油菜BnMKK2基因的克隆及表达分析

通过实验从陇油6号油菜中克隆得到了一种新的MAPK激酶基因BnMKK2基因的cDNA,全长1 344 bp,其中包括5′非翻译区（5′UTR）111 bp,3′非翻译区（3′UTR）165 bp,开放阅读框（ORF）长1 068 bp,编码355个氨基酸,该基因编码的蛋白质分子量为39.3 kDa,理论等电点为6.8。与拟南芥AtMKK2有很高的同源性,因此命名为BnMKK2(GenBank登录号:HQ848661)。该基因实时荧光定量PCR分析表明,BnMKK2基因的表达受低温胁迫诱导。     

Tissue expression and bioinformatics analysis of the vitellogenin gene of Asian arowana (Scleropages formosus)

The RACE technique was used to clone the full‐length vitellogenin (VTG) cDNA sequence of Asian arowana (Scleropages formosus). The full‐length sequence was 5,550 bp with an open reading frame of 5,238 bp, encoding 1,745 amino acids, and 5′ and 3′ UTRs (untranslated regions) of 45 bp and 267 bp, respectively. Phylogenetic analysis showed that S. formosus and silver arowana (Osteoglossum bicirrhosum) share a close evolutionary relationship (bootstrap 100%). The quantitative real‐time PCR results showed that vtg expression was significantly higher in liver and gonads of male and female fish compared with its expression in the other tissues tested (p < 0.01). The relative expression levels of vtg in liver, gland, kidney, heart, head kidney, and brain of female fish were significantly higher than in the corresponding tissues of male fish (p < 0.05).     

Molecular Cloning of cDNA for Trehalase from the European Honeybee,Apis mellifera L., and Its Heterologous Expression in Pichia pastoris

cDNA encoding the bound type trehalase of the European honeybee was cloned. The cDNA (3,001 bp) contained the long 5′ untranslated region (UTR) of 869 bp, and the 3′ UTR of 251 bp including a poly(A) tail, and the open reading frame of 1,881 bp consisting of 626 amino acid residues. The M _r of the mature enzyme comprised of 591 amino acids, excluded a signal sequence of 35 amino acid residues, was 69,177. Six peptide sequences analyzed were all found in the deduced amino acid sequence. The amino acid sequence exhibited high identity with trehalases belonging to glycoside hydrolase family 37. A putative transmembrane region similar to trehalase-2 of the silkworm was found in the C-terminal amino acid sequence. Recombinant enzyme of the trehalase was expressed in the methylotrophic yeast Pichia pastoris as host, and displayed properties identical to those of the native enzyme except for higher sugar chain contents. This is the first report of heterologous expression of insect trehalase.     

Molecular cloning and expression analysis of a F-type lectin gene from Japanese sea perch (<Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>)

The techniques of homology cloning and anchored PCR were used to clone the fucose-binding lectin (F-type lectin) gene from Japanese sea perch (Lateolabrax Japonicus). The full-length cDNA of sea perch F-lectin (JspFL) contained a 5′ untranslated region (UTR) of 39 bp, an ORF of 933 bp encoding a polypeptide of 310 amino acids with an estimated molecular mass of 10.82 kDa and a 3′ UTR of 332 bp. The searches for nucleotides and protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of JspFL was homological to the Fucose-binding lectin in other fish species. In the JspFL deduced amino acid sequence, two tandem domains that exhibit the eel carbohydrate-recognition sequence motif were found. The temporal expressions of gene in the different tissues were measured by real-time PCR. And the mRNA expressions of the gene were constitutively expressed in tissues including spleen, head-kidney, liver, gill, and heart. The JspFL expression in spleen was different during the stimulated time point, 2 h later the expression level became up-regulated, and 6 h later the expression level became down-regulated. The result indicated that JspFL was constitutive and inducible expressed and could play a critical role in the host-pathogen interaction.     

Expression and activity of V‐H+‐ATPase in gill and kidney of marbled eel Anguilla marmorata in response to salinity challenge

The full‐length complementary (c)DNA of vacuolar‐type‐H⁺‐ATPase B1 gene (vhab1) in marbled eel Anguilla marmorata with 1741 base pairs (bp) was identified. It contained a 1512 bp open reading frame encoding a polypeptide with 503 amino acids (55·9 kDa), an 83 bp 5′‐untranslated region (UTR) and a 146 bp 3′‐UTR. The expression levels of A. marmorata vhab1 in gill and kidney of A. marmorata were evaluated at different intervals during the exposure to various salinities (0, 10 and 25). The results indicated that the expression levels of A. marmorata vhab1 messenger (m)RNA in gill and kidney had a significant increase and reached the highest level at 1 h in brackish water (BW, salinity 10) group and 6 h in seawater (SW, salinity 25) group. Therefore, salinity did affect the relative expression level of A. marmorata vhab1 mRNA in gills, which exhibited the enhancement by c. 44 times in SW group when compared with that in fresh water. No remarkable difference in the expression of A. marmorata vhab1 mRNA was observed after 15 days of SW exposure (P > 0·05). V‐H⁺‐ATPase activity exhibited an increase by two‐ to three‐fold when compared with that in gill and kidney from the control group. The consequence primarily suggested that A. marmorata vhab1 gene product in elvers from A. marmorata plays an important role in adaptation response to SW.     

Molecular cloning and characterization of a C-type lectin in roughskin sculpin (Trachidermus fasciatus)

C-type lectins, as the members of pattern-recognition receptors (PRRs), play significant roles in innate immunity responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. In our study, a C-type lectin gene (TfCTL1) was cloned from the roughskin sculpin using expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length of TfCTL1 was 696 bp, consisting of a 95 bp 5′ untranslated region (UTR), a 498 bp open reading frame (ORF) encoding a 165 amino acid protein, and a 103 bp 3′ UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of TfCTL1 contained a signal peptide and a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys⁶¹-Cys¹⁵⁸, Cys¹³⁴-Cys¹⁵⁰) and a Ca²⁺/carbohydrate-binding site (QPD motif). Results from the qRT-PCR indicated that TfCTL1 mRNA was predominately expressed in the liver. The temporal expression of TfCTL1 was obviously up-regulated in the skin, blood, spleen and heart in time dependent manners by lipopolysaccharide (LPS) challenge, whereas in the liver, TfCTL1 was initially down-regulated from 2 h to 48 h followed by an abrupt up-regulation at 72 h. Recombinant TfCTL1 CRD purified from Escherichia coli BL21 was able to agglutinate some Gram-positive bacteria, Gram-negative bacteria and a yeast in a Ca²⁺-dependent manner. Further analysis showed that TfCTL1 can bind to several kinds of microorganisms selectively in a Ca²⁺-independent manner. These results suggested that TfCTL1 might be involved in the innate response as a PRR.     

Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (<Emphasis Type="Italic">Penaeus monodon</Emphasis>)

The techniques of homology cloning and anchored PCR were used to clone the Hsp90 gene from black tiger shrimp. The full length cDNA of black tiger shrimp Hsp90 (btsHsp90) contained a 5′ untranslated region (UTR) of 72 bp, an ORF (open reading frame) of 2160 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 83-kDa and a 3′ UTR of 288 bp. The sequence of the coding region showed 90 and 84% homology with that of the Chiromantes haematocheir and Homo sapiens, respectively. Conserved signature sequences of Hsp90 gene family were found in the btsHsp90 deduced amino acid sequence. The temporal expressions of Hsp90 gene were constitutively in the black tiger shrimp tissues including liver, ovary, muscle, brain stomach, and heart, and their levels were markedly enhanced after 30-min heat treatment at 37°C. In ovarian maturation stages, the expression of btsHsp90 was strongest in the second stage, weaker in the fourth and first stage.     

Molecular cloning and expression analysis of a myosin light chain 1 (MLC‐1) gene from Indian meal moth Plodia interpunctella (Lepidoptera: Pyralidae)

Myosin light chain 1 (MLC‐1) protein acts in the organization, dynamics and transport processes associated with the cytoskeleton. In this work, an MLC‐1 gene was cloned and characterized from the Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae). The isolated PiMLC‐1 cDNA is 913 bp, including a 5′‐untranslated region (UTR) of 79 bp, 3′‐UTR of 381 bp and an open reading frame (ORF) of 453 bp encoding a polypeptide of 150 amino acids, which contains two calcium binding domains (EF‐hands). The deduced PiMLC‐1 protein sequence has 39–94% comparison with other individuals. The qPCR analysis revealed that PiMLC‐1 was expressed in the four developmental stages (egg, larva, pupa and adult) and in all tissues tested, suggesting that it plays an important role in development of P. interpunctella. Based on the MLC‐1 amino acids, phylogenetic analysis showed a similar topology with the traditional classification, suggesting the potential value of the MLC‐1 protein in phylogenetic inference.