Studies on the inheritance of transgenic rice with bar gene

Bar gene driven under the control of CaMV35s promotor was delivered into the immatureembroys of a japonica rice cultivar Jingying 119through biolistic approach. Two putativetransgenic plants were produced, which ex-pressed Basta-resistance. One of the Basta-re-sistance transgenic rices was completely sterile(JY 119-3), the other was self-fertile (JY119-4).

Expression of a barley ABA-responsive protein gene in transgenic rice

A cDNA clone encoding an ABA-responsiveprotein HVA1,was isolated by differentialscreening from barley aleurone layers(Hong etal.).Expression of the HVA1 gene is shownto be developmentally regulated,organ-specif-ic,and ABA-and stress-induced(Hong et

Assessment of Gene Flow Through Detection of SexualCompatibility Between Transgenic Rice with barand Echinochloa crusgalli var.mitis

The possibility of gene flow between two varieties of transgenic rice with bar gene (Y0003 and 99-t) (male) and barnyard grass(Echinochloa crusgalli var. mitis ) (female) was studied by means of reproductive biology. The germination and growth of rice pollen grains on barnyard grass stigmas at 30 min, and 1-4 h after crossing by hand were observed with an optical microscope. The results were compared with the germination and growth of barnyard grass pollen grains at the corresponding time after self-pollination. The results showed that germination and growth of the pollen grains of the two varieties were similar on barnyard grass stigmas, but differed significantly from self-pollination of barnyard grass. Pollen grains germinated and pollen tubes penetrated stigmas normally, and the number of pollen grains being condensing or releasing their inclusions or having released them increased with the time after self-pollination. Pollen grains of transgenic rice on the stigmas of barnyard grass couldn't germinate or grow normally after crossing, neither could they penetrate the stigmas of barnyard grass. Therefore, it could be concluded that the sexual incompatibility between transgenic rice with bar gene and barnyard grass is due to the rice pollen being unable to penetrate the stigma of barnyard grass. Further proof of incompatibility lies in the fact that the emasculated barnyard grass pollinated with the rice pollen grains could not seed.

Transgenic Cotton and Disease Resistance Genes

Success in conventional breeding for resistance to mycotoxin-producing or other phytopathogenic fungi is dependent on the availability of resistance gene(s) in the germplasm.Even when it is available,breeding for disease-resistant crops is very time consuming,especially in perennial crops such

Primary study on the resistance to bacterial blight(X. oryzae)in Cecropin B gene transgenic rices

Bacterial blight (BB) is one of the major dis-eases to rice. Antibacterial Cecropin B genehas been cloned and transformed into rice. Westudied the resistance to bacterial blight inCecropin B gene transgenic rices.Rice variety JYll9 transformed withCecropin B gene by particle bombardment andprogenies were randomly planted in the field in

Improvement of foreign gene expression in transgenic rice(Oryza sativa L.)by matrix attachment regions(MARs)

Matrix attachment regions or matrix associated regions (MARs) were special DNA sequences in chromatin of eukaryotic cells that tightly associated with the nuclear matrix or scaffold in vitro after a combination of nuclease digestion and extraction. They were also called scaffold attachment regions(SARs) . It was found that MARs could improve the expression level and the stability of foreign genes in transgenic plants. The reason might be that a transgene flanked by MARs was transmitted into the plant cells, the MARs would attach the nuclear

An Assessment of Androgenic/Anti-androgenic Effects of GH Transgenic Carp by Hershberger Assay

Objective To evaluate the androgenic and anti-androgenic effects of GH （growth hormone） transgenic carp in male rats.Methods Hershberger assay was carried out in castrated male SD rats aged 4-5 weeks.Testosterone propionate （TP） （0.4 mg/kg BW） was administrated for a positive control,GH transgenic carp （3.0 g/kg BW）＋TP （0.4 mg/kg BW）,parental carp （3.0 g/kg BW） ＋ TP （0.4 mg/kg BW）,and flutamide （Flu） （3.0 g/kg BW） were used for negative controls,and vehicle was administered orally for a blank control.All groups were administrated for 10 consecutive days.At the end of the test,animals were anesthetized,then weights of accessory sex organ were measured.Serum testosterone （T）,luteinizing hormone （LH）,and Follicle-Stimulating Hormone （FSH） levels were detected.Results The weights ratios of the accessory sex organs and body weights showed no significant differences between the solvent control and the GH transgenic carp-treated groups.Serum concentrations of FSH,LH,and T of the rats treated with GH transgenic carp ＋ TP showed no significant changes,compared with those treated with TP only.Conclusion GH transgenic carp does not have any androgenic agonist or antagonist properties in vivo screening tests.

Two Lycopene β-Cyclases Genes from Sweet Orange (Citrus sinensis L. Osbeck) Encode Enzymes With Different Functional Efficiency During the Conversion of Lycopene-to-Provitamin A

Citrus fruits are rich in carotenoids.In the carotenoid biosynthetic pathway,lycopene β-cyclase（LCYb,EC：1.14.-.-） is a key regulatory enzyme in the catalysis of lycopene to β-carotene,an important dietary precursor of vitamin A for human nutrition.Two closely related lycopene β-cyclase cDNAs,designated CsLCYb1 and CsLCYb2,were isolated from the pulp of orange fruits（Citrus sinensis）.The expression level of CsLCYb genes is lower in the flavedo and juice sacs of a lycopeneaccumulating genotype Cara Cara than that in common genotype Washington,and this might be correlated with lycopene accumulation in Cara Cara fruit.The CsLCYb1 efficiently converted lycopene into the bicyclic β-carotene in an Escherichia coli expression system,but the CsLCYb2 exhibited a lower enzyme activity and converted lycopene into the β-carotene and the monocyclic γ-carotene.In tomato transformation studies,expression of CsLCYb1 under the control of the cauliflower mosaic virus（CaMV） 35S constitutive promoter resulted in a virtually complete conversion of lycopene into β-carotene,and the ripe fruits displayed a bright orange colour.However,the CsLCYb2 transgenic tomato plants did not show an altered fruit colour during development and maturation.In fruits of the CsLCYb1 transgenic plants,most of the lycopene was converted into β-carotene with provitamin A levels reaching about 700 μg g-1DW.Unexpectedly,most transgenic tomatoes showed a reduction in total carotenoid accumulation,and this is consistent with the decrease in expression of endogenous carotenogenic genes in transgenic fruits.Collectively,these results suggested that the cloned CsLCYb1 and CsLCYb2 genes encoded two functional lycopene β-cyclases with different catalytic efficiency,and they may have potential for metabolite engineering toward altering pigmentation and enhancing nutritional value of food crops.

Studies on Introduction of Arrowhead Proteinase Inhibitor Gene into N． tobacco Protoplasts

The Arrowhead Proteinase Inhibitor(API)gene was introduced into the protoplasts or mesophyll cells of N.tobacco by PEG-mediated.The transformed protoplasts undergoing dirrerentiation of callus and regeneration of plantlet have been growing into transgenic plants. Restriction endonuclease analysis of products amplificated by PCR indicates the existence of the API gene in the transformed plantlet.The extract of the leaves from the transformed plants shows trypsin inhibitory activity,which indicates the expression of the introduced API gene and the transformed plants can accumulate the inhibitor. However, the variation of the inhlbitory activity of the transformed plants reveals the importance of the integration site of the API gene in the genome.

A preliminary analysis of antineoplastic activity ofparvovirus MVMp NS-1 proteins

Human gastric cancer MKN-45 cells were transfectedwith pULB 3238, a plasmid carrying MVMp NS-1 genewith its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR. After the integration and expression of NS-1 gene, some of the transfectantsdied, while others remained alive, but the growth featuresof survived cells were changed. For further study on theantineoplastic function of parvoviral NS-1 protein in vivo,transgenic mice carrying NS-1 genes were established byconventional method. Among 4 founders, one of them wasfound to be able to transmit the transgene to around 50%of their offsprings. RT-PCR was performed to indicate theexpression of NS-1 gene in transgenic mice and its mRNAappeared in a variety of tissues. The expression of integrated NS-1 gene may correlate with the decreased incidence of tumor induced in vivo by chemical carcinogens.

Early Identification of Stable Transformation Events by Combined Use of Antibiotic Selection and Vital Detection of Green Fluorescent Protein (GFP) in Carrot (Daucus carota L.) Callus

Genetic transformation is a useful technique to complement conventional breeding in crop improvement. Although carrot has been a model organism for in vitro embryogenesis study, genetic transformation of carrot is still lengthy and labor intensive. An efficient transformation and detection system is desirable. Direct infection of Agrobacterium to carrot calli has provided an easy way for carrot genetic transformation. To improve the efficiency of antibiotic selection in this method, we report the combined use of an improved green-fluorescent protein, referred to as smGFP, to establish a versatile selection method for carrot callus transformation system. By combining antibiotic selection with the bright fluorescence observed in the callus tissue, we were able to easily identify stable transformants in early stage of the transformation process. In addition to the GFP expression of the callus cells, the transgenic nature of callus cells was confirmed with Southern and Western analysis. We found we can link the simplicity of carrot-callus-cell transformation, early detection of stable transformants with antibiotic selection, visualization of GFP fluorescence, and molecular analysis （Southern and Western） of callus tissue （non-photosynthetic tissue） to provide a more efficient way in identifying stable transformants at early stage of carrot transformation.

Studies on renin-2 gene in transgenic rats

Objective To determine the function of, in vivo, renin and its role in the pathogenesis of hypertension. Methods A renin 2 gene restriction map was constructed by endonuclease digest ion and Southern blotting hybridization. Transgenic rats were produced via micro injection method.Results The 24 kb fragments containing mouse full length ren 2 and it s flanking sequence were cleaved by single enzymes (EcoRⅠ, KpnⅠ and BamHⅠ) an d combined enzymes (EcoRⅠ/KpnⅠ, KpnⅠ/BamHⅠ and BamHⅠ/EcoRⅠ), respectively. The digests were electrophoresed in 0.8% agarose plates and transferred onto NC membranes. Radioactive 735 bp and 1400 bp probes obtained from half and full l ength renin 1 cDNA were used in southern blotting hybridization. According to t he electrophoresis and hybridization patterns, a ren 2 restriction map was cons tructed. 1603 fertilized rat ova after injection with purified 24 kb renin 2 ge ne were implanted into the oviducts of 81 pseudopregnant recipients in about 2 0 ova per female rat. 306 progenies were obtained from 50 foster mothers (averag e of pregnancies was 56.6%). 248 survived pups were identified by PCR analys is and Southern hybridization, and eight positive rats were found to be the transgenic rats (founder, F). All of them carried long fragments (24 kb) of renin 2 gene with normal blood pressure. Preliminary breeding and screening were carried out in the founder. Total survival pups (17.8%) and overall efficiencies (1%) were h arvested as the same as those reported in the literatures. A systemic observatio n and the problems occurred during production of transgenic rats were also descr ibed besides the technique procedure used in this study.Conclusions Mapping of full length murine ren 2 can be used in invest igation of the structure and function of the gene. The results denoted that the ren 2 tran sgenic rats were successfully established in this study and the technique used i n the production of transgenic rats was proved to be valid in leading to wide s pread application of transgenic technique to many other related researches.

Influence on the immune function of the human peripheral blood mononuclear cells transfected by retrovirus-mediated HSV-tk gene

Graft-versus-host disease （GVHD） is a severe complication and a major source of morbidity and mortality after allogeneic hematopoietic stem cell transplantation （HSCT）. Although T cell depletion of the allogeneic HSCT can efficiently prevent GVHD, it is associated with increased graft rejection and relapse of the malignant disease. To preserve the beneficial effects of donor T cells and avoid their GVHD effects, some approaches have been explored. One of them is to transfer a special suicide gene into the donor T lymphocytes so that they become more sensitive to a specific drug that is ordinarily not toxic. The most commonly used suicide gene is the thymidine kinase-encoding gene of herpes simplex virus （HSV-tk）.