Generation of Antibodies Against DMRT1 and DMRT4 of Oreochromis aurea and Analysis of Their Expression Profile in Oreochromis aurea Tissues

Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood. A single gene known to control somatic sex determination, the DM-domain containing （Doublesex/Mab-3 DNA-binding motif） gene, is highly conserved across species. Vertebrate DMRT1 （DM-related transcription factor 1） expression occurs predominantly in the testis. Here, however, isolated two distinct DM-domain cDNA from Oreochromis aurea ovary and testis have been named DMRT4 （DM-related transcription factor 4） and DMRT1 by BLAST, respectively. Despite high homology in the DM-domain there is little similarity outside the DM-domain.To better understand the structure, function, and possible roles of DMRT4 and DMRT1 as potential candidates for sex differentiation and sex determination, the intact regions encoding DMRT4 and DMRT1 obtained by PCR were sub-cloned into the vector pMAL-c2x and introduced into the Escherichia coli TB1 cell for efficient fusion expression. After purification and cleavage, DMRT4 and DMRT1 proteins were used to immunize adult rabbits following standard protocols. Consequently, it was found by using Western blot analysis that polyclonal antibodies against DMRT4 and DMRT1 had high specificity. The relative expression levels of DMRT4 and DMRT1 mRNA were determined by fluorescent Real-time RT-PCR in female and male Oreochromis aurea with 13-actin as the internal standard. DMRT1 was expressed only in testis, whereas DMRT4 was over expressed in the ovary, but in both female and male, a slight expression in the brain was also detected. Statistical analysis showed that in the brain, mean DMRT4 mRNA levels in female were significantly higher than in male. Meanwhile, the expression of DMRT4 and DMRT1 protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry. It was found that DMRT4 was exclusively expressed in the ovary and DMRT1 in the testis. Study on DMRT4 and DMRT1 expression facilitated the elucidation of their roles and the understanding of sex differentiation of fish.

Cloning of Brassica napus EIN3 Gene and Its Expression Induced by Sclerotinia sclerotiorum

[Objective] The study was to investigate roles of Brassica napus EINB in （ BnEIN3 ） resistance to Sclerotinia sclerotiorum. [ Methods] Genomic PCR and RT-PCR were carded out to isolate genomic DNA and cDNA sequences of BnEIN3 from oilseed rape, based on the highly conserved region of EIN3 gene from Arabidopsis thaliana and the homologous sequences of oilseed rape ESTs. Expression levels of BnEIN3 were detected in three varieties of oilseed rape inoculated with S. sclerotiorum by real-time quantitative PCR.[Results] A 1 947 bp DNA fragment was obtained from oilseed rape. The fragment shared 82% identity to A. thaliana EIIV3, encoded 614 amino acids containing an EIN3 domain, and was named as BnEIN3. Real-time PCR results showed that expression patterns of BnEIN3 were drastically different in different varieties. In highly resistant oilseed rape variety D083, BnEIN3 expression level was significantly increased 72 h after S. sclerotiorum inoculation whereas in middle resistant and susceptible varieties Zhongshuang 9 and 84039, BnEIN3 expression was suppressed. [ Conclusion ] BnEIIV3 may play an important role in oilseed rape resistance to S. sclerotiorum.

Cloning and Analysis of CFL—A LFY_like Gene from Cucumber

LFY and LFY _like genes have been shown to control the initiation of floral meristems in higher plants. The homologous cDNA of LFY, CFL, were cloned from cucumber ( Cucumis sativus L.). Southern blot analysis showed it was a single copy gene in the cucumber genome. Northern blot analysis showed that it expressed in the floral buds and young leaves. The possible role of CFL in the floral and vegetative development of cucumber plant was discussed.

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 Pattern Analysis of Nitrogen- Starvation-induced Genes in Rice

To understand the regulation system of nitrogen X-starvation in higher plants, a cDNA library from N-starved rice (Oryza sativa L.) seedlings was constructed using rapid subtraction hybridization (RaSH) procedure. Through reverse Northern analysis and Northern blotting, 18 unique known genes and two unique unknown genes were identified, which were up-regulated by N-starvation in rice. The known genes are involved in several metabolisms including carbon metabolism, secondary metabolite synthesis, ubiquitylation and protein degradation, phytohormone metabolism, signal transduction, growth regulator and transcription factors. Different induced expression patterns based on spatial and temporal express ions were found for these genes. The results indicate the cross-talks between N-starvation response and various metabolisms in plants.

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, 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 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 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, E. coli Expression and Molecular Analysis of a Novel Sesquiterpene Synthase Gene from Artemisia annua

A 1 886 bp full-length sesquiterpene synthase (AaSES) cDNA was cloned front a high-yield Artemisia annua L. strain 001 by a rapid amplification of cDNA end (RACE) strategy. AaSES is 59% identical to Artemisia cyclase cDNA clone cASC125, 50% identical to epi-cedrol synthase from A. annua , 48% identical to amorpha-4, 11-diene synthase from A. annua, 39% identical to the 5-epi-aristolechene synthase from tabacco, 38 % identical to vetispiradiene synthase front H. muticus, 41 % identical to the, delta-cadinene synthase from cotton. The coding region of the cDNA was inserted into a procaryotic expression vector pET-30a and overexpressed in E. coli BL21 ( DE3). The cyclase proteins extracted front bacterial culture were found largely in an insoluble protein fraction. AaSES expresses in leaves, stems a-rid flowers, not in roots as indicated by Northern blotting analysis.

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.

Cloning and Expression Patterns of a Metallothionein-like GenehtMT2 of Helianthus tuberosus

A novel cDNA sequencehtMT2, which encodes a type 2 metallothionein_like protein, was isolated from Helianthus tuberosus L. tuber cDNA library. The whole sequence is 509 bp, including an open reading frame (ORF) of 240 bp, a 5′ UTR of 62 bp and a 3′ UTR of 207 bp. Two genomic sequences covering the coding region ofhtMT2were cloned by PCR reaction. Sequence analysis revealed that the genomic sequences htMTG_1 of 986 bp and htMTG_2 of 982 bp were both composed of three exons and two introns. The deduced protein consisted of 79 amino acid residues with a predicted molecular weight of 7.8 ku (kD). Amino_terminal and carboxy_terminal domains contained 8 and 7 cysteine residues respectively, separated by a central cysteine free spacer. Sequence alignment revealed that the predicted protein ofhtMT2 was homologous to type 2 metallothioneins (MTs) of plants. Southern blotting analysis indicated that htMT2was encoded by a small multi_gene family in H. tuberosus genome. Northern blotting analysis showed that htMT2 transcripts were detected in stems, leaves and leafstalks, but no transcripts were detected in roots. The expression level in stems was the highest among the above tissues. Transcripts in stems were significantly reduced by Cu 2+ treatment. Judging from the homologies between the deduced HtMT2 and other type 2 plant metallothioneins as well as responses to metal ions, we believe thatwere cloned by PCR reaction. Sequence analysis revealed that the genomic sequences htMTG_1 of 986 bp and htMTG_2 of 982 bp were both composed of three exons and two introns. The deduced protein consisted of 79 amino acid residues with a predicted molecular weight of 7.8 ku (kD). Amino_terminal and carboxy_terminal domains contained 8 and 7 cysteine residues respectively, separated by a central cysteine free spacer. Sequence alignment revealed that the predicted protein ofhtMT2 was homologous to type 2 metallothioneins (MTs) of plants. Southern blotting analysis indicated that htMT2was encoded by a small multi_gene family in H. tuberosus genome. Northern blotting analysis showed that htMT2 transcripts were detected in stems, leaves and leafstalks, but no transcripts were detected in roots. The expression level in stems was the highest among the above tissues. Transcripts in stems were significantly reduced by Cu 2+ treatment. Judging from the homologies between the deduced HtMT2 and other type 2 plant metallothioneins as well as responses to metal ions, we believe that[ShtMT2 encodes a new type 2 metallothionein.

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 Analysis of Violaxanthin De-Epoxidase (VDE) cDNA in Wheat

Violaxanthin de-epoxidase (VDE) is the key enzyme in the xanthophyll cycle and protects plant photosynthetic apparatus from the damage of excessive light. A wheat (Triticum aestivum L cv. Xiaoyan 54) VDE cDNA was obtained using RT-PCR method. Its deduced protein sequence shares high identity with that of Arabidopsis and rice. Southern blot revealed that there are three copies of VDE gene per haploid genome of wheat. VDE transcript levels were higher in green leaf than in root, seed and etiolated leaf. Northern blotting analysis indicated that VDE mRNA level is induced during greening process of etiolated wheat seedling and increased by intense light illumination.

Cloning and Expression of a Profilin Gene from Rapeseed

Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA clone (designated Repro) encoding profilin gene was isolated from rapeseed ( Brassica napus L. cv. canadian Tween) using RT-PCR technique. Sequence analysis showed 82% similarity to Zea mays L. ZmPro3, 85% to Arabidopsis AthPRF1, 82% to Nicotiana tabacum L. NTPRO, 81% to Oryza sativa L. profilin A. A new full-length cDNA was obtained by 5'-RACE and 3'-RACE techniques. Sequence analysis showed that the size of full-length cDNA is 672 bp which contains a major open reading frame of 134 amino, acids, 5' and 3' untranslated regions and a long Poly (A) tail. Northern blot analysis showed that the profilin gene is a pollen and anther specific gene.

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.

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 Differential Gene Expression of Two Catalases in Suaeda salsa in Response to Salt Stress

Two different cDNA clones (Sscat1 and Sscat2) encoding catalase, the primary important H2O2-scavenging enzyme, were isolated from a AZap-cDNA library constructed from a 400 mmol/L NaCl-treated library of Suaeda salsa ( L.) Pall aerial tissue. Sscat1 (1.7 kb) contains a full open reading frame of 492 amino acids and Sscat2 (1.1 kb) is a partial clone. BLAST analysis indicates that the two clones share 71.9% identity in nucleotide sequence and 75% identity in deduced amino acid sequence within the last 287 amino acid residues of Sscat1. Southern blotting analysis showed that Sscat1 is multicopy in S. salsa genome, while Sscat2 is a single copy gene. Northern blotting analysis showed a rapid increase in the steady-level of both genes in roots after 48 It salt treatment, but only Sscat1 was induced in salinity treated leaves. Time-course analysis carried out in leaves confirmed that Sscat1 was induced by salt stress, in contrast to Sscat2. These implied that the expression of Sscat1 and Sscat2 genes are differentially regulated in S. salsa. The activity of total catalase is dramatically increased in response to salt stress.

越南莫侬人的驯象习俗

越南北方的多乐省达克隆河中游是一片宽阔的河谷地带,树林茂密青草茵茵,是大象理想的栖息地。这里栖息着许多大象,有的象群多达上百头。世代居住在达克隆河流域的莫侬人以其熟练的捕象驯象技术著称。据说,莫侬人捕象驯象活动已有近百年历史。起初,为了保护庄稼,人们设陷阱捕获了一头小象。后来,经过精心驯养,小象长大能替人运粮食搬木料,并成为主人出远门代步工具。

Expression and Purification of E.coli NrfA Protein and Preparation of Polyclonal Antibody against NrfA

[Objective] This study aimed to clone the E. coil NrfA gene and construct the pET-28a （＋）-NrfA prokaryotic expression vector for preparation of polyclonal anti- body against E. coil NrfA. [Method] E. coil NrfA gene was cloned from the E. coli genome DNA by PCR and inserted into the vector pET-28a（＋） to construct prokary- otic expression vector pET-28a （＋）-NrfA. E. coil NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by im- munizing rabbit with routine method. The specificity and titer of polyclonal antibody was confirmed by ELISA and Western Blotting. [Result] The constructed prokaryotic expression vector pET-28a（＋）-NrfA was induced by IPTG, the recombinant NrfA pro- tein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained by immunization and purification was about 1：204 900. Western Blotting anal- ysis indicated that the obtained polyclonal antibody against E. coil NrfA protein had high titer and high specificity. [Conclusion] E. coil NrfA gene was cloned and the prokaryotic expression vector pET-28a （＋）-NrfA was constructed successfully, poly- clonal antibody with high titer and high specificity was prepared, which laid the foun- dation for the study of NrfA in different strains of bacteria.