Glutamatergic CYLD deletion leads to aberrant excitatory activity in the basolateral amygdala:association with enhanced cued fear expression

Neuronal activity,synaptic transmission,and molecular changes in the basolateral amygdala play critical roles in fear memory.Cylindromatosis(CYLD)is a deubiquitinase that negatively regulates the nuclear factor kappa-B pathway.CYLD is well studied in non-neuronal cells,yet underinvestigated in the brain,where it is highly expressed.Emerging studies have shown involvement of CYLD in the remodeling of glutamatergic synapses,neuroinflammation,fear memory,and anxiety-and autism-like behaviors.However,the precise role of CYLD in glutamatergic neurons is largely unknown.Here,we first proposed involvement of CYLD in cued fear expression.We next constructed transgenic model mice with specific deletion of Cyld from glutamatergic neurons.Our results show that glutamatergic CYLD deficiency exaggerated the expression of cued fear in only male mice.Further,loss of CYLD in glutamatergic neurons resulted in enhanced neuronal activation,impaired excitatory synaptic transmission,and altered levels of glutamate receptors accompanied by over-activation of microglia in the basolateral amygdala of male mice.Altogether,our study suggests a critical role of glutamatergic CYLD in maintaining normal neuronal,synaptic,and microglial activation.This may contribute,at least in part,to cued fear expression.

Cloning of BjNAC102 Promoter and Construction of Expression Vector in Brassica juncea

Transcription factor NAC102 plays an important role in the abiotic stress responses of plants.In this study,the promoter sequence of 3000 bp located in the upstream of the BjNAC102 gene was cloned from Brassica juncea‘Sichuan Yellow Seed’by using the homologous cloning method.The expression vector of the GUS gene driven by the BjNAC102 promoter was constructed by seamless cloning technology.The results showed that the sequence of the promoter of the BjNAC102 gene contained many cis-acting elements involved in light responsiveness,gibberellinresponsive element,and auxin-responsive element.It was speculated that BjNAC102 played an important role in the abiotic stress response in Brassica juncea.The expression vector of the promoter of the BjNAC102 gene was constructed,which layed a foundation for further studies of the expression pattern of the BjNAC102 gene in Brassica juncea.

Cloning and Prokaryotic Expression of FnBP Ligand Binding Gene of Staphylococcus aureus

[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.

Molecular Cloning and Expression Analysis of FTZ-F1 in the Half-smooth Tongue-sole, Cynoglossus semilaevis

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.

Cloning and Expression of Curcin, a Ribosome-Inactivating Protein from the Seeds of Jatropha curcas

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.

Cloning and Prokaryotic Expression of VP1 Gene of Foot-and-Mouth Disease Virus (FMDV) Type O

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.

MOLECULAR CLONING AND FUNCTIONAL EXPRESSION OF α-BUNGAROTOXIN (V31) FROM CHINESE CONTINENTAL BANDED KRAIT

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.

Molecular Cloning and Expression of RSSG58 Gene in Rice Sperm Cells

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.

Cloning and expression analysis of a long type peptidoglycan recognition protein(PGRP-L) from Xenopus tropicalis

Peptidoglycan recognition proteins（PGRPs） are a family of pattern recognition receptors（PRRs） of the immune system,which bind and hydrolyze bacterial peptidoglycan.Here,a long type PGRP（PGRP-L） was first cloned in the lower vertebrate species Xenopus tropicalis（Xt）.The XtPGRP-L possessed a conserved genomic structure with five exons and four introns.The alignment and phylogenetic analysis indicated that XtPGRP-L might be a type of amidase-like PGRP.The 3-D model showed that XtPGRP-L possessed a conserved structure compared with the Drosophila PGRP-Lb.During embryonic development,XtPGRP-L was not expressed until the 72 h tadpole stage.In adult tissues,it was strongly expressed in the liver,lung,intestine,and stomach.Furthermore,after LPS stimulation,the expression of XtPGRP-L was up-regulated significantly in the liver,intestine and spleen,indicating that XtPGRP-L may play an important role in the innate immunity of Xenopus tropicalis.

Cloning and Differential Expression of a 1-Aminocyclopropane-1-Carboxylate Synthase cDNA from Peach

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.

Cloning of hemoglobin-α1 from half-smooth tongue sole (Cynoglossus semilaevis) and its expression under short-term hypoxia

This study cloned the hemoglobin a1 from the marine teleost, the half-smooth tongue sole （Cynoglossus semilaevis）, and then examined its expression under hypoxia exposure. The full-length of CsHb-a1 （594 bp） cDNA contains an open reading frame encoding 144 amino acids. Sequence analysis shows that the predicted CsHb-a1 amino acids shares high identities with that of other species. Real-time PCR showed that CsHb-a1 was highly expressed in the heart, liver, spleen, kidney and blood. Five to 120 min esposure and long-term （36 h） exposure to hypoxia （1.0 mg/L） significantly increased CsHb-a1 mRNA expression in most tissues compared to those fish held in normoxic conditions （dissolved oxygen （DO）： 6.2 mg/L）. These results suggested that the up-regulation of Hb-a1 is an important component for adaptation of half-smooth tongue sole to short-term hypoxia.

Cloning and Prokaryotic Expression of NS1 Gene of Porcine Parvovirus (PPV) SD1 Strain

[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.

Cloning of Potato POTHR-1 Gene and Its Expression in Response to Infection by Phytophthora infestans and Other Abiotic Stimuli

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.

Cloning and Expression of Conserved Sequences of cry Gene in E.coli Strain Rosetta(DE3)

[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.

Cloning and Expression of Wheat Heat-shock Protein 60 (HSP60) Gene in E.coli

[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.

Molecular Cloning, Escherichia coli Expression and Genomic Organization of Squalene Synthase Gene from Artemisia annua

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.

Cloning and Expression Analysis of PtFATB Gene Encoding the Acyl-acyl Carrier Protein Thioesterase in Populus tomentosa Carr

Acyl-ACP thioesterases （FATs） terminates the fatty acid synthesis and allow the transport of fatty acids out of the plastids, which are the important determinants of cellular metabolism. FATB is a member of FAT enzymes that has been described previously in most of the plants. In silico cloning is a new method that utilizes the bioinformatics on the complete genome and available EST database. In this study, a full-length cDNA clone of PtFATB gene was isolated from Populus tomentosa using this approach. It is 1,450 bp in length and the open reading frame encodes a peptide of 421 amino acids. The predicted amino acid sequence shows significant homology with those from other plant species, which contain typical domains owned by FATB proteins. The transcripts of PtFATB were abundant in leaves, and less in roots detected by using semiquantitative RT-PCR. When the shoots were subjected to the stress treatments （cold, dry, NaC1） and ABA （Abscisic acid）, the expression of PtFATB was only slightly reduced under the treatment of low temperature. This suggests that the expression of PtFATB is in a constitutive fashion. This study provides the basis not only for the identification and characterization of this gene but also for the improvement of cold tolerance by controlling the expression of the PtFATB gene in trees in near future.

Cloning of Rabbit Bone Morphogenetic Protein 15 and Its Expression During in vitro Maturation of Rabbit Oocytes

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.

Cloning and Expression Analysis of Two Wheat cDNAs Encoding Cinnamoyl-CoA Reductase

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.

Cloning of Tap Ⅱ Chalcone Isomerases(CHI1 A) Gene and Construction of Lactococcus Lactis Expression Vector

[Objective]The aim was to clone TapⅡchalcone isomerases（CHI1 A） from soybean specially and construct expression vector of PNZ8149-CHI1 A,and then transform it into Lactococcus Lactis NICE systers.[Method]Chalcone isomerases（CHI1 A） was cloned by RTPCR method,and it was sequenced after cloning into pMD18-T vectors,and recombined to expression vector PNZ8149-CHI1 A,then it was transformed into Lactococcus Lactis NZ3900[Result]The sequencing results indicated that the cloned fragment of CHI1 A contained 670 nucleotides,and shared a sequence homology of 92% with that from Genbank accession number AF595413（CHI1 A）.CHI1 A was transformed into NICE expression system successfully by identification of PCR and digestion.[Conclusion]The foundation of using the microorganism fermentation method to produce flavonoids was laid by construction of efficient induction expression vector with chalcone isomerases CHI1 A.