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.

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.

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.

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.

Enhanced resistance to Botrytis cinerea and Rhizoctonia solani in transgenic broccoli with a Trichoderma viride endochitinase gene

A endochitinase gene（Tch） from the fungus Trichoderma viride was introduced into broccoli（Brassica oleracea var. italica） by Agrobacterium-mediated transformation. Sixty-eight putative transformants were obtained and the presence of the Tch gene was confirmed by both PCR and Southern blot analysis. RT-PCR analysis showed an accumulation of the transcript encoding the endochitinase protein in the transgenic plants. Using real-time quantitative PCR, the expression profiling of endochitinase gene was analyzed. Primary transformants and selfed progeny were examined for expression of the endochitinase using a fluorometric assay and for their resistance to the pathogenic fungi Botrytis cinerea and Rhizoctonia solani. The endochitinase activities in T0 in vitro plants, T0 mature plants and T1 mature plants were correlated with leaf lesions, and the transgenic line T618 had high endochitinse activities of 102.68, 114.53 and 120.27 nmol L–1 MU min–1 mg–1 protein in the three kinds of plants, respectively. The endochitinase activity showed a positive correlation with the resistance to the pathogens. Most transgenic T0 broccoli had increased resistance to the pathogens of B. cinerea and R. solani in leaf assays and this resistance was confirmed to be inheritable. These findings suggested that expression of the Tch gene from T. viride could enhance resistance to pathogenic fungi in Brassica species.

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.

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.

Clinical phenotypes,ALK1 gene mutation and level of related plasma proteins in Chinese hereditary hemorrhagic telangiectasia

Background We determined the diagnosis of hereditary hemorrhagic telangiectasis (HHT) in a suspected HHT family,identified ALK1 gene mutation and established a gene diagnosis method of HHT. The level of related plasma proteins (transforming growth factor β and thrombomodulin) were also analyzed.Methods Bleeding history and family history were collected; Dilatant nasal mucosal capillaries in proband were observed under nasal cavity endoscope; exons 3,7,8 of ALK1 gene in proband and her family members were amplified with polymerase chain reaction (PCR), and the PCR products were analyzed. Using enzyme-linked immunosorbent assay (ELISA),plasma TGF-β1 and TGF-β2 concentrations were measured. Plasma thrombomodulin (TM) level was detected by Western blotting.Results Of all family members,four had epstaxis,two had evident telangiectases on skin or mucosa. Gene screening results showed that C to T substitution at position 1231 in exon 8 of ALK1 gene (CGG→TGG) existed in proband,her affected brother and their father. The mutation did not exist in proband’s sister-in-law and nephew. Plasma TGF-β1 concentrations in the affected HHT was 20538,17194,13131 pg/ml,while that of normal control and unaffected family members was 15950,20297,12836 pg/ml,respectively. Plasma TGF-β2 in HHT patients was 14502,9550,10592 and that of normal controls 8579,20297,7680 pg/ml respectively. Level of plasma TM was in HHT subjects significantly lower than in normal subjects.Conclusions Chinese HHT individuals have mutant ALK1 gene,a C1231T variation on exon 8 of ALK1 is responsible for HHT clinical phenotypes in this family. ALK1 gene analysis,together with special clinical phenotypes and family history,provides a reliable method in diagnosing HHT. In affected HHT subjects,plasma TGFβ levels were not obviously different from those of normal subject; while plasma TM concentration was significantly lower than that in normal subjects. The significance and mechanism remain to be elucidated.

Inhibition of corneal fibrosis by Smad7 in rats after photorefractive keratectomy

Background Haze or corneal subepithelial fibrosis is one of the common complications after refractive surgery procedures, such as photorefractive keratectomy （PRK）, laser epithelial keratomileusis, and epipolis laser in situ keratomileusis, which would result in refractive regression, decreased visual quality, and corneal opacification. Haze directly resulted from corneal fibrosis mediated by transforming growth factor β （TGFβ）. SmadT, an inhibitory Smad, can inhibit TGFβ signal transduction. Recently, the effects of Smad7 on the inhibition of fibrosis in several organs have been studied, while little is known about the effects on cornea after PRK. This study was aimed to determine the effects of lentiviral-mediated Smad7 gene expression on corneal fibrosis in rats after PRK. Methods Four different experimental groups were established using right eyes of Sprague-Dawley rats. Thirty-two eyes underwent de-epithelialization only and served as a sham operation group （group 1）. Ninety-six eyes underwent PRK operation and were further divided into group 2 （the PRK group） without lentivector administration, group 3 （the Lv-blank group） with control lentiviral vector without Smad7 administration, and group 4 （the Lv-Smad7 group） with Smad7 expressing lentiviral vector Smad7 administration. At 1 day, 1 week, 1 month, and 3 months after PRK, the transfection efficiency was determined by measuring the fluorescence signal as well as Smad7 protein and mRNA levels. Corneas were further processed for immunoblotting to assess the phosphorylation of Smad2 as a downstream event of TGFβ/Smad signaling. The expression of fibrotic markers, such as c（-smooth muscle actin （c（-SMA）, Type III collagen （collagen III）, and cell cycle-related marker Ki67, was measured by quantitative real-time reverse transcription polymerase chain reaction （RT-PCR）. Results Lentivirus-mediated exogenous Smad7 gene expression in rat corneal tissue resulted in reduced activation of TGFβ/Smad signaling caused by downregulation of phosphorylation of Smad2. Smad7 also downregulated the expression of TGFβ2. Markers of cell proliferation and fibrosis, including Ki67, c（-SMA, and collagen III, were inhibited by Smad7 up to 3 months after PRK operation. Conclusion Smad7 gene transfer inhibits fibrogenic responses of cornea in rats after PRK.

Down-regulation of mitotic checkpoint in transformed human embryo lung fibroblasts induced by N-methyl-N'-nitro-N-nitrosoguaridine

Background Mutations in mitotic checkpoint genes have been detected in several human cancers, which exhibit chromosome instability. We wanted to know whether mutation of hBub1 could occur in transformed human embryo lung fibroblasts (HELF) cells induced by a chemical carcinogen.Methods HELF cells were transformed by N-methyl-N’-nitro-N- nitrosoguaridine (MNNG), and three flasks of transformed HELF cells (named as T1, T2, and T3) were selected as amplifiers, and mutations of hBub1 in these transformed cells were analyzed by PCR-SSCP and sequencing.Results It was found that any one of three transformed cell lines exhibited aneuploidy with a low mitotic checkpoint function. Subsequent PCR-SSCP and sequence analysis showed an AGT to CGT or ATT mutation at codon 80 in hBub1 gene in T1 cells with a resultant change in amino acid sequence.Conclusion Our study demonstrated that the mitotic checkpoint genes could be targets of MNNG.

Relative expression of PTTG and bFGF in oral squamous cellcarcinoma and Tca8113

The purpose of this study was to investigate the expression of pituitary tumor transforming gene(PTTG)and basicfibroblast growth factor(bFGF)in oral squamous cell carcinoma(OSCC)and tongue cancer cell line Tca8113,as well as their effects on each other.We detected PTTG protein and bFGF in OSCC tissues from 56 cases using the streptavidin-biotin peroxidase(S-P)method;additionally,after being treated with different concentrations of anti bFGF or PTTG antibody,PTTG or bFGF expression in Tca8113 was examined by immuno-cytochemistry.The results were as follows:(1)Positive rates of PTTG protein and bFGF were 78.2%and 67.3%in OSCC,respectively,which were significantly higher than those in normal mucosal tissues(P<0.05).PTTG protein was significantly up-regulated in poorly and moderately differentiated tumors compared to well differentiated tumors(P<0.05),and there was also a significant difference between tumors with lymph node metastasis and tumors without lymph node metastasis(P<0.05).PTTG protein expression was positively correlated with bFGF(r=0.382,P<0.05);(2)PTTG protein emitted strongfluorescence in Tca8113,and it decreased after being treated with anti-bFGF antibody.Anti-PTTG anti-body also had an inhibitive effect on bFGF expression.In summary,the overexpression of PTTG protein is closely related with OSCC differentiation and lymph node metastasis.PTTG protein expression conforms to bFGF in OSCC tissues and Tca8113 cells.Detection of both PTTG and bFGF may help to judge the degree of malignancy and prognosis of patients with OSCC.