Gene cloning, heterologous expression and activity identification of latroeggtoxin-Ⅵ / 生物工程学报

One of the distinct characters of Latrodectus tredecimguttatus is that its toxic components exist not only in the venomous glands, but also in the tissues outside the venomous glands and even in the eggs. Investigation on the toxins outside the venomous glands can deepen our understanding of spider toxins and discover new lead molecules with important application prospects. In order to explore the low-abundance proteinaceous toxins in the L. tredecimguttatus eggs, we used bioinformatic strategies to mine a gene sequence encoding a peptide toxin from the transcriptome of L. tredecimguttatus eggs, and then heterologously expressed the gene successfully with a 3'-RACE combined with nest PCR strategy. Biological activity analyses indicated that the expressed peptide toxin, named latroeggtoxin-Ⅵ (LETX-Ⅵ), could inhibit Na⁺ channel currents in ND7/23 cells and promote dopamine release from PC12 cells, without obvious toxicity against Periplaneta americana and bacteria as well as fungi including Staphylococcus aureus and Candida albicans, demonstrating that LETX-Ⅵ is a mammal-specific neurotoxin with a potential application prospect in development of the tool reagents for neurobiological study and the drugs for treating related diseases.

Obtaining vacuolar protein sorting 34 gene in Sporothrix globosa by RACE method and analysis of its expression during the mycelium-to-yeast transition / 中华微生物学和免疫学杂志

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. -ethods 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 3228 bp in length. The coding sequence was 3000 bp and encoded 999 amino acids with a mo-lecular 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 dimor-phic transformation of S. globosa. The obtainment of the full-length vps34 gene of S. globosa lays the founda-tions for further study on the function of Vps34p.

Full-length cDNA cloning of AsJAZ1 gene and damage-induced expression analysis in Aquilaria sinensis callus / 中草药

Objective: To clone the full-length cDNA of jasmonate-zim-domain protein (JAZ) gene in Aquilaria sinensis to provide the basic information for further study on gene function in sesquiterpenes biosynthesis pathway. Methods: With the total RNA as template, the full-length cDNA of JAZ in A. sinensis was cloned through rapid amplification of cDNA ends (RACE) technique and reverse transcription PCR (qRT-PCR) method. The bioinformatics of the JAZ gene was analyzed as well. The expression of this gene was detected by qRT-PCR method with MeJA and mechanical wounding treatment in A. sinensis callus. Results: The full-length cDNA (1 507 bp) of JAZ gene was named AsJAZ1; GenBank registration number was KP677281. AsJAZ1 was obtained with an open reading frame (ORF) of 990 bp and encoding 330 amino acids. The relative molecular mass of AsJAZ1 calculated was 34 280, and the isoelecric point was 6.89. Real time PCR results indicated that both MeJA treatment and mechanical wounding could stimulate the increase of mRNA expression of AsJAZ1; There was a sharp rise at 0.5 h with about 27 times higher than the control (without MeJA treatment) with MeJA treatment, then dropped significantly. In mechanical wounding treatment, the highest peak presented in 2 h about 17 times compared to the control, then dropped significantly too. The expression of AsJAZ1 gene returned to be normal in 24 h. Conclusion: We have obtained the full-length cDNA sequence of AsJAZ1 gene firstly, which was extremely sensitive to wounding and responded to the early damage.

Cloning and bioinformatics analysis of cytochrome P450 reductase gene in Poria cocos / 中草药

Objective: To clone cytochrome P450 reductase (PcCPR) gene from Poria cocos and to characterize with bioinformatics methods. Methods: According to annotated transcriptome of P. cocos, the PcCPR gene was cloned through RACE, and the genomic DNA sequence was further obtained through PCR. The characteristics of the encoded protein were analyzed using bioinformatics, the 3D structure of the protein was modeled with I-TASSER server, and phylogenetic tree of CPR was carried out with MEGA. Results: The 2 514 bp full-length cDNA sequence (GenBank Accession No. KP768251) and the 5 292 bp genomic DNA sequence (GenBank Accession No. KP896487) of PcCPR was obtained, which contained four exons and three introns. PcCPR encoded a protein with 732 amino acids. The protein was predicted to be an unstable hydrophilic protein with calculated molecular weight of 81 147 and isoelectric point 5.39. PcCPR does not have a signal peptide but has a transmembrane segment (aa residues 7 to 22), and it is anchored to endoplasmic reticulum. There are two flavin binding domains and many predicted FAD and NADP binding sites, these sites are adjacent respectively at 3D structure level. The homologous analysis indicates that PcCPR has a higher similarity with CPR from basidiomycetes than CPR from ascomycetes. Conclusion: The PcCPR was successfully cloned, which will provide a foundation for researches on PcCPR and PcCPR associated metabolic.

Cloning, expression and characterization of COI1 gene (AsCOI1) from Aquilaria sinensis (Lour.) Gilg

Aquilaria sinensis, a kind of typically wounding-induced medicinal plant with a great economical value, is widely used in the production of traditional Chinese medicine, perfume and incense. Coronatine-insensitive protein 1 (COI1) acts as a receptor in jasmonate (JA) signaling pathway, and regulates the expression of JA-responsive genes in plant defense. However, little is known about the COI1 gene in A. sinensis. Here, based on the transcriptome data, a full-length cDNA sequence of COI1 (termed as AsCOI1) was firstly cloned by RT-PCR and rapid-amplification of cDNA ends (RACE) strategies. AsCOI1 is 2330 bp in length (GenBank accession No. KM189194), and contains a complete open frame (ORF) of 1839 bp. The deduced protein was composed of 612 amino acids, with a predicted molecular weight of 68.93 kDa and an isoelectric point of 6.56, and was predicted to possess F-box and LRRs domains. Combining bioinformatics prediction with subcellular localization experiment analysis, AsCOI1 was appeared to locate in nucleus. AsCOI1 gene was highly expressed in roots and stems, the major organs of agarwood formation. Methyl jasmonate (MeJA), mechanical wounding and heat stress could significantly induce the expression level of AsCOI1 gene. AsCOI1 is an early wound-responsive gene, and it likely plays some role in agarwood formation.

Full-length cDNA cloning of AsNINJA1 gene in Aquilaria sinensis and MeJA-induced expression in callus / 中草药

Objective: To clone the full-length cDNA of interacting protein of JAZ (NINJA) gene in Aquilaria sinensis, and to provide the basic information for further study on gene function in sesquiterpenes biosynthesis pathway. Methods: With the total RNA as template, the full-length cDNA of NINJA in A. sinensis was cloned through RACE technique and RT-PCR method. The bioinformatics of cloing NINJA gene was analyzed as well. The expression mode of this gene was with MeJA treatment in A. sinensis callus detected by qRT-PCR method. Results: The full-length cDNA (1 982 bp) of NINJA gene in A. sinensis, named as AsNINJA1 was obtained with an open reading frame of 1 221 bp and encoding 406 amino acids. The relative molecular mass of AsNINJA1 protein calculated was 43 697, and the isoelectric point was 6.02. The qRT-PCR results indicated that MeJA treatment could stimulate the increase of mRNA expression of AsNINJA1; There was a sharp rise at 4 h with nearly 100 times higher than the control (without MeJA treatment), then dropped significantly. Conclusion: The full-length cDNA sequence of AsNINJA1 gene is obtained; AsNINJA1 is extremely sensitive to MeJA treatment, and responded to the early damage.

Cloning of the full-length rat Nor 1 cDNA using T_4 DNA-ligase-mediated 5' RACE / 中国病理生理杂志

AIM: To clone the full-length of 75A EST. METHODS: After the extraction of total RNA from primary cultured rat cerebellar granule cell of 7DIV in the medium containing 25 mmol/L KCl, T_4 DNA ligase-mediated 5' RACE was used to retrieve 5' unknown sequence of 75A EST, and the first round 5' RACE PCR product was subcloned into pGEM-T easy vector for sequence and homogeneous analysis. RESULTS: The first round of 5' RACE produce a 2.5 kb band, and 75A EST was identified to be partial sequence of Neuron-derived orphan receptor (Nor1) gene. After two more rounds RACE, we firstly cloned the full-length of Nor-1 cDNA. CONCLUSION: T_4 DNA ligase mediated 5' RACE is an efficient method to retrieve information about the 5' termini of mRNAs, and lay a foundation for further study which role Nor1 play in the cerebellar granule cell differentiation or survive.

Whole sequence amplification of an expressed sequence tag in immune cells of F344 rats with severe burns / 中国病理生理杂志

AIM: To examine the changes in immune cells which might be involved in systemic immune dysfunction after severe burns, we detected the sequence of expressed sequence tag in lymphocytes and monocytes of F344 rats post-burn. METHODS: Two pairs of primers were designed depending on the sequence of AI764697.1. SMART RACE technology was employed for amplification of whole sequence. Furthermore, the amplified sequence was testified by Northern blot. RESULTS: A 592bp sequence, which was testified by Northern blot, was suceessfally obtained in our experiments. This product was accepted by GenBank and its accession number is AF244895. CONCLUSION: A cDNA whole sequence in immune cells of F344 rats after severe burns was amplified successfully. More research work should be done to identify the resource, location in genome and function of this product.