Research of AtNHX1 Gene Transformation in Brassica napus L. by Agrobacterium tumefaciens

[Objective] The aim was to investigate AtNHX1 gene transformation in Brassica napus L. mediated by Agrobacterium tumefaciens. [Method] By using Agrobacterium-mediated method and cre/lox plant expression vector,the transformation of AtNHX1 gene of Na＋/H＋ antiporter in Brassica napus was studied. [Result] The regeneration rate of cotyledon with petiole was much higher than that of hypocotyl,thus,the cotyledon with petiole was selected as the recipient for transformation. After the cotyledon with petiole was soaked in bacterial solution （OD600=0.4） for 8-10 min,kanamycin-resistant green seeding percentage could reach 3.75%. [Conclusion] The PCR detection of kanamycin-resistant plants proved that NHX1 gene had been inserted into Brassica napus genome. And this research could provide a new way to improve the salt tolerance of Brassica napus.

Agrobacterium tumefaciens-mediated GUS gene transformation of Robinia pseudoacacia 'Idaho'

Based on the plant regeneration system, a GUS gene transformation system to Idaho locust （Robinia pseudoacacia ‘Idaho＇） mediated by Agrobacterium tumefaciens was established. The successful transformation was confirmed by regenerating the shoots fi＇om the infected leaves in the presence of hygromysin; by histochemical X-gluc assays of 15-glucuronidase （GUS） and by PCR and PCR-Southern blotting analysis. The ratio of positive transgenic plants is 5.8% （5 out of 86 plants）. With this system, the target gene DREB was introduced into the leaves of Idaho locust. The transgenic plants regenerated, which was verified by PCR-Southern blot- ting. It is suggested that the transformation system could be a new, simple, reliable and practical route to gene transformation of R. pseudoacacia ＇Idaho＇ mediated with A. tumefaciens.

Construction of a transformation system for the stable expression of foreign genes in Chlorella sp.

A stable transformation system for the expression of foreign genes in the unicellular green marine alga （Chlorella sp. MACC/C95）was established. Using electroporation, the alga was transformed with a plasmid containing the phytase gene under the control of CaMV35S promoter and the neomycin phosphotransferase （ npt ） as a selectable marker gene. The integration of the phytase gene into the Chlorella genome was revealed by PCR and Southern blotting analysis. RT-PCR analysis revealed the expression of phytase gene at the transcript level. The enhanced activity of phytase enzyme in the transformants confirmed the integration and successful expression of phytase gene. The introduced phytase gene and its protein expression were stably maintained for at least 30 generations in media devoid of selectable antibiotics G418. This is an important step toward the production of useful foreign proteins in Chlorella sp. MACC/C95.

Studies on Cloning and Transformation of CBF1 Gene of Maize Grass

[Objective]It is revealed whether the similar maize transcriptional activator in CBF1 gene is regulatory cold resistance gene to lay the foundation for breeding new transgenic Forage Maize Varieties with high cold resistance ability.[Methods]In the present paper,the transcriptional factor gene CBF1 was Successfully cloned by PCR from the leaves of Arabidopsis.The sequence was preliminarily analyzed and plant expression vector was constructed.Then with agrobacterium-mediated transgene technique,CBF1 gene was introduced into maize SAUMZ1.[Results]PCR assay revealed that the CBF1 gene was integrated in the maize grass SAUMZ1 genome.Under different low temperature treatment,the relative electrolyte leakage percentage of transgenic plant was lower than Control.[Conclusion] The results showed that the cold-resistance of maize grass SAUMZ1 enhanced after transforming CBF1 gene.

A callus transformation system for gene functional studies in soybean

Obtaining transgenic plants is a common method for analyzing gene function. Unfortunately, stable genetic transformation is difficult to achieve, especially for plants（e.g., soybean）, which are recalcitrant to genetic transformation. Transient expression systems, such as Arabidopsis protoplast, Nicotiana leaves, and onion bulb leaves are widely used for gene functional studies. A simple method for obtaining transgenic soybean callus tissues was reported recently. We extend this system with simplified culture conditions to gene functional studies, including promoter analysis, expression and subcellular localization of the target protein, and protein-protein interaction. We also evaluate the plasticity of this system with soybean varieties, different vector constructs, and various Agrobacterium strains. The results indicated that the callus transformation system is efficient and adaptable for gene functional investigation in soybean genotype-, vector-, and Agrobacterium strain-independent modes. We demonstrated an easy set-up and practical homologous strategy for soybean gene functional studies.

Cloning and genetic transformation of a novel rice gene OsAPT2

A novel rice gene OsAPT2,which encodes a putative adenine phosphoribosyl transferase(APRT),was cloned.Its full-length cDNA is 1125bp,composing an ORF encoding 212 amino acid residues and a stop cordon,a 5' UTR of 123 bp and a 3' UTR of 363 bp.The sequence data have been submitted to the DDBJ/EMBL/GenBank databases(accession number:AY238894).The deduced amino acid sequence of OsAPT2 is highly homologous to those of previously reported APRTs.The genomic OsAPT2 gene contains 7 exons and 6 introns.Its total length is 4758 bp.Then,an antisense expression vector of the full-length OsAPT2 cDNA was constructed and transformed into rice variety Taibei309 by Agrobacterium tumefaciens mediated transformation method.In total,650 T0 transgenic plants were obtained based on both antibiotic screening and specific PCR identification.One hundred individuals of them were selected and planted in Hainan Island.From those 11 male sterile lines with seed-setting rate lower than 3% in bagged spike were obtained.Results suggest that OsAPT2 is involved in male sterility.Nine of the 11 male sterile lines were constitutive sterile lines;two of the 11 male sterile lines were thermo-sensitive genic male sterile lines,which may be useful in hybride rice breeding.

Overexpression of the Suaeda salsa SsNHX1 gene confers enhanced salt and drought tolerance to transgenic Zea mays

Maize is one of the most important crops worldwide, but it suffers from salt stress when grown in saline-alkaline soil. There is therefore an urgent need to improve maize salt tolerance and crop yield. In this study, the SsNHX1 gene of Suaeda salsa, which encodes a vacuolar membrane Na~+/H~+ antiporter, was transformed into the maize inbred line 18-599 by Agrobacterium-mediated transformation. Transgenic maize plants overexpressing the SsNHX1 gene showed less growth retardation when treated with an increasing NaCl gradient of up to 1%, indicating enhanced salt tolerance. The improved salt tolerance of transgenic plants was also demonstrated by a significantly elevated seed germination rate(79%) and a reduction in seminal root length inhibition. Moreover, transgenic plants under salt stress exhibited less physiological damage. SsNHX1-overexpressing transgenic maize accumulated more Na~+ and K~+ than wild-type(WT) plants particularly in the leaves, resulting in a higher ratio of K~+/Na~+ in the leaves under salt stress. This result revealed that the improved salt tolerance of SsNHX1-overexpressing transgenic maize plants was likely attributed to SsNHX1-mediated localization of Na~+ to vacuoles and subsequent maintenance of the cytosolic ionic balance. In addition, SsNHX1 overexpression also improved the drought tolerance of the transgenic maize plants, as rehydrated transgenic plants were restored to normal growth while WT plants did not grow normally after dehydration treatment. Therefore, based on our engineering approach, SsNHX1 represents a promising candidate gene for improving the salt and drought tolerance of maize and other crops.

Research on Oxalate Oxidase and Its Genes in Plants

This paper introduces the discovery,composition and structure of oxalate oxidase,as well as illustrates the biological functions of this enzyme.With a comprehensive introduction upon previous researches upon gene cloning and heredity transformation of this enzyme,it indicates that heredity transformation can increase the content of oxalate oxidase within the plants and also enhance their resistance.The paper also points out the problems such as lack of gene resources and difficulty in the transformation of heterologous genes,and the focus in later researches should be laid upon the exploration of plant resources relative to this enzyme and selection of resistant species.

Epigenetic changes of pituitary tumor-derived transforming gene 1 in pancreatic cancer

BACKGROUND: Pancreatic cancer is a devastating disease with abnormal genetic changes. The pituitary tumor-derived transforming gene (PTTG) is considered to be implicated in the tumorigenesis of cancers when the gene is epigenetically transformed. In this study, we investigated the relationships between aberrant expression and epigenetic changes of the PTTG1 gene in pancreatic cancer. METHODS: We chose 4 cell lines (PANC-1, Colo357, T3M-4 and PancTu I) and pancreatic ductal adenocarcinoma (PDAC) tissues. After using restriction isoschizomer endonucleases (Msp I /Hpa II) to digest the DNA sequence (5'-CCGG-3'), we performed PCR reaction to amplify the product. And RT-PCR was applied to determine the gene expression. RESULTS: The mRNA expression of the PTTG1 gene was higher in pancreatic tumor than in normal tissue. The gene was also expressed in the 4 PDAC cell lines. The methylation states of the upstream regions of the PTTG1 gene were almost identical in normal, tumor pancreatic tissues and the 4 PDAC cell lines. Some (5'-CCGG-3') areas in the upstream region of PTTG1 were methylated, while some others were unmethylated. CONCLUSIONS: The oncogene PTTG1 was overexpressed in pancreatic tumor tissues and verified by RT-PCR detection. The methylation status of DNA in promoter areas was involved in the gene expression with the help of other factors in pancreatic cancer.

Correlation between ECT2 gene expression and methylation change of ECT2 promoter region in pancreatic cancer

BACKGROUND: Pancreatic cancer is closely related to epigenetic abnormality. The epithelial cell transforming sequence 2 gene (ECT2) plays a critical role in Rho activation during cytokinesis, and thus may play a role in the pathogenesis of pancreatic cancer. In this study, we investigated the relationships between aberrant expression and epigenetic changes of the ECT2 gene in pancreatic cancer. METHODS: Four cell lines (PANC-1, Colo357, T3M-4 and PancTu I) and pancreatic ductal adenocarcinoma (PDAC) tissues were used for mRNA detection. After restriction isoschizomer endonucleases (Msp I/Hpa II) were used to digest the DNA sequence (5'-CCGG-3'), PCR was made to amplify the product. And RT-PCR was applied to determine the expression of the gene. RESULTS: The mRNA expression of the ECT2 gene was higher in pancreatic tumor tissue than in normal tissue. The gene was also expressed in the 4 PDAC cell lines. The methylation states of the upstream regions of the ECT2 gene were almost identical in normal, tumor pancreatic tissues, and the 4 PDAC cell lines. Some of the 5'-CCGG-3' areas in the upstream region of ECT2 were methylated, while others were unmethylated. CONCLUSIONS: The oncogene ECT2 is overexpressed in pancreatic tumor tissues as verified by RT-PCR detection. The methylation status of DNA in promoter areas is involved in the gene expression, along with other factors, in pancreatic cancer.

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.

Effect of insulin-like growth factor-1 on pituitary tumor transforming gene in glioma C6 cells

Objective: To investigate the effect of insulin-like growth factor-1 (IGF-1) on pituitary tumor transforming gene (PTTG) in glioma C6 cells. Methods: Glioma C6 cells were divided into four groups: A group, treated without IGF-1; B group, treated with 0.1 ng/mL dose of IGF-1; C group, treated with 1 ng/mL dose of IGF-1; D group, treated with 10 ng/mL dose of IGF-1. PTTG mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR), western blotting was used to detect the expression of PTTG protein. Results: The expressions of PTTG mRNA were 1.370 ± 0.212, 2.198 ± 0.354, 3.452 ± 0.332, and 4.576 ± 0.387 respectively in the four groups, and there was a significantly difference between any two groups (P < 0.01). The protein expressions of PTTG in the four groups were 1.407 ± 0.334, 1.813 ± 0.465, 2.412 ± 0.576, and 3.128 ± 0.665 respectively, and there was a significantly difference between any two groups (P < 0.01). Conclusion: IGF-1 can up-regulate the expression of PTTG significantly in dosage-dependent manner.

Two mutations in the transforming growth factor beta-induced gene associated with familial Lattice corneal dystrophy

AIM：To report a phenotypic variant pedigree of lattice corneal dystrophy（LCD）associated with two mutations,R124C and A546 D,in the transforming growth factor betainduced gene（TGFBI）.METHODS：A detailed ocular examination was taken for all participants of a LCD family. Peripheral blood leukocytes from each participant were extracted to obtain the DNA. Polymerase chain reaction（PCR）of all seventeen exons of TGFBI gene was performed. The products were sequenced and analyzed. Histological examination was carried out after a penetrating keratoplasty from the right eye of proband. RESULTS：Genetic analysis showed that the proband and all 6 affected individuals harbored both a heterozygous CGC to TGC mutation at codon 124 and a heterozygous GCC to GAC mutation at codon 546 of TGFBI. None of the 100 control subjects and unaffected family members was positive for these two mutations. Ocular examination displayed multiple refractile lattice-like opacities in anterior stroma of the central cornea and small granular deposits in the peripheral cornea. The deposits were stained positively with Congo red indicating be amyloid in nature and situated mainly in the anterior and middle stroma. CONCLUSION：We observed a novel LCD family which carried two pathogenic mutations（R124C and A546D）in the TGFBI gene. The phenotypic features were apparently different from those associated with corresponding single mutations. The result reveals that although the definite mutation is the most important genetic cause of the disease,some different modifier alleles may influence the phenotype.

TRANSFORMING ACTIVITY OF DNA FROM HUMAN ESOPHAGEAL CANCER AND THE IDENTIFICATION OF THE TRANSFORMING GENE

Rat-1 cells were transfected with DNA from human esophageal cancer 2K, 4K, 6K, 7K. 8K. The transforming foci were obtained and the transforming cell lines were established. The cell lines can form larger colony in soft agar. Those nude mice injected subcutaneously with the cells suffered from larger fibrous sarcoma. This indicates that the cell lines have carcinogenicity. The experimental results suggest that human DNA sequence and human Ha-ras special 616Kb (BamHI) band are present in the DNA of the transforming cells. The over-expression of ras gene products P21 were found in the tissues of exophageal cancer, the tissues adjacent to tumor and the transforming cells.

STUDY ON THE RFLPs AND AMPLICATION AND REARRANGEMENT OF THE TRANSFORMING GENES IN PRIMARY HEPATIC CARCINOMA, GASTRIC CARCINOMA AND BRAIN TUMOR WITH SIX HUMAN ONCOGENE PROBES

By using c-Ha-ras-1, N-ras Wigler (left sequence) and P52C.(right sequence), c-sis, v-erbB, c-myc and v-fos oncogenes as probes, restriction fragment length polymorphisms (RFLPs) of tumor tissue DNAs of 95 patients with gastric carcinoma, primary hepatic carcinoma and brain tumor, and those of 90 normal individuals were studied with the techniques of Southern blot and dot blot. Gene amplification and recombination were also examined in some tumors simultaneously. Some alleles of oncogene are reported in Chinese population for the first time. Moreover, the characteristic frequency of some "rare" alleles and genotypes occurred in some tumor samples is significantly higher than that occured in normal individuals. Pedigree analysis for 2 patients showed that some "rare" alleles are also abandant. Besides, gene amplification and recombination were found in some tumors.

THE EFFECT OF db-cAMP ON THE GENE EXPRESSION OF CALMODULIN AND CYTOSKELETON IN THE TRANSFORMED CELLS

We have demonstrated that the distribution of microtubules (MT), mlcrofilaments (MF) and fibronectin (FN) were diminished, while the gene expression of the calmodulin and c- fos enhanced in the transformed C3H10T1/2 cells. After treatment with 1 mM db-cAMP for 1 hour and 2 hours, there was an early and repldly reduced in gene expression of Calmodulin and c-fos respectively. After db-cAMP treatment for 4 -5 days, the number of capping cells of ConA binding decreased significantly and the cell surface microvllll decreased as well. The growth of treated cells was inhibited markedly. By using 4F1 cDNA probe, which is preferentially expressed In G1 phase, we have found that the db- cAMP treated cells were accumulated at G1 phase. Of particular interest is the fact that the distribution of microtubules, mlcrofilaments and fibronectln were recovered after treatment with 1 mM db-cAMP for 6 days. It is suggested that the Inhibition of proliferation, alteration, of phenotype and reco- very of cytoskeleton is transformed cells after treatment with db-cAMP are related to the Inhibition of gene expression of Calmodulin.

Status and Advances of Researches on GA 20-oxidases

GA 20-oxidase, the most important limiting enzyme, can catalyze a series of oxidization of GA biosynthesis pathway from GA12 to GA9 and from GA53 to GA20 in the higher plants. This paper reviews the studies on the characters of GA 20-oxidase, the gene and the protein of GA 20-oxidase and the regulation of GA 20-oxidase gene expression in recent years. At the same time, the prospects for the gene transformation of GA 20-oxidase in agriculture, forestry and horticulture are also discussed.

Expressions and their significance of PTTG and PC proteins in glioma

Objective： To investigate the expressions and their relationship of pituitary tumor transforming gene （PTTG） and proliferating cell nuclear antigen （PCNA） in glioma. Methods： The protein expressions of PTTG and PCNA were detected by immunostaining assay using streptavidin-peroxidase （SP） method in 80 cases of glioma. Results： The positive rates of PTTG in grades Ⅰ-Ⅳ gliomas were 56.3%, 68.2%, 80.8%, and 100.0% respectively, and the protein expression of PTTG increased with the increasing of the pathological grade （X^2= 9.602, P 〈 0.05）; The positive rates of PCNA protein were 37.5%, 54.5%, 69.2%, and 93.8% respectively, and the protein expression of PCNA increased with the increasing of the pathological grade （X2 = 12.147, P 〈 0.01）. The expression of PTTG had positive correlation with the expression of PCNA protein （~s = 0.557, P 〈 0.01）. Conclusion： The expressions of PTTG and PCNA proteins were related to malignant degree of glioma, and may cooperate with each other in the tumorigenesis and progression and can be considered as the indicators of the biological behaviors in glioma.

Transactivation of the TIEG1 confers growth inhibition of transforming growth factor-β-susceptible hepatocellular carcinoma cells

AIM:To investigate the role of transforming growth factor(TGF)-β-inducible early gene 1(TIEG1) in TGF-β-induced growth inhibition in hepatocellular carcinoma(HCC) cells.METHODS:Human hepatocyte and HCC cell lines with varied susceptibilities to TGF-β1 were tested by methylthiazoletetrazolium(MTT) assay.The expression changes of Smad2,Smad3,Smad4,Smad7,TIEG1 and TIEG2 gene following treatment with TGF-β1 in a TGF-β-sensitive hepatocyte cell line(MIHA),a TGF-β-sensitive hepatoma cell line(Hep3B) and two TGF-β-insensitive hepatoma cell lines(HepG2 and Bel7404) were examined.SiRNA targeting TIEG1 was transfected into Hep3B cells and the sensitivity of cells to TGF-β1 was examined.Overexpression of TIEG1 was induced by lentiviral-mediated transduction in TGF-β1-resistant hepatoma cell lines(Bel7404 and HepG2).MTT assay and 4',6-Diamidino-2-phenylindole staining were used to identify cell viability and apoptosis,respectively.The expression level of stathmin was measured by reverse transcriptase polymerase chain reaction and Western-blotting analysis,and stathmin promoter activity by TIEG1 was monitored by a luciferase reporter gene system.RESULTS:TIEG1 was significantly upregulated by TGF-β1 in the TGF-β1-sensitive HCC cell line,Hep3B,but not in the resistant cell lines.The suppression of TIEG1 by siRNAs decreased the sensitivity of Hep3B cells to TGF-β1,whereas the overexpression of TIEG1 mediated growth inhibition and apoptosis in TGF-β1-resistant HCC cell lines,which resembled those of TGF-β1-sensitive HCC cells treated with TGF-β1.Our data further suggested that stathmin was a direct target of TIEG1,as stathmin was signif icantly downregulated by TIEG1 overexpression,and stathmin promoter activity was inhibited by TIEG1 in a dose-dependent manner.CONCLUSION:Our data suggest that transactivation of TIEG1 conferred growth inhibition of TGF-β-susceptible human HCC cells.

Overexpression of the OsPDCD5 Gene Induces Programmed Cell Death in Rice

The primary aim of the present study was to investigate the overexpression of the rice （Oryza sativa L.） programmed cell death 5 （OsPDCD5） gene in rice plant. Constitutive expression of OsPDCD5 from the cauliflower mosaic virus （CaMV） 35S promoter induced programmed cell death （PCD） in transgenic rice. Programmed cell death was accompanied by typical features, including inhibition of developmental growth, a reduction of fresh weight, degradation of total protein content, and fragmentation of genomic DNA. These results suggest that OsPDCD5 plays an essential role in the regulation of PCD in rice plants.