Efficient transformation and expression of gfp gene in the edible mushroom Pleurotus nebrodensis

An efficient transformation method mediated by PEG-protoplasts was developed for the newly commercial edible mushroom Pleu-rotus nebrodensis. Two plasmids were used to co-transform protoplasts of P. nebrodensis. One plasmid is pAN7-1 containing a positive selectable marker gene hph conferring hygromycin B resistance. Another plasmid is pBIue-GFP containing a reporter gene gfp conferring green fluorescent protein. PCR and Southern blot analysis showed that hph gene or/and gfp gene were integrated into the genome of P.nebrodensis transformants. The transformation efficiency of the positive selectable marker gene hph was 3 transformants per microgram of plasmid pAN7-1 DNA, which was about 30 times higher than that previously reported in thoroughly studied Pleurotus species such as Pleurotus ostreatus. The transformation efficiency of the reporter gene gfp was 9 transformants per microgram of plasmid pBlu-GFP DNA. The co-transformation efficiency was 23.68%. This is the first report that a "reporter" gene, green fluorescent protein gene can be successfully stably expressed in this Pleurotus species.     

Expression of green fluorescent protein gene with baculovirus vector in insect cells

The green fluorescence of bioluminescent jellyfishAequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10³ dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus-insect cell expression system. Supported by the National Natural Science Foundation of China Hu Jianhong: born in July, 1972, Master graduate student     

Efficient transformation and expression of gfp gene in the edible mushroom Pleurotus nebrodensis

An efficient transformation method mediated by PEG-protoplasts was developed for the newly commercial edible mushroom Pleurotus nebrodensis. Two plasmids were used to co-transform protoplasts of P. nebrodensis. One plasmid is pAN7-1 containing a positive selectable marker gene hph conferring hygromycin B resistance. Another plasmid is pBlue-GFP containing a reporter gene gfp conferring green fluorescent protein. PCR and Southern blot analysis showed that hph gene or/and gfp gene were integrated into the genome of P. nebrodensis transformants. The transformation efficiency of the positive selectable marker gene hph was 3 transformants per microgram of plasmid pAN7-1 DNA, which was about 30 times higher than that previously reported in thoroughly studied Pleurotus species such as Pleurotus ostreatus. The transformation efficiency of the reporter gene gfp was 9 transformants per microgram of plasmid pBlu-GFP DNA. The co-transformation efficiency was 23.68%. This is the first report that a ＂reporter＂ gene, green fluorescent protein gene can be successfully stably exoressed in this Pleurotus species.     

Two virus-encoded RNA silencing suppressors, P14 of Beet necrotic yellow vein virus and S6 of Rice black streak dwarf virus

Functional analysis for gene silencing suppressor of P14 gene of Beet necrotic yellow vein virus and S6 gene of Rice black streak dwarf virus was carried out by agro- infiltration with recombinant vectors of Potato virus X. The phenotype observation of green fluorescent protein (GFP)expression and Northern blot showed that the gene silencing of gfp transgenic Nicotiana benthamiana induced by homologous sequence was strongly suppressed by the immixture infiltration of either the P14 or the $6. In the suppressed plants, the gfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially the gfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results suggested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors.     

Expression ofChlamydomonas actin-gfp fusion gene in tobacco suspension cell and polymerization of the actin-gfp proteinin vitro

The fusion gene of actin (cDNA ofChlamydomonas reinhardtii) and green fluorescence protein (gfp) had been constructed into two expression vectors which could be expressed inE. coli and tobacco suspension cells BY2. The correct expression was observed inE. coli and BY2 with a fluorescence microscopy. The fusion protein, which took part in the membrane skeleton, was mainly located peripherally along the membrane, specially the fusion protein was distributed around nucleus and cell plate, while the fusion protein also forms F-actin in the cell. The fusion protein was purified from Bl21plus by ammonium sulfate fractionation, ion exchange chromatography and hydrophobic interaction chromatography. The purified production could polymerize into F-actin when the actin polymerizing buffer was added. It was demonstrated that the characteristics and function of actin inChlamydomonas was similar with those of animals and higher plants.     

Cloning and expression of HIV-1 p24 gene in insect cells by using BAC-TO-BAC system

Recombinant transposing vector pFHIV24 was constructed by cloning the HIV-1 p24 gene into the Multiple cloning site (MCS) of the transposing vector pFastBac1 in the correct orientation with respect to the polyhedrin promoter. Recombinant bacmid bHIV24 was obtained by transposing a mini-att Tn7 element from the recombinant pFHIV24 to the mini-att Tn7 attachment site on the bacmid by Tn7 transposition functions provided by the helper plasmid. Minipreparation of recombinant bacmid DNA was transfected intoSpodoptera frugiperda (Sf9) cells to get the recombinant virus. Fresh insect Sf9 cells were infected with the recombinant virus containing p24 to express the target protein. The target protein expressed was analyzed on a 15% polyacrylamide gels and then used as antigen to check HIV-1 positive serum by ELISA. Our positive result shows that the expressed p24 protein could be used as standard antigen for HIV-1 diagnosis by ELISA and other reliable diagnostic methods of HIV-1 infection. Supported by the World Bank Boan Program Mallam Nock Joshua: born in 1967, Master of Science To whom correspondence should be addressed (027-7882712-2938)     

Cloning and expression of HIV-1 p24 gene in insect cells by using BAC-TO-BAC system

Recombinant transposing vector pFHIV24 was constructed by cloning the HIV-1 p24 gene into the Multiple cloning site (MCS) of the transposing vector pFastBac1 in the correct orientation with respect to the polyhedrin promoter. Recombinant bacmid bHIV24 was obtained by transposing a mini-att Tn7 element from the recombinant pFHIV24 to the mini-att Tn7 attachment site on the bacmid by Tn7 transposition functions provided by the helper plasmid. Minipreparation of recombinant bacmid DNA was transfected intoSpodoptera frugiperda (Sf9) cells to get the recombinant virus. Fresh insect Sf9 cells were infected with the recombinant virus containing p24 to express the target protein. The target protein expressed was analyzed on a 15% polyacrylamide gels and then used as antigen to check HIV-1 positive serum by ELISA. Our positive result shows that the expressed p24 protein could be used as standard antigen for HIV-1 diagnosis by ELISA and other reliable diagnostic methods of HIV-1 infection. Supported by the World Bank Boan Program Mallam Nock Joshua: born in 1967, Master of Science To whom correspondence should be addressed (027-7882712-2938)     

Introduction of exogenous DNA into cotton via the pollen-tube pathway with GFP as a reporter

The green fluorescent protein (GFP) gene from the jellyfishAequorea victoria as a vital reporter for gene expression in plants is considered to have several advantages over other reporter genes. The pBIN35S-mGFP4 plasmid DNA has been introduced into cotton embryos by the pollen-tube pathway method. A transformed seedling has been verified according to its GFP-related fluorescence and Southern blotting analysis. The results provided direct and convincing facts in cytology and molecular biology for the pollen-tube pathway method, an efficient transformation technique used in plants.     

Testes and chromosomes in interspecific hybrids betweenHelicoverpa armigera (Hübner) andHelicoverpa assulta (Guenée)

The interspecific hybridization betweenHelicoverpa armigera females andHelicoverpa assulta males yieldedF ₁ hybrids (RS), fertile males and sterile individuals with abnormal genitals. The reverse hybridization betweenH. assulta females andH. armigera males yielded F₁ hybrids (SR)-fertile males and fertile females. The morphology of testes and the karyotype of chromosomes of larvae in the hybrids were investigated. Among the 2d old fifth-instar SR larvae, individuals without testes were fertile females and those with testes were fertile males. The length and breadth of testes between SR and parental species were not significantly different (p>0.05). Among the 2d old fifth-instar RS larvae, the testes were observed in all the individuals, but it could be classified into two types. The length and the breadth of testes in Type 1 larvae were not significantly different from those of their parental species (p>0.05), while those in Type 2 were significantly less than those of their parental species (p<0.01). Mitotic metaphase I of brain cells showed the diploid chromosomes number of both reciprocal hybrids was 2n=62, as many as their parents. The haploid number of 31 was confirmed by counts from spermatocytes at meiotic metaphase from SR male larvae and Type 1 larvae of RS. Meiosis was not observed in spermatocytes of Type 2 larvae of RS. Considering the characteristics of adult hybrids of RS, it was concluded that Type 1 individuals in RS were fertile and those of Type 2 were sterile. The sterility of Type 2 individuals in RS is attributed to the abnormity in development of testes and the failing meiosis of spermatocytes. As a result, the normal spermatozoon could not been produced.     

pEGFP-C1-MCH真核表达载体的构建及其在HEK293 细胞系中的表达

目的:构建pEGFP-C1-MCH真核表达载体,并将其转染入HEK293细胞中,筛选阳性细胞克隆,为研究MCH基因在能量代谢中的功能及机制提供细胞模型.方法:提取脑组织总RNA,反转录为cDNA,参照Genbank中提供的序列设计引物扩增MCH基因全长.再将该基因全长cDNA克隆至质粒pEGFP-C1,经菌落PCR筛选及双酶切和DNA测序鉴定,成功构建了含有目的基因MCH的重组质粒pEGFP-C1-MCH.并利用脂质体2000介导其转染HEK293细胞,用荧光显微镜和RT-PCR检测EGFP和MCH在细胞中的表达.结果:克隆的pEGFP-C1-MCH质粒序列中的MCH与Gen Bank相符;细胞转染72 h后,转染成功的细胞在荧光显微镜下表达较强的绿色荧光,MCH基因稳定表达.结论:pEGFP-C1-MCH真核表达载体的构建及其在HEK293细胞中的稳定表达,为研究MCH基因在能量代谢中的功能及作用机制提供了实验模型.     

Production of human alpha-interferon in silkworm using a baculovirus vector

Microorganisms are generally used for mass production of foreign gene products, but multicellular organisms such as plants have been proposed as an economical alternative. The silkworm may be useful in this context as it can be cultured easily and at low cost. We have therefore developed a virus vector to introduce foreign genes, for example, the gene for human alpha-interferon (IFN-alpha), into silkworms. We used the baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) which has a large (greater than 100 kilobases, kb) double-stranded circular DNA genome within its rod-shaped capsid. Baculoviruses have been used previously as vectors for expression of beta-interferon and beta-galactosidase in established cell lines. Although BmNPV has not been used previously as an expression vector, it has an advantage over the baculovirus Autographa californica NPV in that it has a narrower host range and will not grow in wild insect pests in the field. In the present study, the polyhedrin gene encoding the major inclusion body protein of BmNPV was identified by hybridization with complementary DNA and cloned in a plasmid. For insertion of foreign genes, we constructed a recombinant plasmid carrying a polylinker linked to the promoter of the polyhedrin gene, and inserted the IFN-alpha gene into this plasmid. The resulting plasmid and the BmNPV genomic DNA were co-transfected into BM-N cells, and stable recombinant viruses isolated by plaque assay on BM-N cells. The recombinant virus replicated in silkworm larvae, which synthesized as much as 5 X 10(7) units (approximately 50 micrograms) of interferon in their haemolymph.     

Construction of DNA damage response gene pprI functiondeficient and function-complementary mutants in Deinococcus radiodurans

PprI, a DNA damage response factor from the extraordinary radioresistant bacterium Deinococcus radiodurans, plays a central regulatory role in multiple DNA damage repair. In this study, a fusion DNA fragment carrying kanamycin resistance gene with the D. radiodurans groEL promoter was cloned by PCR amplification and reversely inserted into the pprl locus in the genome of the wild-type strain R1. The resultingpprl-deficient strain, designated YR1,was very sensitive to ionizing radiation. Meanwhile, the recombinant DNA fragment was cloned into the shuttle vector pRADZ3, and resulted in plasmid pRADK with kanamycin resistance in D. radiodurans. The fragments containing complete pprl gene and 3‘-terminal deletion pprIΔ were cloned into plasmid pRADK. The resulted plasmids designated pRADKpprI and pRADKpprIΔ were then transformed to YR1. Results show that YR1 carrying pRADKpprI was able to fully restore the extreme radioresistance to the same level as the wild-type D. raiodurans R1, whereas YR1 pRADKpprIΔ failed to do so. Construction of DNA repair switch PprI function-deficient and function-complementary mutants in D. radiodurans is not only useful to elucidating the relationship between domains and functions of PprI protein, but also opens the door to the further studies of the biological functions of PprI protein in vivo.     

Reconstruction of HaSNPV with helicoverpa hormone receptor 3

In order to develop a more efficient virus for controlling the cotton bollworm Helicoverpa armigera, Helicoverpa hormone receptor 3 （HHR3）, which is involved in the ecdysteroid regulatory pathway, was used to genetically modify wild HaSNPV. HaSNPV-HHR3 budded virus and occlusion body virus were constructed in three steps： preparation of pFastBacHaPhpP10-HHR3 donor plasmid, transposition of HHR3 into the HaBacHZ8 bacmid, and transfection of HzAM1 cells to get HaSNPV-HHR3 virus.HHR3 was proved to be expressed in the HaSNPV-HHR3 virus infected HzAM1 cells by immunoblotting. Results of bioassay indicated that the body weight of the HaSNPV-HHR3 infected larvae was lower than the larvae infected with wild virus and uninfected normal larvae, which suggests that HaSNPV-HHR3 delayed larval growth.     

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic loach (Misgurnus anguillicaudatus Cantor) have been studied. TheGFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of theGFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.     

线粒体定位序列介导的绿色荧光蛋白基因在烟草中表达的分析

由于绿色荧光蛋白可在活组织或细胞中直接检出 ,因而近年已在转基因植物的研究中用作报告基因 ,这样可在植物生长的任何阶段进行活体筛选和鉴定。本研究利用线粒体定位序列对改良 gfp基因在转基因烟草中的表达进行了观察 ,结果表明 :将GFP直接在细胞质中大量表达会对植物细胞产生毒性 ,从而影响植物细胞的分化 ,而将其定位在线粒体中 ,则从转化细胞产生植株的频率明显增高。     

Construction and function of recombinant AcMNPV with double copies of v-cath gene

Two recombinant baculoviruses, dciAcMNPV and dcdAcMNPV in which another copy of the v-cath gene controlled by ie1 promoter and polh promoter was inserted, were respectively constructed by the Bac-to-Bac system. The expression of the v-cath gene of the recombinant baculoviruses in Sf9 cells at different phases was investigated by SDSPAGE and Western blot. The results showed that only recombinant virus dciAcMNPV containing late gene v-cath driven by early gene promoter could express V-CATH protein, cathepsin encoded by virus genome, 12 h post-infection and dcdAcMNPV containing late gene v-cath driven by late and very late gene promoters could express more V-CATH protein. Negative control ncAcMNPV, a mutant deleted vcath gene, could not express V-CATH protein at all. The Spodopera exigua larvae were infected with viruses respectively and the results showed that the toxicity was as follows: dcdAcMNPV>dciAcMNPV>wtAcMNPV>ncAcMNPV. The toxicity of recombinant viruses and the characters of dead larvae showed that the v -cath gene was relative to viral toxicity and host liquefaction. Recombinant baculovirus dcdAcMNPV might be used as a new kind of safe viral-pesticide, because of its high toxicity obtained by adding another gene copy and changing the expression level of its own gene relative to virulence.     

汉滩病毒囊膜糖蛋白g2基因重组腺病毒的构建与表达

获得汉滩病毒G2 基因 ,构建其重组腺病毒并在HEK2 93细胞中包装表达 ,为研究汉滩病毒基因疫苗提供了实验基础。设计引物采用PCR从含汉滩病毒 \|76 1 1 8株M基因的M5 6质粒扩增出糖蛋白G2 基因片段 ,并将其克隆入腺病毒载体Adeno XviralDNA ,筛选获得重组腺病毒DNA ,转染HEK2 93细胞 ,包装、扩增后得到汉滩病毒G2 基因重组腺病毒原种 ;并在感染细胞内初步表达 ,用ELISA检测表达产物。得到了含汉滩病毒G2 基因的重组腺病毒 ,其滴度约为 1 0 10 pfu/mL ,同时在感染的HEK2 93细胞中检测到汉滩病毒糖蛋白G2 的表达。含汉滩病毒糖蛋白G2 基因重组腺病毒的成功构建 ,为研究汉滩病毒基因疫苗提供了实验基础     

Molecular characterization of diapause hormone inHelicoverpa armigera

Diapause hormone (DH) is a neurohormone which is secreted from suboesophageal ganglion and responsible for induction of embryonic diapause in many insects. Using rapid amplification of the cDNA ends (RACE) method, the cDNA encoding diapause hormone inHelicoverpa armigera, a main kind of crops pest was cloned. The nucleotide sequence reveals that the mRNA encodes an open reading frame and the 25-aa DH peptide is localized at N-terminal region just after the signal peptide. A homology search showed thatH. armigera DH has high homology with the diapause hormone ofBombyx mori andHelicoverpa zea; and it also belongs to the FXPRL neuropeptide family. Thus,H. armigera DH seems to be a new type of diapause hormone molecule.     

Construction and characterization of partiallyntrC-deleted mutants inAlcaligenes faecalis

To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially ntrC-deleted mutants of A. faecalis have been generated. To start with, the ntrC gene of A. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. The ntrC gene in pSUM1 was then replaced by a lacZ-Km^r fragment resulted in the generation of a plasmid pSUM2. The lacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type of A. faecalis A1501 by conjugation and two partially ntrC-deleted mutants A15CM1 (ntrC∷lacZ) and A15CM2 (ntrC ^- ) were obtained. To understand the regulatory effect of the NtrC on the expression of nifH and nifA, a nifH-lacZ gene or a nifH-lacZ gene was introduced into the ntrC^- mutant by conjugation. The results indicated that: (ⅰ) although the ntrC^-mutant was nif ⁺ , its nitrogen fixation activity was only 20% that of the wild type; (ⅱ) the ntrC^- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (ⅲ) the regulation of ntrC gene expression did not require its own product; (ⅳ) the expression of nifH in A . faecalis was positively regulated by the ntrC. Deletion of the ntrC resulted in the reduction of nifH expression or even totally inactivated nitrogen fixation; (ⅴ) there was no obvious influence on the expression of nifA in A. faecalis if the ntrC gene was deleted.