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.

cDNA Cloning, Bioinformatic and Tissue-specific Expression Analysis of Porcine JARID1C Gene

Jumonji, AT-rich interactive domain 1C （JARID1C） protein belongs to the highly conserved ARID protein family, which is involved in chromatin remodeling and transcriptional regulation during cell growth, differentiation, and development. In humans, this gene plays a vital role in normal brain development and function. Using an in silico approach in combination with 5＇ rapid amplification of cDNA ends （5＇ RACE）, the full-length cDNA of JARIDIC （GenBank accession No. EF139241） from porcine ovary, which contains 5,908 bp nucleotides, with an open reading frame （ORF） of 4,548 bp, has been cloned. The putative porcine JARID 1C protein, which is located in the nucleus, encodes 1,516 amino acids with a molecular weight of 170 kDa and a pI of 5.44. Bioinformatic prediction indicates that the protein contains several conserved domains： a JmjN domain, an ARID domain, a JmjC domain, a C5HC2 zinc finger domain, and a PHD zinc finger domain. Similarity comparisons for nucleic and amino acid sequences reveal that the porcine JARID1C protein shares a high identity with its dog, mouse, rat, and human counterparts. The phylogenetic tree of the JARID1 subfamily proteins has been constructed to reveal the evolutionary relationship of various species. Real-time PCR analysis shows that the JARIDIC gene is expressed in various tissues, but at different levels. The expression levels of this gene are higher in the brain and gonad than in other tissues, suggesting that the JARID1C protein plays a role in porcine brain and gonad functions.

Gene Cloning and Expression Analysis of G Protein αq Subunit from Helicoverpa assulta (Guenée)

The cDNA encoding the G protein αq subunit was isolated from the antennae of Helicoverpa assulta （Guen6e） by reverse transcription polymerase chain reaction （RT-PCR） and named as HassGαq. Sequencing analysis showed that the fulllength of HassGαq open reading frame （ORF） is 1 062 bp, 353 amino acid residues are encoded. The predicted molecular weights （MW） and isoelectric point （PI） are 41.5 kD and 5.15, respectively. HassGαq gene was then constructed into expression vector pGEX-4T-2 for over expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that induced by Isopropyl-β-D-Thiogalactoside （IPTG）, the GST-HassGαq fusion protein is expressed in Escherichia coil BL21, and its MW was found to be about 66 kD nearly equal to the predicted. In addition, RT-PCR analysis showed that the expressions of HassGαq are not tissue specific.

Molecular Cloning and Expression Analysis of an Actin Gene from Chinese Licorice(Glycyrrhiza uralensis Fisch)

A PCR-based homologous cloning strategy was used to identify an actin gene from the roots of Chinese licorice（Glycyrrhiza uralensis Fisch）. Results of sequence analysis indicate that a 1137 bp cDNA with an open reading frame encoding 377 amino acids, actin ortholog, GuActin, was successfully cloned and characterized（GenBank accession No. EU190972）. Thus far, GuActin is the first actin of Chinese licorice that has been identified at a molecular level. Analysis by Northern blot shows that GuActin was expressed strongly in the roots, particularly in radicles than in stems and leaves. These results suggest that GuActin may be a member of the vegetative subfamily of the actin family.

Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

Molecular Cloning and Expression Analysis of a Cys2/His2 Type Zinc Finger Protein Gene in Upland Cotton

The zinc finger proteins belong to the largest family of transcription factors.But there is little research of Cys2/His2 type zinc finger proteins in cotton,and there is no submission of correlating

Molecular cloning and expression analysis of Crustin-like gene from Chinese shrimp Fenneropenaeus chinensis

A new member of antimicrobial protein genes of the Crustin family was cloned from haemocytes of the Chinese shrimp Fennero- penaeus chinensis by 3 ′and 5′ RACE. The full-length cDNA of Crustin-like gene contains a 390 bp open reading frame, encoding 130 amino acids. The deduced peptide contains a putative signal peptide of 17 amino acids and mature peptide of 113 amino acids. The molecular mass of the deduced mature peptide is 12. 3 ku. It is highly cationic with a theoretical isoelectric point of 8.5. The deduced amino acids sequence of this Crustin showed high homology with those of Penaeus （ Litopenaeus ） setferus. Northern blotting showed that the cloned Crustin gene was mainly expressed in haemocytes, gill, intestine, and RNA in situ hybridization indicated that the Crustin gene was constitutively expressed exclusively in haemocytes of these tissues. Capillary elec- trophoresis RT-PCR analysis showed that Crustin was up-regulated dramatically from 12 to 48 h after a brief decrease of mRNA during first 6 h in response to microbe infection. The level of Crustin mRNA began to restore at 72 h post-challenge. This indicated that Crustin gene might play an important role when shrimps are infected by bacterial pathogen.

Gene cloning and prokaryotic expression of recombinant flagellin A from Vibrio parahaemolyticus

The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene （tTaA） from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can he used for further functional and structural studies.

Gene cloning and expression analyses of WBC genes in the developing grapevine seeds

White-brown complex （WBC） transporters, also called half-size ATP binding cassette G （ABCG） transporters, are involved in many biological processes, including seed development; however, the WBC transporters in grapevines received less attention to date. To reveal the molecular characteristics of WBCs and the connection between WBCs and agronomic traits of stenospermocarpic （seedless） grapevine, we carried out a genomic census and analysis of ovule-associated expression for VvWBC genes in grapevine. We identified 30 VvWBC genes and cloned full-length complementary DNAs （cDNAs） for 20 of these. The tissue or organ-specific expression analysis showed that several VvWBCs exhibited distinct expression patterns with some showing tissue specificity. Twelve VvWBC genes were found to be expressed in the developing ovules. Moreover, the results of quantitative real-time PCR （qRT-PCR） suggested that four of twelve ovule-expressed VvWBCs have distinct expression profiles during the development of ovules between seeded and stenospermocarpic grapevines. These four genes might be involved in ovule abortion. Meanwhile, chromosome mapping, multiple sequence alignments, exon/intron structure analyses and synteny analyses were preformed on VvWBC genes. Our experiments provide a new perspective on the mechanism of stenospermocarpic seedlessness and put forward a framework for further study of WBC transporters.

Analysis of Cloning and Expression Characteristics of Capsicum chinense Jacq. CcMYB Gene

In order to discuss the role of MYB gene in capsaicine synthesis process, one CcMYB gene was cloned from Capsicum chinense Jacq. by RT-PCR. Its cDNA has a total length of 1 038 bp, and was speculated to code 345 amino acids, comprising an complete open reading frame. The isoelectric point is 8.57, and the molecular weight is 38.2 KD. The protein is a neutral hydrophobin without transmentbrane structure. There are two MYBDNA domains at the N terminal. The fluorescence quantitative PCR results showed that CcMYB gene was expressed in all the root, stem, leaf, flower, placenta and fruit tissue of pepper, and the expression level was the highest in fruit ; and CcMYB was expressed in fruit at the highest level at turning stage, and at the second highest level at expansion stage, which accords with the expression profile of punl gene in fruit development period. It is speculated that CcMYB gene plays an important role in the regulation of capsaicine synthesis in C. chinense fruit.

Molecular cloning and expression under abiotic stresses and hormones of the ethylene response factor Ⅶ gene FmRAP2.12 from Fraxinus mandshurica

RELATED TO AP2.12(RAP2.12)is one of the Ethylene Response Factors(ERF)transcription factor and plays a key role in controlling plant root bending and responding to multiple abiotic stresses including hypoxia stress.In this study,FmRAP2.12 gene was isolated and characterized from Fraxinus mandshurica Rupr.The open reading frame(ORF)of FmRAP2.12 was 1170 bp and encoded a protein of 389 amino acids.The conserved domains,three-dimensional phylogenetic relationship of FmRAP2.12 was also investigated.Quantitative real-time(qRT-PCR)analyzed the expression of FmRAP2.12 in different tissues.The expression level of FmRAP2.12 was highest in roots followed by leaves,and lowest in male flowers.Abiotic stress and hormone signal-induced expression was established using qRT-PCR.Salt stress induced FmRAP2.12 to a double peak pattern:the first peak value was at 6 h and the second at 72 h.Drought stress also induced FmRAP2.12 to a double peak pattern:the first at6 h and the second at 48 h.FmRAP2.12 was up-regulated after initiation of gibberellic acid(GA3)treatment,with a one peak pattern at 24 h.FmRAP2.12 may not respond to cold stress and Abscisic acid(ABA)treatment.The transient overexpression of FmRAP2.12 caused the up-expression of downstream key genes of abiotic stress response and gibberellin pathway.Our research reveals the molecular characteristic and expression patterns under abiotic stress and hormone condition of FmRAP2.12,providing support for the genetic improvement of F.mandshurica at a molecular level.

Molecular Cloning and Expression Analysis of a FT Homologous Gene from Solanum tuberosum

A homologue of flowering locus T gene, designated StFT, was isolated from Solanum tuberosum by reverse transcriptasepolymerase chain reaction （accession no. GU223211）. The DNA sequence of StFT was 1 626 bp long and contained four exons and three introns. The open reading frame of the gene was 522 bp long and encoded a putative protein of 173 amino acids with a molecular weight of 19.75 kD and a theoretical pI of 7.76. StFT protein had a conserved PBP domain and a higher degree of identity with FT homologous members from other species. Analysis on the mRNA levels of StFT showed that it was highly expressed in leaves, apical buds, flowers, and swelling stolons. Further analysis indicated that its expression was regulated by CONSTANS gene in StCOL-antisense transgenic potato plants.

Molecular cloning, sequence analysis, and cadmium stress-rated expression changes of BTG1 in freshwater pearl mussel(Hyriopsis schlegelii)

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.

cDNA Cloning, Prokaryotic Expression of Two Splicing Products of mLRG, a Mouse Gene of Lipopolysaccharide Response

Aim: To clone two splicing products of the mouse mLRG-cDNA and to express mLRG protein. Methods: The sequence obtained was compared human lrg to mouse genome with a comparative BLAST genome search and found completely identical. We spliced some fragments to a whole mouse lrg-cDNA sequence and designed a pair of primers at completely homologous fragments in 5’-UTR and 3’-UTR, we amplified mouse lrg-cDNA by RT-PCR. Then the sequence encoding the mLRG protein was amplified by RT-PCR from the total RNA of NIH3T3 cell stimulated by lps (lipopolysaccharide), and we got two splicing products of mLRG (mLRGW, mLRGS) and two sequences encoding protein were cloned into the prokaryotic expression vector pTAT so as to construct the recombinant expression vector pTAT-MLRGW and pTAT-MLRGS. The proteins were expressed in E. coli BL21 (DE3). Results: We got a cDNA fragment with the length of 1905 bp. Its location is at chromosome X qF4 site and we amplified two encoding regions covered 1554 bp and 1404 bp respectively (mlrgW mlrgS). His-TAT-mLRGW and His-TAT-mLRGS fusion protein were expressed successfully. mlrgW is consist of 10 exons and 9 introns;mlrgS is consist of 11exons and 10 introns. Conclusion: Cloning of two splicing products of mouse novel gene MLRG and prokaryotic protein expressions are of help in the further study of this gene.

Molecular cloning and expression analysis of interleukin - 1β from Japanese sea perch(Lateolabrax japonicus )

The technology of homology cloning and anchored PCR was used to clone the IL-1β gene from the Japanese sea perch （Lateolabrax iaponicus）. The full-length cDNA of sea perch IL-1β was 1 310 bp, including a 5＇ untranslated regiop （UTR） of 136 bp, a 3＇ UTR ot 430 bp, and an ORF of 774 bp encoding a polypeptide of 258 amino acids with an estimated molecular mass of 29.31 kDa. The searches for nucleotides and protein sequence similarities with the BLAST analysis indicated that the deduced amino acid sequence of sea perch IL-1β was homological to the IL-1β in other fish species and even the mammalian. Conserved signature sequences of the IL-1β gene family were found in the sea perch IL-1β deduced amino acid sequence. Temporal expressions of the IL-1β gene in LPS or iridovirus challenged group and in control group were measured by the semi-quantitative RT-PCR. The mRNA transcripts of IL-1β could be detected in head-kidney, spleen, liver, gill and heart of the healthy individuals, and the expression level of IL-1β in head-kidney, spleen and gill was higher than that in liver and heart, but it was hard to be detected in the brain. After being stimulated by the LPS or iridovirus, the IL-1β expression in most of examined tissues was up-regulated, and also could be detected in the brain. These results indicated that the expression of sea perch IL-1β was constitutive and could be up-regulated by immune effector stimulation. Therefore the sea perch IL-1β could play a critical role in the host-pathogen interaction.

Molecular Cloning of IGFBP-1 Gene and Developmental Expression of Its mRNA in Different Tissues of Nanjiang Mongolian Gazelles

The objective of the present study was to investigate the developmental expression patterns of Insulin-like growth factor-binding protein 1 (IGFBP-1) gene in different tissues of postnatal Nanjiang Mongolian Gazelles. Samples of heart, liver, spleen, lung, longissimus dorsi, semimembranosus, m. triceps brachii and biceps muscle of thigh were collected from a total of 36 Nanjiang Mongolian Gazelles at the age of 0, 15, 30, 60, 90 and 120 days after birth (3 males and 3 females at each age). The CDS was sequenced and ontogeny of mRNA levels of IGFBP-1 were measured by real-time fluorescence quantitative RT-PCR. The size of IGFBP-1 ORF was 792 bp encoding 263 amino acid residues, and displayed higher nucleotide/amino acid sequence identities with other ruminants compared to non-ruminants. The levels of IGFBP-1 mRNA in liver were highest (P<0.01), levels were medium in lung, spleen and heart, and the lowest in the muscles; there were no significant differences among the muscles (P>0.05). Three expression patterns of IGFBP-1 mRNA during postnatal growth from birth to day 60 were found: consistently decreasing (liver), fluctuating as increasing then decreasing (heart) or as decreasing then increasing then decreasing (spleen, lung and muscles). The results indicate that the IGFBP-1 gene is highly conserved among species, and liver has the highest expression. It was concluded that IGFBP-1 plays important roles in early postnatal growth and is expressed in a developmental-tissue-dependent manner.

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,Characterization and Expression Profile of Myf5 and Myf6 During Growth and Development in the Seriola lalandi

Myogenic Regulatory Factors(MRFs)is involved in the muscle growth and differentiation.In this study,the cDNA sequence of yellowtail kingfish MRFs genes were cloned by rapid amplification of cDNA ends(RACE)method;then,the character-istics of these genes and the predicted protein sequences were analyzed by bioinformatics methods,the tissue and embryonic stages differential expression pattern were detected by the quantitative real-time PCR.Our results showed that the yellowtail kingfish(YTK)Myf5 cDNA has a full length of 951 bp,encoding 266 amino acids.The yellowtail kingfish Myf6 cDNA has a full length of 1105bp,encoding 250 amino acids.The proteins containα-helix,β-strand,and loops.The Neighbour-joining tree revealed that YTK Myf5 and Myf6 are closely related to Seriola dumerili.The yellowtail kingfish Myf5 and Myf6 gene expressed significantly higher in mus-cle than in other tissues(P<0.05).In addition,Myf5 and Myf6 in muscle was significantly expressed in 400g and 500 g fish but not in 50 g,suggesting that myogenic regulatory factors expression had a great relationship with the fish size.Our results also indicated that Myf5 and Myf6 have different functions during embryonic development,because Myf5 showed highest expression level at the neuroembryo period,but Myf6 had the highest expression level at embryo coverage yolk 70%stage.Myf5 gene showed highest ex-pression at 30 d of age,suggesting it played key roles in myogenic period.However,the Myf6 gene was significantly highly ex-pressed at 60 d,revealing this gene functioned in the later muscle formation period.

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 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.