectives of developing vaccines to prevent malaria transmission are malaria control and preventing the reemergence of the disease in endemic regions. Molecular and in silico characterization of a candidate molecule is the first step in the vaccine design process. Determining the sequence and amplification of full-length cDNA copies from the mRNA transcripts is often challenging. The methods in this chapter provide a protocol for the rapid amplification of cDNA ends (RACE) and genome walking. Carboxypeptidase B2 enzyme from A. stephensi (CPBAs-2) was selected as the target molecule and the steps in its characterization and in silico analysis are explained in this chapter.

Overexpression of BnPCS1, a Novel Phytochelatin Synthase Gene From Ramie (Boehmeria nivea), Enhanced Cd Tolerance, Accumulation, and Translocation in Arabidopsis thaliana.

Phytochelatins (PCs) play important roles in the detoxification of and tolerance to heavy metals in plants. The synthesis of PCs is catalyzed by phytochelatin synthase (PCS), which is activated by heavy metal ions. In this study, we isolated a PCS gene, BnPCS1, from the bast fiber crop ramie (Boehmeria nivea) using the RACE (rapid amplification of cDNA ends) method. The full-length BnPCS1 cDNA is 1,949 bp in length with a 1,518 bp open reading frame (ORF) that encodes a 505 amino acid protein. The deduced BnPCS1 protein has a conserved N-terminus containing the catalytic triad Cys58, His164, Asp182, and a flexible C-terminal region containing a C371C372QETC376VKC379 motif. The BnPCS1 promoter region contains several cis-acting elements involved in phytohormone or abiotic stress responses. Subcellular localization analysis indicates that the BnPCS1-GFP protein localizes to the nucleus and the cytoplasm. Real-time PCR assays show that the expression of BnPCS1 is significantly induced by cadmium (Cd) and the plant hormone abscisic acid (ABA). Overexpression lines of BnPCS1 exhibited better root growth and fresh weight, lower level of MDA and H2O2, and higher Cd accumulation and translocation factor compared to the WT under Cd stress. Taken together, these results could provide new gene resources for phytoremediation of Cd-contaminated soils.

Identification of tung tree FATB as a promoter of 18:3 fatty acid accumulation through hydrolyzing 18:0-ACP

Tung oil extracted from tung tree (Vernicia fordii) seeds is very industrially useful due to its drying quality given by α-eleostearic acid (ESA). Previous comparative transcriptome analysis revealed that a putative tung tree FATB gene (VfFATB) encoding fatty acyl-ACP (acyl carrier protein) thioesterase B (FATB) was significantly unregulated during oil synthesis stage. Fatty acyl-ACP thioesterases determine the length of the fatty acid carbon chains through releasing acyl chain from acyl-ACP. For a better understanding of the mechanism of α-eleostearic acid biosynthesis, it would be desirable to further characterize the function of VfFATB gene involved in fatty acid synthesis pathways in tung tree seeds. In view of this, VfFATB gene was cloned by RACE (rapid amplification of cDNA ends) method to further characterize its functions. Expression profiles of VfFATB were positively correlated with oil contents in tung tree seeds. VfFATB is localized in chloroplasts and restored the abnormal development of Arabidopsis fatb mutants. Fatty acid profiles in VfFATB-transformed E. coli (Escherichia coli) cells and Arabidopsis fatb mutant plants showed that the presence of VfFATB led to accumulation of 18:0 and 18:3 fatty acids in vivo. In vitro thioesterase assay using crude extract from seed tissue of tung tree and purified VfFATB protein further indicated that 18:0-ACP rather than 18:3-ACP was the main substrate of VfFATB. The above results strongly suggest that VfFATB, an 18:0-ACP-preferring thioesterase, is essential for plant normal development and promotes the accumulation of 18:3 fatty acid through hydrolyzing 18:0-ACP to produce free 18:0 fatty acid as the substrate of downstream related enzymes. VfFATB functions in chloroplast. VfFATB expression level is positively correlated with tung oil content. VfFATB is essential for plant normal development. VfFATB is an 18:0-ACP-preferring thioesterase.

Characterization of the stearoyl-ACP desaturase gene (PoSAD) from woody oil crop Paeonia ostii var. lishizhenii in oleic acid biosynthesis

Paeonia ostii var. lishizhenii has been approved as a woody oil crop with the outstanding characteristic of abundant α-linolenic acid (C18:3, ALA) in its seed oil. The stearoyl-ACP desaturase gene (SAD) regulates the first key step from stearic acid (C18:0, SA) to oleic acid (C18:1, OA) in the ALA biosynthetic pathway, but its functional characterization in P. ostii var. lishizhenii is absent to date. In this study, a key PoSAD gene (1719 bp in length) was acquired from endosperm of P. ostii var. lishizhenii by transcriptome sequencing analysis and the RACE (rapid-amplification of cDNA ends) method. Bioinformatic analysis of the PoSAD protein showed high homology (ranging from 90.4% to 94.4%) and similar physical and chemical properties to SAD from other higher plants, indicating that it encodes a putative stearoyl-ACP desaturase. Analysis of cis-acting elements found several endosperm tissue-specific motifs; i.e., one Prolamin box, thirteen DOFCOREs and one RY repeat in its promoter. The results of the qRT-PCR experiments verified that PoSAD was most highly expressed in developing endosperm at 59 days after pollination (53.7 times that in shoots) compared with that in roots (1.4 times), stems (2.5 times), leaves (3.1 times), petals (13.1 times) and stamens (46.0 times). Meanwhile, the fatty acid contents in P. ostii var. lishizhenii endosperm at seven growth stages were compared with variation in PoSAD expression. Heterologous expression of PoSAD significantly decreased SA and increased OA content, which effectively reduced the ratios of SA to OA in Saccharomyces cerevisiae and Arabidopsis thaliana. However, contents and ratios of palmitic acid (C16:0) and palmitoleic acid (C16:1) were stable in transgenic yeast, and palmitoleic acid remained absent in transgenic A. thaliana seeds. These results illustrate that PoSAD plays an essential role in endosperm development of P. ostii var. lishizhenii, strictly in catalysis of SA desaturation and OA biosynthesis but without functioning in PA desaturation. The results contribute to our understanding of the characterization of PoSAD in OA biosynthesis in P. ostii var. lishizhenii.

Full-length 5'RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum

Covalently closed circular DNA (cccDNA) is the episomal form of the HBV genome that stably resides in the nucleus of infected hepatocytes. cccDNA is the template for the transcription of 6 major viral RNAs, i.e. preC, pg, preS1/2, S and HBx RNA. All viral transcripts share the same 3' end and are all to various degrees subsets of each other. Especially under infection conditions, it has been difficult to study in depth the transcription of the different viral transcripts. We thus wanted to develop a method with which we could easily detect the full spectrum of viral RNAs in any lab. We set up an HBV full-length 5'RACE (rapid amplification of cDNA ends) method with which we measured and characterized the full spectrum of viral RNAs in cell culture and in chronically infected patients. In addition to canonical HBx transcripts coding for full-length X, we identified shorter HBx transcripts potentially coding for short X proteins. We showed that interferon-β treatment leads to a strong reduction of preC and pgRNAs but has only a moderate effect on the other viral transcripts. We found pgRNA, 1 spliced pgRNA variant and a variety of HBx transcripts associated with viral particles generated by HepAD38 cells. The different HBx RNAs are both capped and uncapped. Lastly, we identified 3 major categories of circulating RNA species in patients with chronic HBV infection: pgRNA, spliced pgRNA variants and HBx. This HBV full-length 5'RACE method should significantly contribute to the understanding of HBV transcription during the course of infection and therapy and may guide the development of novel therapies aimed at targeting cccDNA. Especially under infection conditions, it has been difficult to study the different hepatitis B virus transcripts in depth. This study introduces a new method that can be used in any standard lab to discriminate all hepatitis B viral transcripts in cell culture and in the serum of patients.

Obtaining vacuolar protein sorting 34 gene in Sporothrix globosa by RACE method and analysis of its expression during the mycelium-to-yeast transition

Objective To obtain the full-length sequence of the vacuolar protein sorting 34 coding gene (vps34) of Sporothrix globosa (S.globosa) and to investigate the role of vps34 gene during the phase transition from mycelium to yeast in S. globosa. Methods The 3′ end and 5′ end of the vps34 gene of S. globosa were amplified by rapid amplification of cDNA ends (RACE). The obtained sequences were spliced and analyzed by bioinformatics software. Quantitative reverse transcription PCR (qRT-PCR) was used to analyze the expression of vps34 gene in mycelial and yeast phases. Results The vps34 gene of S. globosa was 3 228 bp in length. The coding sequence was 3 000 bp and encoded 999 amino acids with a molecular mass of 111.49×103 and an isoelectric point of 6.38. It contained three domains including C2_PI3K_class_Ⅲ, PI3Ka_Ⅲ and PI3Kc_Ⅲ. The results of qRT-PCR showed that the expression of vps34 gene in yeast-phase S. globosa was higher than that in mycelial phase at 24 h (P<0.05), and the greatest difference between them was observed at 48 h (P<0.01). Conclusions Vps34p participates in the process of dimorphic transformation of S. globosa. The obtainment of the full-length vps34 gene of S. globosa lays the foundations for further study on the function of Vps34p. Key words: Sporothrix globosa; Sporotrichosis; Vacuolar protein sorting 34; Rapid amplification of cDNA ends; Quantitative reverse transcription PCR; Dimorphic transformation

Cloning and characterization of MdGST1 from red apple leaves

Glutathione S-transferase (GST) is involved in the downstream steps of the anthocyanin biosynthesis pathway in plants. However, the gene(s) encoding this enzyme have not been isolated from apple (Malus × domestica Borkh.) yet. We isolated a gene encoding GST from leaves of the red-fleshed apple variety ‘Royalty’ by full cDNA library sequencing and the 3′ rapid-amplification of cDNA ends method, and designated it MdGST1. In total, seven different MdGST1 transcripts were found. These had three different untranslated but identical protein-coding regions. Phylogenetic analysis showed that MdGST1 is a TT19-type GST, which is involved in anthocyanin transport. qRT-PCR analyses showed that the transcript level of MdGST1 was much higher in red leaves than in bagged or green leaves. When MdGST1 was introduced into a non-pigmented mutant of Arabidopsis, the transformants showed a visible purple phenotype in leaves and stems. Our results suggest that MdGST1 plays a role in anthocyanin biosynthesis. This information will help to improve the understanding of the mechanism of apple coloration.

Molecular Cloning of the B4GALNT2 Gene and Its Single Nucleotide Polymorphisms Association with Litter Size in Small Tail Han Sheep.

Simple SummaryIn French Lacaune sheep, the B4GALNT2 (beta-1, 4-N-acetyl-galactosaminyl transferase 2) gene was considered as the potential gene for a FecL (mutation), which regulates the ovine ovulation rate. Three specific mutation sites linked with the FecL mutation have not been previously found in 11 sheep breeds. However, two mutations of g.36946470C > T and g.36933082C > T in the exon of B4GALNT2 were found to have had a significant effect on the litter size in the first parity for Small Tail Han (STH) Sheep (p < 0.05). B4GALNT2, which is mainly expressed in ovine ovary, also plays an important role in sheep reproduction. Furthermore, we discovered two transcription start sites (TSS) of B4GALNT2 in its 5′-flanking region in ovine granule cells in vitro.A new fecundity gene named the FecL (mutation), which regulates the ovulation rate, was discovered in French Lacaune sheep. The B4GALNT2 (beta-1, 4-N-acetyl-galactosaminyl transferase 2) gene was considered as the potential FecL mutation gene. This study explores whether the effect of the FecL mutation exists in other sheep breeds, and the features of the B4GALNT2 gene in terms of the molecular structure and its expression profile. Using Sanger sequencing, we found that high and low fecundity breeds from among 11 measured sheep breeds all had no variation in the three specific mutation sites, which were linked with the FecL mutation. However, two mutations of g.36946470C > T and g.36933082C > T in the exon of B4GALNT2 had a significant effect on litter size in the first parity for Small Tail Han (STH) Sheep (p < 0.05). Two transcription start sites (TSS) of B4GALNT2 in its 5′-flanking region were discovered in ovine granule cells in vitro, through the RACE (Rapid amplification of cDNA ends) method. Except for in the kidney and oviduct, no significant difference in expression levels had been found between STH sheep and Tan sheep breeds. The B4GALNT2 gene, as a candidate for FecL, may have a relationship with the differences in litter size in STH sheep. B4GALNT2 is mainly expressed in the ovine ovary, which also suggests that B4GALNT2 plays an important role in sheep reproduction.

Capping-RACE: a simple, accurate, and sensitive 5' RACE method for use in prokaryotes.

Rapid amplification of cDNA ends (RACE) is a prevalent technique used to obtain the 5′ ends of transcripts. Several different 5′ RACE methods have been developed, and one particularly simple and efficient approach called CapFinder relies on the 5′ cap-dependent template-switching that occurs in eukaryotes. However, most prokaryotic transcripts lack a 5′ cap structure. Here, we report a procedure to capture primary transcripts based on capping the 5′ triphosphorylated RNA in prokaryotes. Primary transcripts were first treated with vaccinia capping enzyme to add a 5′ cap structure. First-strand cDNA was then synthesized using Moloney murine leukaemia virus reverse transcriptase. Finally, a template-switching oligonucleotide with a tail containing three ribonucleic acid guanines was hybridized to the cDNA 3′ poly(C) and further used as template for reverse transcriptase. It is oligonucleotide sequence independent and is more sensitive compared to RLM-RACE. This approach specifically identified the transcription start sites of ompA, sodB and shiA in Escherichia coli and of ompA, rne and rppH in Brucella melitensis. Furthermore, we also successfully identified the transcription start sites of small noncoding genes ryhB and micC in E. coli and bsnc135 and bsnc149 in B. melitensis. Our findings suggest that Capping-RACE is a simple, accurate, and sensitive 5′ RACE method for use in prokaryotes.

Expression and Antitumor Function of Latcripin-4 RCC1 and ANK Domain Protein on HepG2 from the Shiitake Medicinal Mushroom, Lentinus edodes C91-3 (Agaricomycetes), Transcriptome.

The recombinant protein of Latcripin-4 regulator of chromosome condensation 1 (RCC1) and ankyrin (ANK) domains were expressed and the antitumor activity of Latcripin-4 on HepG2 cells was studied. First, the Latcripin-4 transcript was selected from the medicinal mushroom Lentinus edodes C91-3 transcriptome by bioinformatics. Then the full-length gene of Latcripin-4 was isolated with 3'-full rapid amplification of cDNA ends (RACE) and 5'-full RACE methods according to the transcriptome. The RCC1 and ANK domains from the full-length gene were selected and inserted into the expression vector pET-32a (+) and expressed in Escherichia coli Rosetta-gami (DE3). Western blotting indicated that the protein was expressed successfully. The biological function of Latcripin-4 RCC1 and ANK domain protein on HepG2 cells was studied with the CCK-8 assay. All results demonstrated that Latcripin-4 RCC1 and ANK domain protein can inhibit the growth of human HepG2 liver cancer cells, which brings new insights to identifying antitumor proteins from medicinal food for cancer therapy.

Grass carp (Ctenopharyngodon idellus) invariant chain of the MHC class II chaperone protein associates with the class I molecule

The invariant chain (Ii) is an important immune molecule, as it assists major histocompatibility complex (MHC) class II molecules to present antigenic peptides. The relationship between the Ii and MHC molecules in teleosts remains poorly understood. This study focused on the molecular structure of grass carp Ii (gIi), its organ distribution, correlations with gene transcription, and the association with MHC. gIi cDNA was cloned using designed degenerate primers and the rapid amplification of cDNA ends method (RACE). The gIi sequence was 92%–96% similar to that of other teleosts, but only 52%–67% similar to that of mammals, respectively. The gIi gene was distributed in all 12 organs examined by PCR. The gIi gene transcription levels were markedly higher in organs enriched with immune cells than in other organs (P < 0.01). Moreover, positive correlations were detected between transcription levels of the gIi and gMhcI or II genes in different organs (r = 8.415–8.523, P = 0.001). The gIi co-localized on endomembrane systems with either class I or II molecules in co-transfected cells observed by a laser confocal. Further testing confirmed that the gIi bound gMHCI and II molecules. Taken together, these results indicate that the gIi is associated with MHC class I and II molecules, suggesting homology of both MHC molecules.

Molecular cloning, sequence identification, polymorphism and association of the porcine &amp;lt;i&amp;gt;SPATS2L&amp;lt;/i&amp;gt; gene

Abstract. Spermatogenesis-associated, serine-rich 2-like (SPATS2L) is an important reproduction-related gene. In this study, the full-length cDNA sequence of the porcine SPATS2L gene was cloned through the RACE (rapid amplification of cDNA ends) method. The porcine SPATS2L gene encodes a protein of 559 amino acids which shares high homology with the SPATS2L proteins of seven species: dog (94 %), white-tufted-ear marmoset (91 %), human (92 %), rhesus monkey (92 %), horse (92 %), rat (88 %) and mouse (88 %). This novel porcine gene was assigned to GeneID: 100415809. The phylogenetic analysis revealed that the porcine SPATS2L gene has a close genetic relationship with the canine SPATS2L gene. PCR-Pst I-RFLP was established to detect GU474997:c.1687 C &gt; T substitution of porcine SPATS2L gene mRNA, and eight pig breeds displayed obvious genotype and allele frequency differences at this mutation locus. Association of this single-nucleotide polymorphism (SNP) with litter size traits was assessed in Large White (n = 100) and Landrace (n = 100) pig populations, and results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White sows and Landrace sows (P &lt; 0.01). Therefore, SPATS2L gene could be an useful candidate gene in selection for increasing litter size in pigs.

De Novo Sequencing and Analysis of the Safflower Transcriptome to Discover Putative Genes Associated with Safflor Yellow in Carthamus tinctorius L.

Safflower (Carthamus tinctorius L.), an important traditional Chinese medicine, is cultured widely for its pharmacological effects, but little is known regarding the genes related to the metabolic regulation of the safflower’s yellow pigment. To investigate genes related to safflor yellow biosynthesis, 454 pyrosequencing of flower RNA at different developmental stages was performed, generating large databases.In this study, we analyzed 454 sequencing data from different flowering stages in safflower. In total, 1,151,324 raw reads and 1,140,594 clean reads were produced, which were assembled into 51,591 unigenes with an average length of 679 bp and a maximum length of 5109 bp. Among the unigenes, 40,139 were in the early group, 39,768 were obtained from the full group and 28,316 were detected in both samples. With the threshold of “log2 ratio ≥ 1”, there were 34,464 differentially expressed genes, of which 18,043 were up-regulated and 16,421 were down-regulated in the early flower library. Based on the annotations of the unigenes, 281 pathways were predicted. We selected 12 putative genes and analyzed their expression levels using quantitative real time-PCR. The results were consistent with the 454 sequencing results. In addition, the expression of chalcone synthase, chalcone isomerase and anthocyanidin synthase, which are involved in safflor yellow biosynthesis and safflower yellow pigment (SYP) content, were analyzed in different flowering periods, indicating that their expression levels were related to SYP synthesis. Moreover, to further confirm the results of the 454 pyrosequencing, full-length cDNA of chalcone isomerase (CHI) and anthocyanidin synthase (ANS) were cloned from safflower petal by RACE (Rapid-amplification of cDNA ends) method according to fragment of the transcriptome.

Novel 5'/3'RACE Method for Amplification and Determination of Single-Stranded RNAs Through Double-Stranded RNA (dsRNA) Intermediates.

To acquire the full-length sequences and to determine the 5'/3'ends of the RNA genomes and mRNA transcripts using the rapid amplification of cDNA ends (RACE) protocols-via cDNA or mRNA templates-are a great challenge. This 4-steps RNA-based RACE method uses different ways to determine the RNA ends through a double-stranded (ds) RNA intermediate (dsRNA-RACE). In the first step a complementary RNA strand is synthesised by Phi6 RNA replicase enzyme next to the template ssRNA forming a dsRNA intermediate. The following steps include adapter ligation, nucleic acid purification and two classical methods with minor modifications reverse transcription and polymerase chain reaction. The dsRNA-RACE protocol could be used in wide variety of ssRNA (cellular, viral, bacterial, etc.) templates in the field of microbiology and cellular biology and suitable for the amplification of full-length RNAs including the 5'/3'ends. This is a novel, expansively utilizable molecular tool with fewer disadvantages than the existing 5'/3'RACE approaches.

A partial genomic characterization and up-regulation of HSP90 by imidazole derivative KK-42 in the shrimp Penaeus vannumei

Aim: HSP90, one of the most important and multifunctional stress proteins in cells, plays important role in cell self-protection and environmental adaptability in crustacean. Our pre- vious study showed that application of imidazole derivative KK-42 can increase survival rate of Penaeus vannumei. In an attempt to research the possible molecular mechanism of KK-42 increasing survival rate in P. vannumei, one HSP90 cDNA sequence from the hepatopancreas was first isolated and then its spatiotemporal expressions were analyzed. Material and Methods: The HSP90 was cloned from the hepatopancreas of P. vannumei using the rapid amplification of cDNAends methods and its sequence was analyzed with biological software. Expression level was determined by real-time PCR. Results: The full-length cDNA contains a 2163 bp open reading frame encoding a predicted protein of 721 amino acids. The P. vannumei HSP90 protein sequence is most closely related to other HSP90 enzymes, the higher mRNA level is detected in hepatopancreas. The hepato- pancreas HSP90 expression is significantly induced by KK-42 and increases more than 69.1 %, even up to 505.5 % and 481.7 % on day 2 and day 3, respectively. Conclusion: The up-regulation of HSP90 expression induced by KK-42 is beneficial to imp- rove adaptability that is likely one of the molecular mechanisms of KK-42 increasing survival

Cloning and expression analysis of the gastric carcinoma-related gene, ELCOX3

Gastric cancer is a pathological process of an accumulation of multigene and multistage mutations. A new gene segment, MDSCBC11, has been previously obtained using a gene chip and is negatively associated with gastric cancer. The present study aimed to clone the full cDNA sequence of the MDSCBC11 segment and to detect its expression in gastric carcinomas and normal gastric mucosa. Multiple-tissue northern blots revealed that the new MDSCBC11-represented gene was expressed as two transcripts that were 0.8 kb and 1.5 kb in size. The cDNA sequence of the smaller transcript was 822 bp, created by 5′ rapid amplification of cDNA ends (RACE) and 3′ RACE methods. A bioinformatics analysis indicated that the deduced amino acid sequence of MDSCBC11 had a 99% homology with the cytochrome c oxidase III (COX3) gene in the mitochondria. A total of 46 cases of gastric carcinomas, adjacent gastric mucosa and normal gastric mucosa were individually collected, and the mRNA expression of the ELCOX3 gene was detected by RT-PCR. ELCOX3 mRNA was expressed in all 46 cases of the normal gastric mucosa. The expression levels of ELCOX3 mRNA in the gastric carcinomas were lower compared with that of the adjacent and normal gastric mucosa (P<0.05), with the percent of downregulation at 23.91% (11/46 cases). The downregulation of ELCOX3 gene expression was associated with the development of human gastric carcinomas.

Molecular Cloning and Characteristics of Catalase cDNA from Chinese Soft-shelled Turtle (Pelodiscus sinensis)

A catalase cDNA was cloned from the liver of the Chinese soft-shelled turtle(Pelodiscus sinensis) using reverse transcription-polymerase chain reaction(RT-PCR) with degenerate primers. Both 3’-and 5’-untranslated regions were isolated by the rapid amplification of cDNA ends method(RACE). Analysis of nucleotide sequence revealed that the catalase cDNA clone consisted of 2173 bp with an open reading frame of 1587 bp encoding a protein of 528 amino acids. The calculated molecular mass of the mature protein is 59.8 kDa with an estimated pI of 6.84. The peroxisomal targeting signal SNL at the C-terminal and two putative N-glycosylation sites NLSV and NVSQ were found in the catalase. Sequence comparison showed that this catalase, deduced by the amino acid sequence, had high similarity and identity with those of vertebrates recorded in GenBank. Four functional domains and conserved amino acids responsible for binding heme and NAPDH including four essential residues were observed. The 3-D homology model of the turtle catalase was predicted by SwissModel based on the relative domains of bovine catalase structure(PDB ID: 3rgp). The mRNA expression and enzyme activities in liver, brain, spleen, kidney, heart, gut, lung and muscle were investigated, and the results showed that the mRNA and enzyme activities of catalase in these tissues were species-specific.

A Novel Apoptosis Correlated Molecule: Expression and Characterization of Protein Latcripin-1 from Lentinula edodes C91–3

An apoptosis correlated molecule—protein Latcripin-1 of Lentinula edodes C91–3—was expressed and characterized in Pichia pastoris GS115. The total RNA was obtained from Lentinula edodes C91–3. According to the transcriptome, the full-length gene of Latcripin-1 was isolated with 3′-Full Rapid Amplification of cDNA Ends (RACE) and 5′-Full RACE methods. The full-length gene was inserted into the secretory expression vector pPIC9K. The protein Latcripin-1 was expressed in Pichia pastoris GS115 and analyzed by Sodium Dodecylsulfonate Polyacrylate Gel Electrophoresis (SDS-PAGE) and Western blot. The Western blot showed that the protein was expressed successfully. The biological function of protein Latcripin-1 on A549 cells was studied with flow cytometry and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-tetrazolium Bromide (MTT) method. The toxic effect of protein Latcripin-1 was detected with the MTT method by co-culturing the characterized protein with chick embryo fibroblasts. The MTT assay results showed that there was a great difference between protein Latcripin-1 groups and the control group (p < 0.05). There was no toxic effect of the characterized protein on chick embryo fibroblasts. The flow cytometry showed that there was a significant difference between the protein groups of interest and the control group according to apoptosis function (p < 0.05). At the same time, cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. The work demonstrates that protein Latcripin-1 can induce apoptosis of human lung cancer cells A549 and brings new insights into and advantages to finding anti-tumor proteins.

Differential expression of cysteine protease inhibitor (CPI) gene of Polygonum sibiricum Laxm. leaves, stems and rhizomes in response to NaHCO3

A cDNA clone which encodes a cysteine protease inhibitor gene, named PsCPI, has been identified in Polygonum sibiricum Laxm. by the rapid amplification of cDNA ends method (RACE). Analysis of the nucleotide sequence reveals that the PsCPI gene cDNA clone consists of 437 bp, containing 38 bp in the 5' untranslated region, 336 bp in the open reading frame (ORF) encoding 111 amino acids and 63 bp in 3' untranslated region. The gene accession nucleotide sequence number in GenBank is GU562861. Expression analysis by real-time quantitative PCR reveals that the PsCPIgene is expressed in leaves, stems and rhizomes. The result shows that PsCPI gene expression can be induced by 3% NaHCO3 in the three earlier mentioned tissues, but there are different expression modes in leaves, stems and rhizomes under the salinity-alkalinity stress. Keywords: Polygonum sibiricum Laxm., Polygonum sibiricum Laxm cysteine protease inhibitor, rapid amplification of cDNA ends, real-time polymerase chain reaction, gene expression.

Identification and expression pattern of ribosomal L5 gene in common wheat ( Triticum aestivum L.)

The full-length cDNA sequence (1158 bp) encoding a ribosomal L5 protein, designated as TaL5, was firstly isolated from common wheat ( Triticum aestivum L.) using the rapid amplification of cDNA ends method (RACE). The open reading frame (ORF) of TaL5 gene was 906 bp, and its deduced amino acid sequence (301 residues) shared high similarity to those of other higher plant L5 proteins. TaL5 protein contained a putative 5S binding region (74 amino acids). TaL5 DNA sequence was further cloned, and sequence analysis showed that it contained 7 introns and 8 exons. Predicated using TargetP software, TaL5 protein was putatively located in mitochondria and contains a transit peptide of 12 amino acids. During grain filling period, temporal expression pattern of TaL5 gene was approximately consistent with the rates of starch accumulation in grains. Additionally, TaL5 gene was dramatically induced by salt, drought and freezing stresses, exogenous abscisic acid (ABA) and salicylic acid (SA) in wheat seedlings. These implied that TaL5 gene could function in growth, development and abiotic stresses in wheat plants.