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1.
Cui Lijie Chen Yuhui Shen Guoan Zhao Lingxia Tang Kexuan 《Plant Molecular Biology Reporter》2011,29(2):466-472
Thymosin α1 (Tα1) was widely used for the treatment of hepatitis (B and C) and several cancers. However, current production
of Tα1 is difficultly meeting clinical needs. To develop a novel and safety approach for Tα1 production, we synthesized a
Tα1 gene (124 bp) based on the plant codon usage bias and constructed a four-copy Tα1 gene concatemer (408 bp) by using isocaudamer technique. This 4 × Tα1 structure was cloned into plant binary expression vector
pCAMBIA2300 with twin transfer deoxyribonucleic acids (T-DNAs) and integrated into lettuce genome via Agrobacterium-mediated
transformation. Thirteen positive plants were identified by polymerase chain reaction and confirmed by Southern blot analysis,
and 11 marker-free lettuce plants were obtained in T2 generation. The content of recombined Tα1 (rTα1) protein reached 1798.317 ± 87.312 ng/g
in fresh leaves of transgenic lettuce. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay demonstrated that rTα1 protein stimulated mouse splenic lymphocyte proliferation in vitro.
These data suggest that biologically active rTα1 was successfully expressed in marker-free transgenic lettuce, and this method
could provide an alternative choice for large-scale production of Tα1 in the future. 相似文献
2.
3.
Xinbo Guo Rui Hai Liu Xueqing Fu Xiaofen Sun Kexuan Tang 《Plant Cell, Tissue and Organ Culture》2013,114(2):225-236
Epidemiological studies have shown that regular consumption of fruits and vegetables is associated with reduced risk of chronic diseases. Vegetables can provide vitamins, phenolics, flavonoids, minerals and dietary fibers for optimal health benefits. However, some nutrients contained in many fruits and vegetables cannot meet of the complete nutrition need in the human body. Biotechnology has the potential to improve the nutritional value of crops. Considering the high consumption of romaine lettuce in human diet worldwide, the objective of study is to enhance the contents of vitamin C, phenolics and antioxidant activity in lettuce leaves by genetic engineering techniques. The gene expression level, vitamin C content, total phenolics, as well as total and cellular antioxidant activities were analyzed by real-time PCR, HPLC, Folin–Ciocalteu, Hydro-PSC and CAA methods, respectively. The bio-fortification of genetically engineered lettuce increased vitamin C up to 48.94 ± 1.34 mg/100 g FW following the increased over-expression of At GLDH. This is almost a 3.2-fold increase as the content when compared with wild type lettuce (p < 0.05). In addition, phenolic compounds in transgenic lettuce contained 120.4 ± 1.62 mg GA equiv./100 g FW, almost double the phenolic content of the wild type. Total antioxidant activities were 735.4 ± 47.7 μmol vitamin C equiv./100 g FW, cellular antioxidant activities were 7.33 ± 0.86 μmol quercetin equiv./100 g FW (PBS wash) and 18.14 ± 0.68 μmol quercetin equiv./100 g FW (No PBS wash) in transgenic lettuce, respectively, 1.5, 4 and twofold increases when compared with the wild type. This study suggests that bio-fortification by genetic engineering has great potential to improve vitamin C, phenolic contents and antioxidant activity in lettuce. 相似文献
4.
Tian Zhang Zhen Qi Yueyue Wang Fangyuan Zhang Renyong Li Qingsheng Yu Xiangbin Chen Huojun Wang Xin Xiong Kexuan Tang 《Microbiological research》2013,168(3):130-137
Lipase produced by Penicillium expansum is widely used in laundry detergent and leather industry; however, the absence of an efficient transformation technology sets a major obstacle for further enhancement of its lipase productivity through advanced gene engineering. In this work, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for P. expansum PE-12 transformation, using hygromycin phosphotransferase (hph) as a selectable marker gene. As a result, we revealed that the frequency of transformation surpassed 100 transformants/105 condida, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and all the transformants showed mitotic stability. Facilitated by this newly established method, for the first time, P. expansum PE-12 was genetically engineered to improve the lipase yield, through a homologous expression vector carrying the endogenous lipase gene (PEL) driven by the strong constitutive promoter of the glyceraldehydes-3-phosphate dehydrogenase gene (gpdA) from Aspergillus nidulans. The highest expression level of the engineered strain reached up to 1700 U/mL, nearly 2-fold of the original industrial strain (900 U/mL). Our reproducible ATMT system has not only revealed the great potential of homologous expression-directed genetic engineering, which is more efficient and specific compared to traditional mutagenesis, but also provided new possibilities and perspectives for any other practical applications of P. expansum-related genetic engineering in the future. 相似文献
5.
Wang X Liu L Liu S Sun X Deng Z Pi Y Sun X Tang K 《Journal of biochemistry and molecular biology》2004,37(5):538-545
A new CRT binding factor (CBF) gene designated Cbcbf25 was cloned from Capsella bursa-pastoris, a wild grass, by the rapid amplification of cDNA ends (RACE). The full-length cDNA of Cbcbf25 was 898 bp with a 669 bp open reading frame (ORF) encoding a putative DRE/CRT (LTRE)-binding protein of 223 amino acids. The predicted CbCBF25 protein contained a potential nuclear localization signal (NLS) in its N-terminal region followed by an AP2 DNA-binding motif and a possible acidic activation domain in the C-terminal region. Bioinformatic analysis revealed that Cbcbf25 has a high level of similarity with other CBF genes like cbf1, cbf2, and cbf3 from Arabidopsis thaliana, and Bncbf5, Bncbf7, Bncbf16, and Bncbf17 from Brassica napus. A cold acclimation assay showed that Cbcbf25 was expressed immediately after cold triggering, but this expression was transient, suggesting that it concerns cold acclimation. Our study implies that Cbcbf25 is an analogue of other CBF genes and may participate in cold-response, by for example, controlling the expression of cold-regulated genes or increasing the freezing tolerance of plants. 相似文献
6.
Wang J Wu W Zuo K Fei J Sun X Lin J Li X Tang K 《Cellular & molecular biology letters》2004,9(3):465-473
A full-length cDNA of a new serine/threonine (Ser/Thr) protein kinase gene, designated as BnSOS2 (GenBank Acc. No.AY310413), was cloned from Brassica napus by rapid amplification of cDNA ends (RACE). The full-length cDNA of BnSOS2 was 1779 bp and contained a 1539-bp open reading frame encoding a protein of 512 amino acids. Homology analysis shows that BnSOS2 strongly resembles other Ser/Thr protein kinase genes, and that its putative protein belongs to a typical Ser/Thr kinase family. Northern blot analysis reveals that BnSOS2 is salt-inducible. Our results indicate that BnSOS2 is a new member of the plant SOS2 gene family, which may play an important role in salt tolerance of plants. 相似文献
7.
Cloning and functional analysis of a cDNA encoding Ginkgo biloba farnesyl diphosphate synthase 总被引:3,自引:0,他引:3
Farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2. 5.1.10) catalyzes the synthesis of farnesyl diphosphate, and provides precursor for biosynthesis of sesquiterpene and isoprenoids containing more than 15 isoprene units in Ginkgo biloba. Here we report the cloning, characterization and functional analysis of a new cDNA encoding FPS from G. biloba. The full-length cDNA (designated GbFPS) had 1731 bp with an open reading frame of 1170 bp encoding a polypeptide of 390 amino acids. The deduced GbFPS was similar to other known FPSs and contained all the conserved regions of trans-prenyl chain-elongating enzymes. Structural modeling showed that GbFPS had the typical structure of FPS, the most prominent feature of which is the arrangement of 13 core helices around a large central cavity. Southern blot analysis revealed a small FPS gene family in G. biloba. Expression analysis indicated that GbFPS expression was high in roots and leaves, and low in stems. Functional complementation of GbFPS in an FPS-deficient strain confirmed that GbFPS mediates farnesyl diphosphate biosynthesis. 相似文献
8.
Lei?Zhang Tiefeng?Xu Xiaofen?Sun Hanming?Zhang Tianlin?Tang Kexuan?TangEmail author 《In vitro cellular & developmental biology. Plant》2003,39(5):459-462
Summary An efficient in vitro plant regeneration system from cotyledons was established in tetraploid Isatis indigotica Fort. Factors influencing shoot regeneration from cotyledons, including culture medium type, combinations of plant growth
regulators, and sucrose concentrations in the medium, as well as illumination were investigated. Murashige and Skoog's (MS)
medium was found to be best for promoting shoot regeneration, followed by Gamborg's B5 and White's medium. The highest shoot regeneration frequency was achieved from cotyledons cultured on MS medium supplemented
with 2.0 mgl−1 (8.9 μM) 6-benzyladenine and 1.0 mgl−1 (5.4 μM) α-naphthaleneacetic acid (NAA), with 97.9% regeneration, associated with a high number of multiple shoots developed per
explant (8.6 shoots per explant). A sucrose concentration of 3% present in the medium and light conditions were beneficial
for shoot regeneration. The shoots developed were rooted in a half-strength MS medium supplemented with 1.0 mgl−1 (5.4 μM) NAA and successfully transplanted in soil in pots with over 85% survival. The establishment of an efficient plant regeneration
procedure from cotyledons provides a basis for the rapid in vitro multiplication of tetraploid Isatis indigotica Fort., one of the most extensively used medicinal plants in China currently under great shortage. 相似文献
9.
A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root. 相似文献
10.
Geranylgeranyl diphosphate synthase (GGPPS) [EC 2.5.1.29] catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP),
which is a key precursor for diterpenes such as taxol. Herein, a full-length cDNA encoding GGPPS (designated as CgGGPPS) was cloned and characterized from hazel (Corylus avellana L. Gasaway), a taxol-producing angiosperms. The full-length cDNA of CgGGPPS was 1515 bp with a 1122 bp open reading frame (ORF) encoding a 373 amino acid polypeptide. The CgGGPPS genomic DNA sequence
was also obtained, revealing CgGGPPS gene was not interrupted by an intron. Southern blot analysis indicated that CgGGPPS belonged to a small gene family. Tissue expression pattern analysis indicated that CgGGPPS expressed the highest in leaves.
RT–PCR analysis indicated that CgGGPPS expression could be induced by exogenous methyl jasmonate acid. Furthermore, carotenoid
accumulation was observed in Escherichia coli carrying pACCAR25ΔcrtE plasmid carrying CgGGPPS. The result revealed that cDNA encoded a functional GGPP synthase. 相似文献