Curcin, a ribosome-inactivating protein with a molecular weight of about 28.2 kD, which strongly inhibits the protein synthesis in rabbit reticulocyte lysate system with an IC50 value of about (0.19 +/- 0.01) nmol/L, ...Curcin, a ribosome-inactivating protein with a molecular weight of about 28.2 kD, which strongly inhibits the protein synthesis in rabbit reticulocyte lysate system with an IC50 value of about (0.19 +/- 0.01) nmol/L, was purified from the seeds of Jatropha curcas L. The protein has the activity of rRNA N-glycosidase. Degenerate primers were designed based on the N-terminal partial sequence from purified curcin. The full-length curcin cDNA by RT-PCR and 5'-RACE was cloned. The deduced amino acids sequence indicates that a preprotein with 20 amino acid residues is first translated and then processed to a mature protein with 251 amino acids. The deduced amino acids sequence shares homology of 33% and 57% to those of type I ribosome-inactivating proteins (RIPs) and A chain of type II RIPs, respectively. The sequence encoding mature curcin was integrated into the pQE-30 vector for expression in Escherichia coli strain M15 (pREP4). The purified recombinant curcin was able to inhibit protein synthesis in rabbit reticulocyte lysate system.展开更多
The cDNA encoding a variant of α bungarotoxin was cloned from the venom glands of Bungarus multicinctus by RT PCR.The deduced protein precursor contained a 21 amino acid signal peptide and a following 74 amino ...The cDNA encoding a variant of α bungarotoxin was cloned from the venom glands of Bungarus multicinctus by RT PCR.The deduced protein precursor contained a 21 amino acid signal peptide and a following 74 amino acid mature protein.The signal peptide is very similar to those of short chain neurotoxins,κ neurotoxins and cardiotoxins.The amino acid sequence of the mature protein is identical to α bungarotoxin (V31),a minor variant of α bungarotoxin identified by protein sequencing technique.Furthermore,the cDNA encoding the deletion precursor of α bungarotoxin was also cloned.By use of pMAL p2,the variant was overexpressed in E coli as a soluble fusion protein and purified by sepharose 6B amylose affinity chromatography,which was confirmed by western blotting with the antisera agai nst α bungarotoxin.The recombinant variant was achieved after digestion by factor X a.It displayed about 1/6 in vivo toxicity of natural α bungarotoxin.The successful cloning and functional expression of α bungarotoxin provided a basis for the future study of structure function of long neurotoxins.展开更多
The ACC synthase is the key enzyme in ethylene biosynthesis and fruit ripening. To study the mechanism of ACC synthase in peach Prunus persica (L.) Batsch) fruit ripening, we cloned a full_length cDNA of ACC synthase ...The ACC synthase is the key enzyme in ethylene biosynthesis and fruit ripening. To study the mechanism of ACC synthase in peach Prunus persica (L.) Batsch) fruit ripening, we cloned a full_length cDNA of ACC synthase pacs from peach using 5′/3′ RACE PCR. The nucleic acid sequence of pacs was 1 848 bp, containing 177 bp of 5′untranslated sequence, 1 449 bp of an open reading frame, and 219 bp of 3′untranslated sequence (excluding the stop codon TAA). The pacs open reading frame encoded a 483_amino acid polypeptide with a predicted size of 54 kD and a calculated PI of 6.43. The deduced protein from ACC synthase cDNA pacs had 65%, 70%, 75%, and 90% homology with the other deduced proteins from tomato (S19677), plum (AB031026), papaya (U68216) and apple (AB034993), which contained the active site of ACC synthase SLSKDMGFPGFR conserved among these plant ACC synthases. RNA_based PCR amplification combined with hybridization analysis with pacs and another ACC synthase cDNApacs12 (AF467782) cloned by us before as probes, indicated that expression patterns of both clones were very similar. mRNAs of both clones expressed in the alabastrum and petal, and were induced after ethylene treatment. Wounding and IAA treatments could induce ACC synthase expression of both clones in the leaves. However, the wounding treatment of leaves has induced more abundant pacs ACC synthase expression than that ofpacs12. Pacs mRNA expressed in both green mature and ripening fruit, whilepacs12mRNA was little or undetectable in green mature fruit, but apparent in ripening fruit. Both clone mRNAs accumulated more in leaves (following wounding and IAA treatments) and flowers than in fruits.展开更多
[Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus（PPV）. [ Method] One pair of primers were designed according to PPV genome sequences on...[Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus（PPV）. [ Method] One pair of primers were designed according to PPV genome sequences on GenBank website and the sequences of prokaryotic expression vector pET30a （ ＋ ） with multiple cloning sites. The whole sequence of NS1 gene in PPV SD1 strain was amplified by using PCR technology and the positive recombinant plasmid was analyzed by sequencing and homology comparison. The prokaryotic expression recombinant plasmid PET30a/NS1 was constructed to make its induction expression in Escherichia coll. [ Result] The target fragment with the length of 2 208 bp was obtained from PCR amplification. The nucleotide homologies between the cloned NS1 gene and the reported relevant PPV genes were from 97.3 % to 99.4 %, which indicated that NS1 gene had high conservation. But it had a 12-basepair successive deletion near the hydroxyl end. The cloned PPV NS1 gene was successfully expressed in prokaryotic cell, and its expression products existed mostly in inclusion bodies. [ Conclusion] The results of SDS-PAGE detection showed that the molecular weight of PPV NS1 protein was 86 KD.展开更多
[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta （DE3）. [Method] Specific primers were designed according to NCBI database information and the conserved sequences of c...[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta （DE3）. [Method] Specific primers were designed according to NCBI database information and the conserved sequences of cry gene were amplified by PCR from Bt transgenic cotton. Then recombinant plasmids were constructed and expressed in E. coil strain Rosetta （DE3）. Finally, the effects of different concentrations and inducing time of IPTG on the expression level of protein were investigated. [Result] Two conserved sequences （304 and 853 bp respectively） of cry gene were amplified. The result of SDS-PAGE confirmed that the recombinant plasmids pGEX-4t-I-304 and pGEX-4t-1-853 could express fusion proteins by IPTG induction and the molecular weight of protein products was 39 and 62.4 kDa respectively, which was in accordance with predicted result. The optimal protein ex- pression conditions were confirmed as induction with 0.15 mmol/L IPTG for 7 h. [Conclusion] This study prepared the ground for the further detection of Bt transgenic crops.展开更多
[Objective]The aim was to construct the wheat heat-shock protein 60 （HSP60） gene in prokaryotic expression vector and express HSP60 efficiently in E.coli. [Method]According to the wheat HSP60 gene sequence in GenBan...[Objective]The aim was to construct the wheat heat-shock protein 60 （HSP60） gene in prokaryotic expression vector and express HSP60 efficiently in E.coli. [Method]According to the wheat HSP60 gene sequence in GenBank,a pair of primers P1/P2 were designed and synthesized. The wheat HSP60 gene fragment was amplified from the wheat RNA by RT-PCR and inserted into bacterial expression vector of pGEX-4T-1. The construct of pGEX-4T-1-HSP60 was subsequently transformed into E.coli BL21. [Result]The construct of pGEX-4T-1-HSP60 was verified by restriction endonuclease digestion and sequenced. Compared with the sequences of wheat HSP60 genes in GenBank,homology accounted to 100%. Expression of the GST-HSP60 fusion protein was induced with IPTG. Its molecular weight was about 90 kD. The result was identified by electrophoresis of SDS-PAGE. Expression of the protein bands was consistent with the expected size. [Conclusion]The recombinant prokaryotic expression vector in pGEX-4T-1-HSP60 was constructed successfully and expressed stably in E.coli BL21. This will lay the foundation for further study on the functions of the protein and its mechanism.展开更多
The 12-oxophytodienoate reductase(OPR),a flavin mononucleotide-dependent oxidoreductase,regulates plant responses to stress conditions such as heavy metals,drought,saline-alkali,pests,and diseases by participating in ...The 12-oxophytodienoate reductase(OPR),a flavin mononucleotide-dependent oxidoreductase,regulates plant responses to stress conditions such as heavy metals,drought,saline-alkali,pests,and diseases by participating in the synthesis of plant hormones such as jasmonic acid(JA).In this study,homologous cloning was employed to obtain the Brassica napus‘Zhongshuang 11’OPR1 gene(BnOPR1).Bioinformatics analysis was conducted for the deduced protein BnOPR1,and a plant expression vector for BnOPR1 was constructed.The bioinformatics analysis revealed that BnOPR1 was 1125 bp in length,encoding 375 amino acid residues.The deduced protein had a molecular weight of 41.604 kDa,an isoelectric point of 6.05,a molecular formula of C1853H2855N511O550S16,an aliphatic index of 35.47,a lipophilicity index of 74.87,and an instability index of 39.49.It had 49 phosphorylation sites and lacked transmembrane domains and signal peptides.The phylogenetic analysis indicated that BnOPR1 had a close relationship with the OPR1 protein from Brassica rapa since they shared the same clade,while it had a distant relationship with the OPR1 protein from Raphanus sativus.In this paper,the expression vector for BnOPR1 was successfully constructed by seamless cloning and named pBnOPR1.The findings laid a foundation for further studying the roles of BnOPR1 in the response to antimony stress.展开更多
[Objective] The aim was to study the cDNA cloning of SMAT gene from Chimonanthus praecox and its expression. [Method] A novel cDNA was cloned by PT-PCR using the total RNA as template, and amplified to the desirable s...[Objective] The aim was to study the cDNA cloning of SMAT gene from Chimonanthus praecox and its expression. [Method] A novel cDNA was cloned by PT-PCR using the total RNA as template, and amplified to the desirable size. The RT-PCR products were reclaimed and transformed into E. coli DH 5o together with the PMD18-T vector after ligating by T-A cloning. Identified by colony PCR and EcoRI and Notl digestion, the recombinant plasmid with target gene was screened out and conducted the sequence analysis. [Result] Results of the sequence analysis showed that the ORF fragment of the SAMT cDNA was successfully cloned from Chimonanthus praecox gene, with the length of 1 196 bp and encoding 380 amino acids fragment which shared 99.2% homology to that of previously reported SAMT cDNA from Chimonanthus praecox （ABU88887）. The SAMT cDNA fragment was sub-cloned into the prokaryotic expression vector PGEX1-4T-1, and the obtained re- combinant plasmid was named PGSAMT. After inducting by 0.01mol/L IPTG, the re- sult of the SDS-PAGE analysis showed that the molecular weight of the fusion ex- pression SAMT protein was about 66 kDa, which was close to the predicted fusion protein derived from the 26 kDa GST band and 42.3 kDa SAMT gene of Chimo- nanthus praecox encoded protein. [Conclusion] This study successfully cloned and expressed the SAMT gene of Chimonanthus praecox.展开更多
[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding...[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S.aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a(+)were digested by BamH Ⅰ and EcoR Ⅰ double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21(DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S.aureus. [Conclusion] The FnBP ligand binding gene of S.aureus was successfully cloned and expressed in prokaryotic cells.展开更多
To investigate the expression characteristics of sex related gene of FTZ-F 1 in the half-smooth tongue-sole （Cynoglossus semilaevis）, the homologue FTZ-F1 （hsFTZ-F1） full-length cDNA was isolated from the testis b...To investigate the expression characteristics of sex related gene of FTZ-F 1 in the half-smooth tongue-sole （Cynoglossus semilaevis）, the homologue FTZ-F1 （hsFTZ-F1） full-length cDNA was isolated from the testis by homologous cloning, and the cDNA included the open reading frame and a 66bp 5＇-UTR, along with a 1619bp 3＇-UTR, encoding a predicted 485 amino acid protein. Sequence, tissue distribution and phylogenic analyses of the FTZ-F1 showed that the hsFTZ-F1 belonged to SF-1/Ad4BP group. The hsFTZ-F1 transcripts were highly abundant in the gonads, kidneys, brain and head-kidneys, but weakly in other tissues. However, the expression level in the brain and head-kidney of female was highly abundant than in the male. The hsFTZ-F1 expression was highly abundant in the embryo than in the larvae, which suggested that the hsFTZ-F1 may be involved in the organogenesis in the tongue sole.展开更多
According to the complete genome of foot-and-mouth disease virus(FMDV)type O,a pair of special primers was designed to amplify VP1 gene.The VP1 gene was amplified by RT-PCR and subsequently inserted into the expressio...According to the complete genome of foot-and-mouth disease virus(FMDV)type O,a pair of special primers was designed to amplify VP1 gene.The VP1 gene was amplified by RT-PCR and subsequently inserted into the expression vector pGEX-6p-1 and induced by IPTG.Then SDS-PAGE showed the expressed protein was 51 kD in molecular weight.Then the product was purified by GSTrap FF columns.The product was detected through Western-blot that showed the protein has antigenicity.It provided fundamental data and materials for further investigation on diagnosis method of FMDV.展开更多
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 proce...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.展开更多
[Objective] This study was conducted to investigate cloning and expression of Pun1 gene controlling pungency of pepper. [Method] With Capsicum annuum L as a material, the cDNA sequence of Capunl gene was obtained, wit...[Objective] This study was conducted to investigate cloning and expression of Pun1 gene controlling pungency of pepper. [Method] With Capsicum annuum L as a material, the cDNA sequence of Capunl gene was obtained, with a total length of 1 457 bp, coding 440 amino acids. [Result] Phylogenetic analysis showed that Capunl was closest to Pun1 of C. chinense, with a genetic distance of 0.019 3. Plant expression vector pCAM-Punl-GFP was constructed and transformed into to- bacco, and it was found that the protein coded by fusion gene Punl：：GFP was lo- cated on cell membrane. Prokaryotic expression vectors were constructed, and by SDS-PAGE and Western Blot detection, an induced protein with a molecular weight of 63 ku was obtained. It was found by real-time fluorescence quantitative expres- sion that Pun1 gene was expressed at the highest level 30 d after flowering, de- creased then, and could not be detected substantially 40 and 45 d after flowering. [Conclusion] This study provides information and reference for molecular regulation mechanism of Pun1 gene.展开更多
A complete cDNA of potato Phytophthora infestans-induced hypersensitive response-related protein gene (POTHR-1) was cloned using rapid amplification of cDNA ends (RACE) strategy according to a fragment sequence which ...A complete cDNA of potato Phytophthora infestans-induced hypersensitive response-related protein gene (POTHR-1) was cloned using rapid amplification of cDNA ends (RACE) strategy according to a fragment sequence which we had cloned using suppression subtractive hybridization (SSH) technique. The potato POTHR-1 gene encodes a protein of 225 amino acids, which shares 81% identity with tobacco hin1 gene-enoded protein (harpin-induced protein). Southern blot revealed that there are two to three copies of POTHR-1 in potato genome. The POTHR-1 gene expression in potato leaves showed that its transcripts accumulated remarkably in leaves after 36 h inoculation with P. infestans. Mechanical wounding and jasmonic acid (JA) could induce the POTHR-1 gene expression and osmotic stress just induce a slight accumulation of POTHR-1 gene mRNA, while salicylic acid (SA) had no detectable function on the induction accumulation of POTHR-1 gene transcripts. The potato POTHR-1 gene may preferentially associate with hypersensitive response (HR) or biotic cell death during interaction between host and pathogen.展开更多
[Objective] This study aimed to clone accD gene from Brassica campestris （Yunnan small rapeseed） with different oil content and analyze the effect of accD gene expression level on rapeseed oil content. [Method] The ...[Objective] This study aimed to clone accD gene from Brassica campestris （Yunnan small rapeseed） with different oil content and analyze the effect of accD gene expression level on rapeseed oil content. [Method] The accD gene encoding β-CT Ⅱsubunit of acetyl-CoA carboxylase was cloned from Yunnan small rapeseeds with different oil content. Then, sequence alignment and the influence upon seed oil content by the expression of accD gene were analyzed. [Result] There were a few base pair substitutes among the accD genes from different Yunnan small rapeseeds, and their homology was up to 98.6%. The expression of accD gene was increased with reproduction development, and reached peak in late-stage siliques. The seed oil content was positively influenced by the expression of accD gene in middle-stage and late-stage siliques. [Conclusion] This study provides a certain theoretical basis for illustrating the molecular mechanism about rapeseed oil content and breeding new high-oil rapeseed cultivars.展开更多
A 1 539 by squalene synthase (AaSQS) cDNA was cloned from a high-yield Artemisia annua L. strain 001 by reverse transcription-polymerise chain reaction (RT-PCR). The amino acid sequence of AaSQS is 70%, 77%, 44% and 3...A 1 539 by squalene synthase (AaSQS) cDNA was cloned from a high-yield Artemisia annua L. strain 001 by reverse transcription-polymerise chain reaction (RT-PCR). The amino acid sequence of AaSQS is 70%, 77%, 44% and 39%a identical to that of squalene synthases from Arabidopsis thaliana, tobacco, human and yeast, respectively. The AaSQS genomic DNA has a complex organization containing 14 exons and 13 introns. Full-length or C-terminal truncated cDNA was subcloned into prokaryotic expression vector pET30a and the constructed plasmid was introduced to Escherichia coli strain BL21 (DE3) for induced overexpression. No squalene synthase protein with expected molecular mass was observed in E. cola containing the putative full-length squalene synthase cDNA, however, overexpression in E. coli was achieved by truncating 30 amino acids of hydrophobic region at the carboxy terminus.展开更多
The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different（from Ⅰ to Ⅵ） concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase（BAD...The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different（from Ⅰ to Ⅵ） concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase（BADH： EC 1.2.1.8） activities have a direct relation with increased stressing time in the same treatment; both exhibit a single peak with increasing the concentration of saline-alkali solution, and number V shows the highest value. The BADH gene of Leyrnus chinensis was cloned by RT-PCR and RACE technology and was designated as LcBADH. The cDNA sequence of LcBADH was 1774bp including the open reading frame（ORF） of 1521bp（coding 506 amino acids）. The vector of prokaryotic expression was constructed by inserting the LcBADH gene fragmcnt into pET30a（＋） and transformed into E. coli BL21（DE3）. The result of SDS-PAGE shows that the idio-protein with a molecular mass of 56.78 kDa was effectively expressed in the recombinant bacteria induced by isopropyl fl-D-thiogalactoside（IPTG）.展开更多
The B cells translocation gene 1 （BTG1） is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among vertebrates. Here, ...The B cells translocation gene 1 （BTG1） is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among vertebrates. Here, for the first time we cloned the full-length eDNA sequence of Hyriopsis schlegelii （Hs-BTG1）, an economically important freshwater shellfish and potential indicator of environmental heavy metal pollution, for the first time. Using rapid amplification of eDNA ends （RACE） together with splicing the EST sequence from a haemocyte eDNA library, we found that Hs-BTG1 contains a 525 bp open reading frame （ORF） encoding a 174 amino-acid polypeptide, a 306 bp 5＇ untranslated region （5＇ UTR）, and a 571 bp 3＇ UTR with a Poly（A） tail as well as a transcription termination signal （AATAAA）. Homologne searching against GenBank revealed that Hs-BTG1 was closest to Crassostrea gigas BTG1, sharing 50.57% of protein identities. Hs-BTG1 also shares some typical features of the BTG/TOB family, possessing two well-conserved A and B boxes. Clustering analysis of Hs-BTG1 and other known BTGs showed that Hs-BTG1 was also closely related to BTG1 of C. gigas from the invertebrate BTG1 clade. Function prediction via homology modeling showed that both Hs-BTG1 and C. gigas BTG1 share a similar three-dimensional structure with Homo sapiens BTG1. Tissue-specific expression analysis of the Hs-BTG1 via real-time PCR showed that the transcripts were constitutively expressed, with the highest levels in the hepatopancreas and gills, and the lowest in both haemocyte and muscle tissue. Expression levels of Hs-BTG1 in hepatopancreas （2.03-fold）, mantle （2.07-fold）, kidney （2.2-fold） and haemocyte （2.5-fold） were enhanced by cadmium （Cd2＋） stress, suggesting that Hs-BTG 1 may have played a significant role in H, schlegelii adaptation to adverse environmental conditions.展开更多
Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15...Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.展开更多
Cinnamoyl-CoA reductase (CCR) is responsible for the first committed reaction in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understandi...Cinnamoyl-CoA reductase (CCR) is responsible for the first committed reaction in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of the lion biosynthesis in wheat development, two cDNAs encoding CCR were identified from wheat (Triticum aestivum L. cv. H4564). DNA sequence analyses indicated that the two cDNAs represent two classes of CCR. RT-PCR and Northern blot hybridization demonstrated that one of them, W-cr6, was expressed actively in stem and leaf tissue, the other one, W-cr19, was expressed in root and stem tissue. The results suggested that there are at least two genes encoded for CCR existing in wheat genome.展开更多
文摘Curcin, a ribosome-inactivating protein with a molecular weight of about 28.2 kD, which strongly inhibits the protein synthesis in rabbit reticulocyte lysate system with an IC50 value of about (0.19 +/- 0.01) nmol/L, was purified from the seeds of Jatropha curcas L. The protein has the activity of rRNA N-glycosidase. Degenerate primers were designed based on the N-terminal partial sequence from purified curcin. The full-length curcin cDNA by RT-PCR and 5'-RACE was cloned. The deduced amino acids sequence indicates that a preprotein with 20 amino acid residues is first translated and then processed to a mature protein with 251 amino acids. The deduced amino acids sequence shares homology of 33% and 57% to those of type I ribosome-inactivating proteins (RIPs) and A chain of type II RIPs, respectively. The sequence encoding mature curcin was integrated into the pQE-30 vector for expression in Escherichia coli strain M15 (pREP4). The purified recombinant curcin was able to inhibit protein synthesis in rabbit reticulocyte lysate system.
基金the"95"great program of Chinese Academy of Sciences! (KY95 1-A1-3 0 1-0 2 )
文摘The cDNA encoding a variant of α bungarotoxin was cloned from the venom glands of Bungarus multicinctus by RT PCR.The deduced protein precursor contained a 21 amino acid signal peptide and a following 74 amino acid mature protein.The signal peptide is very similar to those of short chain neurotoxins,κ neurotoxins and cardiotoxins.The amino acid sequence of the mature protein is identical to α bungarotoxin (V31),a minor variant of α bungarotoxin identified by protein sequencing technique.Furthermore,the cDNA encoding the deletion precursor of α bungarotoxin was also cloned.By use of pMAL p2,the variant was overexpressed in E coli as a soluble fusion protein and purified by sepharose 6B amylose affinity chromatography,which was confirmed by western blotting with the antisera agai nst α bungarotoxin.The recombinant variant was achieved after digestion by factor X a.It displayed about 1/6 in vivo toxicity of natural α bungarotoxin.The successful cloning and functional expression of α bungarotoxin provided a basis for the future study of structure function of long neurotoxins.
文摘The ACC synthase is the key enzyme in ethylene biosynthesis and fruit ripening. To study the mechanism of ACC synthase in peach Prunus persica (L.) Batsch) fruit ripening, we cloned a full_length cDNA of ACC synthase pacs from peach using 5′/3′ RACE PCR. The nucleic acid sequence of pacs was 1 848 bp, containing 177 bp of 5′untranslated sequence, 1 449 bp of an open reading frame, and 219 bp of 3′untranslated sequence (excluding the stop codon TAA). The pacs open reading frame encoded a 483_amino acid polypeptide with a predicted size of 54 kD and a calculated PI of 6.43. The deduced protein from ACC synthase cDNA pacs had 65%, 70%, 75%, and 90% homology with the other deduced proteins from tomato (S19677), plum (AB031026), papaya (U68216) and apple (AB034993), which contained the active site of ACC synthase SLSKDMGFPGFR conserved among these plant ACC synthases. RNA_based PCR amplification combined with hybridization analysis with pacs and another ACC synthase cDNApacs12 (AF467782) cloned by us before as probes, indicated that expression patterns of both clones were very similar. mRNAs of both clones expressed in the alabastrum and petal, and were induced after ethylene treatment. Wounding and IAA treatments could induce ACC synthase expression of both clones in the leaves. However, the wounding treatment of leaves has induced more abundant pacs ACC synthase expression than that ofpacs12. Pacs mRNA expressed in both green mature and ripening fruit, whilepacs12mRNA was little or undetectable in green mature fruit, but apparent in ripening fruit. Both clone mRNAs accumulated more in leaves (following wounding and IAA treatments) and flowers than in fruits.
基金Supported by the Natural Science Foundation Program of Shandong Province(2007ZRA16001)~~
文摘[Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus（PPV）. [ Method] One pair of primers were designed according to PPV genome sequences on GenBank website and the sequences of prokaryotic expression vector pET30a （ ＋ ） with multiple cloning sites. The whole sequence of NS1 gene in PPV SD1 strain was amplified by using PCR technology and the positive recombinant plasmid was analyzed by sequencing and homology comparison. The prokaryotic expression recombinant plasmid PET30a/NS1 was constructed to make its induction expression in Escherichia coll. [ Result] The target fragment with the length of 2 208 bp was obtained from PCR amplification. The nucleotide homologies between the cloned NS1 gene and the reported relevant PPV genes were from 97.3 % to 99.4 %, which indicated that NS1 gene had high conservation. But it had a 12-basepair successive deletion near the hydroxyl end. The cloned PPV NS1 gene was successfully expressed in prokaryotic cell, and its expression products existed mostly in inclusion bodies. [ Conclusion] The results of SDS-PAGE detection showed that the molecular weight of PPV NS1 protein was 86 KD.
基金Supported by Scientific Research Fund for Doctoral Program of Wuhan Polytechnic University (2006696)~~
文摘[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta （DE3）. [Method] Specific primers were designed according to NCBI database information and the conserved sequences of cry gene were amplified by PCR from Bt transgenic cotton. Then recombinant plasmids were constructed and expressed in E. coil strain Rosetta （DE3）. Finally, the effects of different concentrations and inducing time of IPTG on the expression level of protein were investigated. [Result] Two conserved sequences （304 and 853 bp respectively） of cry gene were amplified. The result of SDS-PAGE confirmed that the recombinant plasmids pGEX-4t-I-304 and pGEX-4t-1-853 could express fusion proteins by IPTG induction and the molecular weight of protein products was 39 and 62.4 kDa respectively, which was in accordance with predicted result. The optimal protein ex- pression conditions were confirmed as induction with 0.15 mmol/L IPTG for 7 h. [Conclusion] This study prepared the ground for the further detection of Bt transgenic crops.
基金Supported by National Natural Science Foundation of China(30870109)~~
文摘[Objective]The aim was to construct the wheat heat-shock protein 60 （HSP60） gene in prokaryotic expression vector and express HSP60 efficiently in E.coli. [Method]According to the wheat HSP60 gene sequence in GenBank,a pair of primers P1/P2 were designed and synthesized. The wheat HSP60 gene fragment was amplified from the wheat RNA by RT-PCR and inserted into bacterial expression vector of pGEX-4T-1. The construct of pGEX-4T-1-HSP60 was subsequently transformed into E.coli BL21. [Result]The construct of pGEX-4T-1-HSP60 was verified by restriction endonuclease digestion and sequenced. Compared with the sequences of wheat HSP60 genes in GenBank,homology accounted to 100%. Expression of the GST-HSP60 fusion protein was induced with IPTG. Its molecular weight was about 90 kD. The result was identified by electrophoresis of SDS-PAGE. Expression of the protein bands was consistent with the expected size. [Conclusion]The recombinant prokaryotic expression vector in pGEX-4T-1-HSP60 was constructed successfully and expressed stably in E.coli BL21. This will lay the foundation for further study on the functions of the protein and its mechanism.
文摘The 12-oxophytodienoate reductase(OPR),a flavin mononucleotide-dependent oxidoreductase,regulates plant responses to stress conditions such as heavy metals,drought,saline-alkali,pests,and diseases by participating in the synthesis of plant hormones such as jasmonic acid(JA).In this study,homologous cloning was employed to obtain the Brassica napus‘Zhongshuang 11’OPR1 gene(BnOPR1).Bioinformatics analysis was conducted for the deduced protein BnOPR1,and a plant expression vector for BnOPR1 was constructed.The bioinformatics analysis revealed that BnOPR1 was 1125 bp in length,encoding 375 amino acid residues.The deduced protein had a molecular weight of 41.604 kDa,an isoelectric point of 6.05,a molecular formula of C1853H2855N511O550S16,an aliphatic index of 35.47,a lipophilicity index of 74.87,and an instability index of 39.49.It had 49 phosphorylation sites and lacked transmembrane domains and signal peptides.The phylogenetic analysis indicated that BnOPR1 had a close relationship with the OPR1 protein from Brassica rapa since they shared the same clade,while it had a distant relationship with the OPR1 protein from Raphanus sativus.In this paper,the expression vector for BnOPR1 was successfully constructed by seamless cloning and named pBnOPR1.The findings laid a foundation for further studying the roles of BnOPR1 in the response to antimony stress.
文摘[Objective] The aim was to study the cDNA cloning of SMAT gene from Chimonanthus praecox and its expression. [Method] A novel cDNA was cloned by PT-PCR using the total RNA as template, and amplified to the desirable size. The RT-PCR products were reclaimed and transformed into E. coli DH 5o together with the PMD18-T vector after ligating by T-A cloning. Identified by colony PCR and EcoRI and Notl digestion, the recombinant plasmid with target gene was screened out and conducted the sequence analysis. [Result] Results of the sequence analysis showed that the ORF fragment of the SAMT cDNA was successfully cloned from Chimonanthus praecox gene, with the length of 1 196 bp and encoding 380 amino acids fragment which shared 99.2% homology to that of previously reported SAMT cDNA from Chimonanthus praecox （ABU88887）. The SAMT cDNA fragment was sub-cloned into the prokaryotic expression vector PGEX1-4T-1, and the obtained re- combinant plasmid was named PGSAMT. After inducting by 0.01mol/L IPTG, the re- sult of the SDS-PAGE analysis showed that the molecular weight of the fusion ex- pression SAMT protein was about 66 kDa, which was close to the predicted fusion protein derived from the 26 kDa GST band and 42.3 kDa SAMT gene of Chimo- nanthus praecox encoded protein. [Conclusion] This study successfully cloned and expressed the SAMT gene of Chimonanthus praecox.
基金Supported by National Natural Science Foundation of China(30771596)Ph.D.Programs Foundation of Ministry of Education of China(20060183010)~~
文摘[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S.aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a(+)were digested by BamH Ⅰ and EcoR Ⅰ double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21(DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S.aureus. [Conclusion] The FnBP ligand binding gene of S.aureus was successfully cloned and expressed in prokaryotic cells.
基金Supported by grants from State 863 High-Technology R&D Project of China(2006AA10A403)Shandong Genetic Improvement Key Project for Agricultural OrganismDoctor Initial Funding of Guangdong Ocean University(0712103)
文摘To investigate the expression characteristics of sex related gene of FTZ-F 1 in the half-smooth tongue-sole （Cynoglossus semilaevis）, the homologue FTZ-F1 （hsFTZ-F1） full-length cDNA was isolated from the testis by homologous cloning, and the cDNA included the open reading frame and a 66bp 5＇-UTR, along with a 1619bp 3＇-UTR, encoding a predicted 485 amino acid protein. Sequence, tissue distribution and phylogenic analyses of the FTZ-F1 showed that the hsFTZ-F1 belonged to SF-1/Ad4BP group. The hsFTZ-F1 transcripts were highly abundant in the gonads, kidneys, brain and head-kidneys, but weakly in other tissues. However, the expression level in the brain and head-kidney of female was highly abundant than in the male. The hsFTZ-F1 expression was highly abundant in the embryo than in the larvae, which suggested that the hsFTZ-F1 may be involved in the organogenesis in the tongue sole.
基金Supported by the Science and Technology Foundation from Science&Technology Department of Guangxi Autonomous Region(0779001)~~
文摘According to the complete genome of foot-and-mouth disease virus(FMDV)type O,a pair of special primers was designed to amplify VP1 gene.The VP1 gene was amplified by RT-PCR and subsequently inserted into the expression vector pGEX-6p-1 and induced by IPTG.Then SDS-PAGE showed the expressed protein was 51 kD in molecular weight.Then the product was purified by GSTrap FF columns.The product was detected through Western-blot that showed the protein has antigenicity.It provided fundamental data and materials for further investigation on diagnosis method of FMDV.
文摘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.
基金Supported by College Student Innovation Fund Project of Jilin University(2015821243)~~
文摘[Objective] This study was conducted to investigate cloning and expression of Pun1 gene controlling pungency of pepper. [Method] With Capsicum annuum L as a material, the cDNA sequence of Capunl gene was obtained, with a total length of 1 457 bp, coding 440 amino acids. [Result] Phylogenetic analysis showed that Capunl was closest to Pun1 of C. chinense, with a genetic distance of 0.019 3. Plant expression vector pCAM-Punl-GFP was constructed and transformed into to- bacco, and it was found that the protein coded by fusion gene Punl：：GFP was lo- cated on cell membrane. Prokaryotic expression vectors were constructed, and by SDS-PAGE and Western Blot detection, an induced protein with a molecular weight of 63 ku was obtained. It was found by real-time fluorescence quantitative expres- sion that Pun1 gene was expressed at the highest level 30 d after flowering, de- creased then, and could not be detected substantially 40 and 45 d after flowering. [Conclusion] This study provides information and reference for molecular regulation mechanism of Pun1 gene.
文摘A complete cDNA of potato Phytophthora infestans-induced hypersensitive response-related protein gene (POTHR-1) was cloned using rapid amplification of cDNA ends (RACE) strategy according to a fragment sequence which we had cloned using suppression subtractive hybridization (SSH) technique. The potato POTHR-1 gene encodes a protein of 225 amino acids, which shares 81% identity with tobacco hin1 gene-enoded protein (harpin-induced protein). Southern blot revealed that there are two to three copies of POTHR-1 in potato genome. The POTHR-1 gene expression in potato leaves showed that its transcripts accumulated remarkably in leaves after 36 h inoculation with P. infestans. Mechanical wounding and jasmonic acid (JA) could induce the POTHR-1 gene expression and osmotic stress just induce a slight accumulation of POTHR-1 gene mRNA, while salicylic acid (SA) had no detectable function on the induction accumulation of POTHR-1 gene transcripts. The potato POTHR-1 gene may preferentially associate with hypersensitive response (HR) or biotic cell death during interaction between host and pathogen.
基金Supported by the National Key Technology Research and Development Program(2011BAD35B04)the National High Technology Research and Development Program of China (863 Program, 2011AA10A104)~~
文摘[Objective] This study aimed to clone accD gene from Brassica campestris （Yunnan small rapeseed） with different oil content and analyze the effect of accD gene expression level on rapeseed oil content. [Method] The accD gene encoding β-CT Ⅱsubunit of acetyl-CoA carboxylase was cloned from Yunnan small rapeseeds with different oil content. Then, sequence alignment and the influence upon seed oil content by the expression of accD gene were analyzed. [Result] There were a few base pair substitutes among the accD genes from different Yunnan small rapeseeds, and their homology was up to 98.6%. The expression of accD gene was increased with reproduction development, and reached peak in late-stage siliques. The seed oil content was positively influenced by the expression of accD gene in middle-stage and late-stage siliques. [Conclusion] This study provides a certain theoretical basis for illustrating the molecular mechanism about rapeseed oil content and breeding new high-oil rapeseed cultivars.
文摘A 1 539 by squalene synthase (AaSQS) cDNA was cloned from a high-yield Artemisia annua L. strain 001 by reverse transcription-polymerise chain reaction (RT-PCR). The amino acid sequence of AaSQS is 70%, 77%, 44% and 39%a identical to that of squalene synthases from Arabidopsis thaliana, tobacco, human and yeast, respectively. The AaSQS genomic DNA has a complex organization containing 14 exons and 13 introns. Full-length or C-terminal truncated cDNA was subcloned into prokaryotic expression vector pET30a and the constructed plasmid was introduced to Escherichia coli strain BL21 (DE3) for induced overexpression. No squalene synthase protein with expected molecular mass was observed in E. cola containing the putative full-length squalene synthase cDNA, however, overexpression in E. coli was achieved by truncating 30 amino acids of hydrophobic region at the carboxy terminus.
基金Supported by the National Natural Science Foundation of China(Nos30590382 and 30570273)
文摘The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different（from Ⅰ to Ⅵ） concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase（BADH： EC 1.2.1.8） activities have a direct relation with increased stressing time in the same treatment; both exhibit a single peak with increasing the concentration of saline-alkali solution, and number V shows the highest value. The BADH gene of Leyrnus chinensis was cloned by RT-PCR and RACE technology and was designated as LcBADH. The cDNA sequence of LcBADH was 1774bp including the open reading frame（ORF） of 1521bp（coding 506 amino acids）. The vector of prokaryotic expression was constructed by inserting the LcBADH gene fragmcnt into pET30a（＋） and transformed into E. coli BL21（DE3）. The result of SDS-PAGE shows that the idio-protein with a molecular mass of 56.78 kDa was effectively expressed in the recombinant bacteria induced by isopropyl fl-D-thiogalactoside（IPTG）.
基金supported by the Key Scientific and Technological Programme of Jiangxi Province,China(20121BBF60036)the Special Fund for Agro-scientific Research in the Public Interest,State Agriculture Ministry of China(200903028)+2 种基金the Science and Technology Landing Project of Jiangxi Province,China(KJLD12001)the Youth Fund of the Education Department of Jiangxi Province,China(GJJ14219)the National Natural Science Foundation of China(31160534)
文摘The B cells translocation gene 1 （BTG1） is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among vertebrates. Here, for the first time we cloned the full-length eDNA sequence of Hyriopsis schlegelii （Hs-BTG1）, an economically important freshwater shellfish and potential indicator of environmental heavy metal pollution, for the first time. Using rapid amplification of eDNA ends （RACE） together with splicing the EST sequence from a haemocyte eDNA library, we found that Hs-BTG1 contains a 525 bp open reading frame （ORF） encoding a 174 amino-acid polypeptide, a 306 bp 5＇ untranslated region （5＇ UTR）, and a 571 bp 3＇ UTR with a Poly（A） tail as well as a transcription termination signal （AATAAA）. Homologne searching against GenBank revealed that Hs-BTG1 was closest to Crassostrea gigas BTG1, sharing 50.57% of protein identities. Hs-BTG1 also shares some typical features of the BTG/TOB family, possessing two well-conserved A and B boxes. Clustering analysis of Hs-BTG1 and other known BTGs showed that Hs-BTG1 was also closely related to BTG1 of C. gigas from the invertebrate BTG1 clade. Function prediction via homology modeling showed that both Hs-BTG1 and C. gigas BTG1 share a similar three-dimensional structure with Homo sapiens BTG1. Tissue-specific expression analysis of the Hs-BTG1 via real-time PCR showed that the transcripts were constitutively expressed, with the highest levels in the hepatopancreas and gills, and the lowest in both haemocyte and muscle tissue. Expression levels of Hs-BTG1 in hepatopancreas （2.03-fold）, mantle （2.07-fold）, kidney （2.2-fold） and haemocyte （2.5-fold） were enhanced by cadmium （Cd2＋） stress, suggesting that Hs-BTG 1 may have played a significant role in H, schlegelii adaptation to adverse environmental conditions.
文摘Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.
文摘Cinnamoyl-CoA reductase (CCR) is responsible for the first committed reaction in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of the lion biosynthesis in wheat development, two cDNAs encoding CCR were identified from wheat (Triticum aestivum L. cv. H4564). DNA sequence analyses indicated that the two cDNAs represent two classes of CCR. RT-PCR and Northern blot hybridization demonstrated that one of them, W-cr6, was expressed actively in stem and leaf tissue, the other one, W-cr19, was expressed in root and stem tissue. The results suggested that there are at least two genes encoded for CCR existing in wheat genome.
