The Novel Use of a Combination of Sonication and Vacuum Infiltration in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated Transformation of Kidney Bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) with <Emphasis Type="Italic">lea</Emphasis> Gene

An efficient gene transfer system without tissue culture steps was developed for kidney bean by using sonication and vacuum infiltration assisted, Agrobacterium-mediated transformation. Transgenic kidney bean with a group 3 lea (late embryogenesis abundant) protein gene from Brassica napus was produced through this approach. Among 18 combinations of transformation methods, Agrobacterium-mediated transformation combined with 5 min sonication and 5 min vacuum infiltration turned to be optimal, resulting in the highest transformation efficiency. Transgenic kidney bean plants demonstrated enhanced growth ability under salt and water deficit stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by drought stress. Transgenic lines with high level of lea gene expression showed higher stress tolerance than lines with lower expression level. Stress tolerance of transgenic kidney bean correlated much better with lea gene expression levels than with gene integration results. There is no prior report on the production of transgenic kidney bean using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation.     

Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and <Emphasis Type="Italic">Arabidopsis</Emphasis>

Erythropoietin (EPO) is a glycoprotein used for curing human anemia by regulating the differentiation of erythroid progenitors and the production of red blood cells. To examine the expression of recombinant EPO in plants, pPEV-EP21, in which human epo cDNA under the control of the CaMV 35S promoter, was introduced into tobacco and Arabidopsisvia Agrobacterium tumefaciens-mediated transformation. The RNA expression level of epo in the transgenic lines was initially estimated by Northern blot analysis. Two transgenic lines, which exhibited a high expression level of epo mRNA determined by Northern analysis, were chosen for Western blot analysis to examine the production of EPO proteins. Those two lines, EP21-12 and EP21-14, revealed detectable bands on the immunoblot. Interestingly, constitutive expression of the human epo gene affected the morphologies in transgenic plants such that vegetative growth of transgenic tobacco was retarded, and male sterility was induced in transgenic tobacco and ArabidopsisThese authors contributed equally to this work     

Functional expression of bovine growth hormone gene in Pleurotus eryngii

The expression of the bovine growth hormone (bGH) gene was examined in Pleurotus eryngii, which belongs to the family of oyster mushrooms. The region encoding mature bGH, which has a variety of regulatory effects on growth and metabolic processes, was amplified using designed primers containing initiation and termination codons and then subcloned into pPEV binary expression vector. The recombinant vector (pPEVbGH) was introduced in P. eryngii via Agrobacterium tumefaciens-mediated transformation. Recombinant bGH was expressed in P. eryngii harboring pPEVbGH vector under control of the cauliflower mosaic virus (CaMV) 35S promoter up to a level of approximately 26% of total cell proteins after 6 days of cultivation, after which the recombinant protein was analyzed by SDS-PAGE and Western blotting. Interestingly, the growth rate of P. eryngii mycelia harboring pPEVbGH vector was approximately three times faster than that of control P. eryngii, suggesting that bGH affected the growth of P. eryngii. Biological activities were examined in Sprague-Dawley rats, which were administered regular feed mixed with mycelial extracts containing bGH (0.1 or 0.2 μg of bGH per g of animal feed). Mycelial extracts containing bGH significantly affected growth rates and lipid profiles; total cholesterol, triglyceride, HDL, and LDL levels were improved in rats fed mycelial extracts compared with those administered regular feed containing nontransgenic P. eryngii. This result indicates that P. eryngii harboring pPEVbGH vector could produce biologically active bGH. Further, levels of all growth-related factors increased, resulting in faster growth rates in bGH-treated groups. Accordingly, these data suggest that P. eryngii can be applied to the production of industrially useful proteins using a plant expression vector as an efficient mushroom host system.     

Growth hormone and gene expression of in vitro‐matured rhesus macaque oocytes

Growth hormone (GH) in rhesus macaque in vitro oocyte maturation (IVM) has been shown to increase cumulus expansion and development of embryos to the 9–16 cell stage in response to 100 ng/ml recombinant human GH (r‐hGH) supplementation during IVM. Although developmental endpoints for metaphase II (MII) oocytes and embryos are limited in the macaque, gene expression analysis can provide a mechanism to explore GH action on IVM. In addition, gene expression analysis may allow molecular events associated with improved cytoplasmic maturation to be detected. In this study, gene expression of specific mRNAs in MII oocytes and cumulus cells that have or have not been exposed to r‐hGH during IVM was compared. In addition, mRNA expression was compared between in vitro and in vivo‐matured metaphase II (MII) oocytes and germinal vesicle (GV)‐stage oocytes. Only 2 of 17 genes, insulin‐like growth factor 2 (IGF2) and steroidogenic acute regulator (STAR), showed increased mRNA expression in MII oocytes from the 100 ng/ml r‐hGH treatment group compared with other IVM treatment groups, implicating insulin‐like growth factor (IGF) and steroidogenesis pathways in the oocyte response to GH. The importance of IGF2 is notable, as expression of IGF1 was not detected in macaque GV‐stage or MII oocytes or cumulus cells. Mol. Reprod. Dev. 77: 353–362, 2010. © 2009 Wiley‐Liss, Inc.     

Expression profiles of growth-related genes in two Nile tilapia strains and their crossbred provide insights into introgressive breeding effects

The Nile tilapia (Oreochromis niloticus) is a prominent farmed fish in aquaculture worldwide. Crossbreeding has recently been carried out between the Red-Stirling and the wt Chitralada strains of Nile tilapia, producing a heterotic hybrid (7/8 Chitralada and 1/8 Red-Stirling) that combines the superior growth performance of the Chitralada with the reddish coloration of the Red-Stirling strain. While classical selective breeding and crossbreeding strategies are well known, the molecular mechanisms underlying the phenotypic expression of economically advantageous traits in tilapia remain largely unknown. Molecular investigations have shown that variable expression of growth hormone (gh), insulin-like growth factors (igf1 and 2) and somatolactin (smtla) – components of the growth hormone/insulin-like growth factor (GH/IGF) axis – and myostatin (mstn) genes can affect traits of economic relevance in farmed animals. The aim of this study was to assess and compare the gene expression signature among Chitralada, Red-Stirling and their backcross hybrid in order to gain insights into the effects of introgressive breeding in modulation of the GH/IGF axis. Gene expression analyses in distinct tissues showed that most genes of the GH/IGF axis were up-regulated and mstn was down-regulated in backcross animals in comparison with Red-Stirling and Chitralada animals. These gene expression profiles revealed that backcross animals displayed a distinctive expression signature, which attests to the effectiveness of the introgressive breeding technique. Our findings also suggest that the GH/IGF axis and mstn genes might be candidate markers for fish performance and prove useful within genetic improvement programs aimed at the production of superior-quality tilapia strains using introgressive breeding.     

Secretion of human growth hormone in Bacillus subtilis using prepropeptide coding region of Bacillus amyloliquefaciens neutral protease gene

Using a secretion vector plasmid pES150, which contained a region coding for promoter and prepropeptide of Bacillus amyloliquefaciens neutral protease gene (Honjo et al. (1985) J. Biotechnol. 2, 73–84), a hybrid plasmid pES150GH was constructed. The plasmid pES150GH possesses a mature human growth hormone gene following the prepropeptide coding region. Bacillus subtilis N325 harboring pES150GH produced growth hormone in the medium. The secreted hormone was indistinguishable immunologically from human pituitary growth hormone on double immunodiffusion test. The level of secretory production of growth hormone by B. subtilis reached 40–50 mg l⁻¹ of the culture. The size of the hormone secreted by B. subtilis was approximately 23 K on SDS-PAGE. This value was rather large in comparison with that of natural growth hormone (22 K). It was observed that 99% of synthesized growth hormone was excreted into the medium and that the mode of secretion was co-translational. The secreted growth hormone (23 K) is suggested to be degraded to a 15 K form in the culture medium by proteolysis.     

Characterization of the specific and sustained <Emphasis Type="Italic">GH1</Emphasis> expression induced by rAAV2/1 in normal adult male rats

Our aim was to investigate the long-term effects of intramuscular injection of rAAV2/1-CMV-GH1 viral particles on GH1 expression in normal adult male rats. We found that specific and sustained GH1 expression did not improve muscle exercise performance despite inducing local muscle hypertrophy. Injection of rAAV2/1-CMV-GH1 had some systemic effects on the liver and heart and on lipid metabolism in the healthy rats. Serum levels of hGH (human growth hormone), insulin, glucose and leptin increased significantly, which might induce insulin resistance. The serum concentration of IGF-1 (insulin-like growth factor 1), IGF-BP3 (insulin-like growth factor binding protein 3) and PIIINP (N-terminal propeptide of type III procollagen) markedly increased at 24 weeks after injection of GH1. In conclusion, GH1 expression driven by AAV2/1 in normal animals did not improve muscle strength but did increase muscle mass and may have systemic effects in healthy animals.     

Genetic introduction of foreign genes to <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> by restriction enzyme-mediated integration

Pleurotus eryngii was transformed via restriction enzyme-mediated integration. In order to construct the transformation plasmid, the enhanced cyan fluorescent protein (ECFP) gene was ligated next to the gpd promoter of the plasmid pAN7-1. Transformation was facilitated via the heat treatment of a transformation mixture containing 1 μg of the HindIII-digested plasmid DNA and 10⁶ mushroom protoplasts in 40% polyethyleneglycol solution, resulting in 10–40 hygromycin-resistant transformants. Successful transformation was evidenced by PCR, Southern blot, and confocal fluorescence microscopic analyses on the selected transformants. To date, this is the first report on the transformation of P. eryngii by REMI technique.     

Growth hormone differentially regulates growth and growth-related gene expression in closely related fish species

Zebrafish (Danio rerio) have become an important model organism for developmental biology and human health studies. We recently demonstrated differential growth patterns between the zebrafish and a close relative the giant danio (Danio aequipinnatus), where the giant danio appears to exhibit indeterminate growth similar to most fish species important for commercial production, while zebrafish exhibit determinate growth more similar to mammalian growth. This study focused on evaluating muscle growth regulation differences in adult zebrafish and giant danio utilizing growth hormone treatment as a mode of growth manipulation. Growth hormone treatment resulted in increased overall growth in giant danio, but failed to increase growth in the zebrafish. Growth hormone treatment increased muscle IGF-I and GHrI gene expression in both species, but to a larger degree in the giant danio. In contrast, zebrafish exhibited a larger increase in IrA and IGF-IrB gene expression in muscle in response to GH treatment. In addition muscle myostatin levels were differentially regulated between the two species, with a down-regulation observed in rapidly growing, GH-treated giant danio and an up-regulation in zebrafish not actively growing in response to GH. This is the first report of differential expression of growth-regulating genes in closely related fish species exhibiting opposing growth paradigms. These results further support the role that the zebrafish and giant danio can play important model organisms for determinate and indeterminate growth.     

Enhancement of tolerance to soft rot disease in the transgenic Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>) inbred line,Kenshin

We developed a transgenic Chinese cabbage (Brassica rapa L. ssp. pekinensis) inbred line, Kenshin, with high tolerance to soft rot disease. Tolerance was conferred by expression of N-acyl-homoserine lactonase (AHL-lactonase) in Chinese cabbage through an efficient Agrobacterium-mediated transformation method. To synthesize and express the AHL-lactonase in Chinese cabbage, the plant was transformed with the aii gene (AHL-lactonase gene from Bacillus sp. GH02) fused to the PinII signal peptide (protease inhibitor II from potato). Five transgenic lines were selected by growth on hygromycin-containing medium (3.7% transformation efficiency). Southern blot analysis showed that the transgene was stably integrated into the genome. Among these five transgenic lines, single copy number integrations were observed in four lines and a double copy number integration was observed in one transgenic line. Northern blot analysis confirmed that pinIISP-aii fusion gene was expressed in all the transgenic lines. Soft rot disease tolerance was evaluated at tissue and seedling stage. Transgenic plants showed a significantly enhanced tolerance (2–3-fold) to soft rot disease compared to wild-type plants. Thus, expression of the fusion gene pinIISP-aii reduces susceptibility to soft rot disease in Chinese cabbage. We conclude that the recombinant AHL-lactonase, encoded by aii, can effectively quench bacterial quorum-sensing and prevent bacterial population density-dependent infections. To the best of our knowledge, the present study is the first to demonstrate the transformation of Chinese cabbage inbred line Kenshin, and the first to describe the effect of the fusion gene pinIISP-aii on enhancement of soft rot disease tolerance.     

Production of recombinant great sturgeon (Huso huso,Linnaeus, 1758) growth hormone (GH) by Pichia pastoris (Guillierm, 1956)

In this research, the encoding cDNA of growth hormone (GH) was cloned from the pituitary gland of great sturgeon Huso huso (three adults: two females and one male, 7–9 years old, 70–90 kg, reared in concrete ponds). In order to obtain the great sturgeon recombinant GH expression in Pichia pastoris, the mature encoding cDNA was first cloned in TA vector PTZ57R and then sequenced. After confirmation of the correct GH sequence, the GH coding sequence was subcloned into pHILS1 expression vector. The yeast Pichia pastoris GS115 strain was transformed with the expression plasmid. Results obtained from this study showed that great sturgeon GH recombinants were expressed upon induction with methanol and exported into the medium. The level of expression was examined using RNA analysis, SDS‐PAGE, and western blot analysis. RNA analysis of the recombinant strains showed a sharp, specific band in 800 bp. The specific band in transformants indicated the presence of GH RNA in the yeast. SDS‐PAGE and western blot analysis showed a specific 21 kDa band for the growth hormone. Culture conditions were optimized for pH = 6 and incubation time (after 24 h induction, peaking at 72 h) for maximal protein production. The results provide useful information for the future production of recombinant growth hormones in other sturgeon species.     

Composition analysis of tropical white oyster mushroom (Pleurotus citrinopileatus)

The protein content of the mushroom (Pleurotus citrinopileatus) was determined at different stages of its growth cycle and compared with that of the tropical oyster mushroom Pleurotus sajor-caju. In both species, maximum protein content was obtained at the bud stage but fell thereafter. All essential amino acids were present in adequate amounts, as were K, P, Fe, Ca, Na, Mg and Mn. The heavy metals, Cu and Zn, were present but in very low concentrations, much below the tolerance limit of human beings.     

Cloning and differential expression of the growth hormone in Pampus argenteus

The growth hormone (GH) is a pluripotent hormone produced by the pituitary in vertebrates. It plays important roles in the growth, development, and metabolism of vertebrates.We cloned GH cDNA sequence of Pampus argenteus (GenBank: KT257176). Multi‐sequence analysis revealed P. argenteus GH cDNA contained four conservative cysteine residues positions (Cys⁶⁹, Cys¹⁷⁷, Cys¹⁹⁴, and Cys²⁰²) and shared more than 51.5% identity with homologues from other reported bony fish GHs, except that of Lepisosteus osseus. We used semi‐quantitative RT‐PCR and quantitative real‐time PCR to detect GH expression in 10 tissues and GH expression levels in the pituitary at six different growth stages, and also detected GH content in serum at different growth stages . qPCR showed that GH mRNA was detected in the liver, muscle, kidney, intestine, pituitary, olfactory bulb, stomach, heart, gill, and ovary. The highest level of P. argenteus GH mRNA was observed in the pituitary (P < 0.01, n = 3). At different growth stages, P. argenteus GH expression first increased, decreased, and increased again. GH gene expression levels and the variations of serum GH levels of P. argenteus were consistent with the growth rate and associated with the sexual maturity. In addition, in situ hybridization was used to locate the GH expression in pituitary. In situ hybridization showed that the GH‐positive cells were round, oval, or irregular and often gathered into groups or presented branches along the nerve fibers.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Cry1C,Cry2A</Emphasis> and <Emphasis Type="Italic">Cry9C</Emphasis> genes into <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> and plant regeneration

Three constructs harbouring novel Bacillus thuringiensis genes (Cry1C, Cry2A, Cry9C) and bar gene were transformed into four upland cotton cultivars, Ekangmian10, Emian22, Coker201 and YZ1 via Agrobacterium-mediated transformation. With the bar gene as a selectable marker, about 84.8 % of resistant calli have been confirmed positive by polymerase chain reaction (PCR) tests, and totally 50 transgenic plants were regenerated. The insertions were verified by means of Southern blotting. Bioassay showed 80 % of the transgenic plantlets generated resistance to both herbicide and insect. We optimized conditions for improving the transformation efficiency. A modified in vitro shoot-tip grafting technique was introduced to help entire transplantation. This result showed that bar gene can replace antibiotic marker genes (ex. npt II gene) used in cotton transformation.     

Derivation of a growth hormone gene cassette for goat by mutagenesis of the corresponding bovine construct and its expression in Pichia pastoris

Recombinant bovine growth hormone (rbGH), a 191-aa polypeptide that affects animal growth and lactation, has been used for several years to increase milk production in dairy cattle. It has also been used in goats (Capra hircus) instead of their own hormone (chGH), which is still not available in the market. Since both hormones differ in only one amino acid residue, a strategy based on PCR mediated site-directed mutagenesis, was used to convert the bGH expression cassette harbored by an integration plasmid for Pichia pastoris into a chGH. Transformation by homologous recombination of Pichia pastoris GS115 strain with the linearized new plasmid resulted in transformants that, upon fermentation and induction with methanol, secreted a band with the expected size and immunoreactivity for GH. Production of total proteins secreted into culture medium (50 ml) was 20 µg/ml, of which 60% was chGH as judged by densitometry in SDS-PAGE. Its biological activity was confirmed in vitro when 3T3 pre-adipocytes exposed to the induced culture medium differentiated into adipocytes in cell culture.     

Enhancement of Heterologous Gene Expression in Flammulina velutipes Using Polycistronic Vectors Containing a Viral 2A Cleavage Sequence

Agrobacterium tumefaciens-mediated transformation for edible mushrooms has been previously established. However, the enhancement of heterologous protein production and the expression of multi-target genes remains a challenge. In this study, heterologous protein expression in the enoki mushroom Flammulina velutipes was notably enhanced using 2A peptide-mediated cleavage to co-express multiple copies of single gene. The polycistronic expression vectors were constructed by connecting multi copies of the enhanced green fluorescent protein (egfp) gene using 2A peptides derived from porcine teschovirus-1. The P2A peptides properly self-cleaved as shown by the formation of the transformants with antibiotic resistant capacity and exciting green fluorescence levels after introducing the vectors into F. velutipes mycelia. The results of western blot analysis, epifluorescent microscopy and EGFP production showed that heterologous protein expression in F. velutipes using the polycistronic strategy increased proportionally as the gene copy number increased from one to three copies. In contrast, much lower EGFP levels were detected in the F. velutipes transformants harboring four copies of the egfp gene due to mRNA instability. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming.     

Ectopic Expression of the Tomato <Emphasis Type="Italic">Mi-1</Emphasis> Gene Confers Resistance to Root Knot Nematodes in Lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis>)

The full genomic region of the root knot nematode (Meloidogyne spp.) resistance gene Mi-1 was cloned from tomato and transformed into lettuce to investigate its function in a heterologous system. Transgenic lettuce lines containing the Mi-1 gene were developed using Agrobacterium-mediated transformation. Ectopic expression of the Mi-1 gene was observed in transgenic lines, and resistance to root knot nematode was improved.     

Expression of the IL-2 gene in the potato (Solanum tuberosum cv. Superior)

We intended to examine the expression of the T-cell growth factor (human interleukin-2) so that a binary vector, pSSK-1, carrying the IL-2 gene was constructed and transferred intoA. tumefaciens for the purpose of the transformation of the potato (Solanum tuberosum cv. Superior). All of theAgrobacterium-infected potato explants were regenerated to shoots in modified MS medium and 81% of them rooted on the medium containing kanamycin (200 mg/L). Southern and Northern analysis were performed to verify the transformation events. EL-ISA test indicated that IL-2 protein was produced from IL-2-transformed potatoes. These results suggested expression and production of the IL-2 protein from the transgenic potato.