Chloroplast Genetic Engineering in Higher Plants

Chloroplast genetic engineering, with several advantages over nuclear genetic engineering, is now regarded as an attractive new technology in basic and applied research, including deepening our understanding of plastid genome, engineering plant metabolic system, generating transplastomic plants with higher resistance to insect, disease, drought and herbicide and bioproducing of antibodies and vaccines. In this review, the principle and operating system for chloroplast genetic engineering and its application in higher plants have been discussed.

Research Progress on Biological Risks of Agricultural GM Laboratory

The paper summarizes the research status on biological risks of agricultural GM laboratory and the risk factors of bio-safety in GM crops laboratory,and then proposes corresponding safety management suggestions.

Advances in Plant Science and Its Prospects

Focusing on the global food security and analyzing the challenges now confronting human being of global climate change,sustainable development of agriculture,energy and environment,plant scientists should take the social responsibility and play the role in solving the global problems like food security from the developmental status and potential of plant science and technology. Moreover,by working with agronomists, ecologists and environmental experts,plant scientists do provide novel ideas and technologies for developing low carbon agriculture,for breeding new crop varieties with high yield and quality and stress resistance,for developing new type of energy plants,for supplying healthy foods and drugs,reasonably using land and water resources,and for ameliorating environment,making better contribution to the sustainable development of the globe and human being ourselves.

The Expression and Functional Analysis of the Gene At2G34450 in High Mobility Group B Proteins from Arabidopsis thaliana

[Objective]To better understand the functions of High Mobility Group B （HMGB） proteins in the transcriptional regulation of plant stress responses.[Method]We cloned the At2G33450 gene encoding At2G34450 protein in Arabidopsis thaliana.Binary vectors carrying the above gene were transformed into Arabidopsis to detect the influences of environmental stimuli to transgenic Arabidopsis.[Result] Under salt or drought stress the transgenic Arabidopsis plants over-expressed At2G33450 displayed retarded germination and subsequent growth compared with wild-type plants.[Conclusion]Our results provide a novel basis for understanding the biological functions of HMGB protein family members that differently affect germination and seedling growth of Arabidopsis plants under various stress conditions.

Impact of tiny miRNAs on cancers

miRNAs are a class of small, ∽22nt, non-coding RNAs that negatively regulate gene expression at the posttranscriptional level. They play profound and pervasive roles in manipulating gene expression involved in cell development, proliferation and apoptosis in various eukaryotes, which, in theory, could provide an access to many human diseases in theory. Recent evidence demonstrates that aberrant miRNA expression is a hallmark of tumor development, revealing that miRNA genes could function as potential oncogenes and repressors in the human body. miRNAs can affect tumorigenesis mainly by interrupting the cell cycle at the cellular level and by interacting with signaling, oncogenes and with the response to environmental factors at the molecular level. The established miRNA expression signature could be a potent tool to diagnose and treat human cancers in the future.

NAT2＊6A, a haplotype of the N-acetyltransferase 2 gene, is an important biomarker for risk of anti-tuberculosis drug-induced hepatotoxicity in Japanese patients with tuberculosis

AIM: To investigate an association between N -acetyltransferase 2 (NAT2 )-haplotypes/diplotypes and adverse effects in Japanese pulmonary tuberculosis patients. METHODS: We studied 100 patients with pulmonary TB treated with anti-TB drugs including INH. The frequencies and distributions of single nucleotide polymorphisms, haplotypes, and diplotypes of NAT2 were determined by the PCR-restriction fragment length polymorphism method, and the results were compared between TB patients with and without adverse effect, using multivariate logistic regression analysis.RESULTS: Statistical analysis revealed that the frequency of a variant haplotype, NAT2*6A , was signifi cantly increased in TB patients with hepatotoxicity, compared with those without hepatotoxicity [P = 0.001, odds ratio (OR) = 3.535]. By contrast, the frequency of a wild-type (major) haplotype, "NAT2*4", was signif icantly lower in TB patients with hepatotoxicity than those without hepatotoxicity (P < 0.001, OR = 0.265). There was no association between NAT2-haplotypes and skin rash or eosinophilia. CONCLUSION: The present study shows that NAT2 is one of the determinants of anti-TB drug-induced hepatotoxicity. Moreover, the haplotypes, NAT2*4 and NAT2*6A, are useful new biomarkers for predicting anti- TB drug-induced hepatotoxicity.

HDACs,histone deacetylation and gene transcription: from molecular biology to cancer therapeutics

Histone deacetylases （HDACs） and histone acetyl transferases （HATs） are two counteracting enzyme families whose enzymatic activity controls the acetylation state of protein lysine residues, notably those contained in the N-terminal extensions of the core histones. Acetylation of histones affects gene expression through its influence on chromatin conformation. In addition, several non-histone proteins are regulated in their stability or biological function by the acetylation state of specific lysine residues. HDACs intervene in a multitude of biological processes and are part of a multiprotein family in which each member has its specialized functions. In addition, HDAC activity is tightly controlled through targeted recruitment, protein-protein interactions and post-translational modifications. Control of cell cycle progression, cell survival and differentiation are among the most important roles of these enzymes. Since these processes are affected by malignant transformation, HDAC inhibitors were developed as antineoplastic drugs and are showing encouraging efficacy in cancer patients.

Effect of deleted pancreatic cancer locus 4 gene transfection on biological behaviors of human colorectal carcinoma cells

AIM: To investigate the effect of deleted pancreatic cancer locus 4 (DPC4) gene transfection on biological behaviors of human colorectal carcinoma cells and the role of DPC4 gene in colorectal carcinogenesis. METHODS: PcDNA3.1-DPC4 plasmid was re-constructed by gene-recombination technology. SW620 cells, a human colorectal carcinoma cell line, were transfected with PcDNA3.1-DPC4 plasmid using lipofectamine transfecting technique. Transfected cells were selected with G418. Expression of Smad4 protein was detected in cells transfected with DPC4 gene by immunohistochemistry and Western blot. Biological characterristics of transfected cells were evaluated by population-doubling time and cloning efficiency. Alterations of percentage of S phage cells (S%) and apoptosis rate were determined by flow-cytometry. RESULTS: PcDNA3.1-DPC4 plasmid was constructed successfully. SW620 cells transfected with PcDNA3.1-DPC4 plasmid (DPC4+-SW620 cells) showed a strong intracellular expression of Smad4 protein, and the positive signal was localized in cytoplasm and nuclei, mainly in cytoplasm, where the expressions of Smad4 protein in SW620 cells transfected with PcDNA3.1 plasmid (PcDNA3.1-SW620 cells) and non-transfected SW620 cells (SW620 cells) were weaker than those in DPC4+-SW620 cells. The population-doubling time in DPC4+-SW620 cells (116 h) was significantly longer than that in SW620 cells (31 h) and PcDNA3.1-Sw620 cells (29 h) (P<0.01). The cloning efficiencies of DPC4+-SW620 cells (12%) were markedly lower than those of SW620 cells (69%) and PcDNA3.1-Sw620 cells (67%) (P<0.01). Compared with SW620 cells and PcDNA3.1-Sw620 cells, the Go-G1% of DPC4+-SW620 cells was obviously higher and the S% was markedly lower (P<0.05). Apoptosis rate of DPC4+-SW620 cells was significantly higher than that of SW620 cells and PcDNA3.1-SW620 cells. CONCLUSION: PcDNA3.1-DPC4 plasmid can be successfully re-constructed and stably transfected into human SW620cells, thereby the cells can steadily express Smad4. DPC4 protein may regulate proliferation of colorectal carcinoma cells by inhibiting cell growth and inducing cell apoptosis.

Determination of Selected Toxic Elements in Apples from Organic Farming

Due to the elevated ecological awareness nowadays the consumption of products of organic agriculture is increasing. Organic farming means excluding the use of synthetic inputs, such as synthetic fertilizers and pesticides or genetically modified organisms, but there are only a few regulations regarding heavy metal concentrations in soils. Thus a not negligible uptake of metals from the soil where the apple trees are grown may occur. Furthermore inorganic copper compounds being traditional fertilizers for apple trees are not considered as synthetic fertilizers, thus they are still used in organic apple farming for soil or foliar application. Thus also apples produced by organic agriculture may contain toxic elements, such as cadmium, chromium, copper, and lead. The concentrations of these elements were determined in whole apples, as well as in the flesh and peel in order to estimate the possible risk for human health. Prior to analysis using ICP-AES samples underwent a microwave assisted digestion. The LODs obtained are below the recommended maximum levels in vegetables/fruits by WHO. In all samples no Cu, Cr, and Pb could be detected. Only Ca was found in the peel of about a third of samples investigated in concentrations between LOD and LOQ （1-3 μg/g）.

Soybean GmMYB76, GmMYB92, and GmMYB177 genes confer stress tolerance in transgenic Arabidopsis plants

MYB-type transcription factors contain the conserved MYB DNA-binding domain of approximately 50 amino acids and are involved in the regulation of many aspects of plant growth, development, metabolism and stress responses. From soybean plants, we identified 156 GmMYB genes using our previously obtained 206 MYB unigenes, and 48 were found to have full-length open-reading frames. Expressions of all these identified genes were examined, and we found that expressions of 43 genes were changed upon treatment with ABA, salt, drought and/or cold stress. Three GmMYB genes, GmMYB76, GmMYB92 and GmMYB177, were chosen for further analysis. Using the yeast assay system, GmMYB76 and GmMYB92 were found to have transactivation activity and can form homodimers. GmMYB177 did not appear to have transactivation activity but can form heterodimers with GmMYB76. Yeast onehybrid assay revealed that all the three GmMYBs could bind to cis-elements TAT AAC GGT TTT TT and CCG GAA AAA AGG AT, but with different affinity, and GmMYB92 could also bind to TCT CAC CTA CC. The transgenic Arabidopsis plants overexpressing GmMYB 76 or GmMYB177 showed better performance than the GmMYB92-transgenic plants in salt and freezing tolerance. However, these transgenic plants exhibited reduced sensitivity to ABA treatment at germination stage in comparison with the wild-type plants. The three GmMYB genes differentially affected a subset of stress-responsive genes in addition to their regulation of a common subset of stress-responsive genes. These resuits indicate that the three GmMYB genes may play differential roles in stress tolerance, possibly through regulation of stress-responsive genes.

MicroRNAs in pancreatic ductal adenocarcinoma

Ductal adenocarcinoma of the pancreas is a lethal cancer for which the only chance of long-term survival belongs to the patient with localized disease in whom a potentially curative resection can be done. Therefore, biomarkers for early detection and new therapeutic strategies are urgently needed. miRNAs are a recently discovered class of small endogenous non-coding RNAs of about 22 nucleotides that have gained attention for their role in downregulation of mRNA expression at the post-transcriptional level. miRNAs regulate proteins involved in critical cellular processes such as differentiation, proliferation, and apoptosis. Evidence suggests that deregulated miRNA expression is involved in carcinogenesis at many sites, including the pancreas. Aberrant expression of miRNAs may upregulate the expression of oncogenes or downregulate the expression of tumor suppressor genes, as well as play a role in other mechanisms of carcinogenesis. The purpose of this review is to summarize our knowledge of deregulated miRNA expression in pancreatic cancer and discuss the implication for potential translation of this knowledge into clinical practice.

Application of Genetically Modified Technology in Maize Breeding

The achievements of genetical y modified （GM） maize in herbicide toler-ance, insect resistance, disease resistance and alkali resistance were reviewed in this paper so as to provide some reference for the researches on molecular breed-ing of maize in China.

MicroRNAs identification and bioinformatics analysis in large yellow croaker Larimichthys crocea using an integrated comparative and ab initio approach

MicroRNAs(miRNAs) are a group of small, endogenous, single-stranded non-coding RNAs that post-transcriptionally regulate gene expression levels. Previous studies have revealed that miRNAs play key roles in multiple biological processes, such as growth and development in both animals and plants. Computational identification is an efficient method for miRNA prediction in organisms with a reference genome before high-throughput miRNA sequencing experiments. In this study, we employed an integrated strategy combining the homology-based and ab initio approaches to predict miRNAs from the genome and transcriptome of large yellow croaker, one of the most commercially important marine fish in China and East Asia. A total of 418 miRNA molecules, including 287 miRNAs by the homology-based method and 131 miRNAs by the ab initio approach, were identified for large yellow croaker. Additionally, 16 053 target genes were predicted and annotated for Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) databases. Meanwhile, we analysed single nucleotide polymorphisms(SNPs) around large yellow croaker miRNA and found that the miRNA seed regions were significantly less prone to mutations, indicating that the miRNA sequences were under strict natural selection based on their essential regulation functions in living cells. Twenty-two SNPs were identified in large yellow croaker miRNA seed regions, which dramatically influenced the miRNA-gene regulation networks. This is the first reported miRNA detection from both the genome and transcriptome using the integrated strategy for large yellow croaker species, and the miRNA and SNP analyses in this work provide important resources and a reference for subsequent miRNA functional investigations in large yellow croaker.

Effect of Salinity on the Biosynthesis of Amines in Litopenaeus vannamei and the Expression of Gill Related Ion Transporter Genes

This study examined the effect of salinity on the expression of Na+/K+-ATPase(NKA) α-subunit and vacuolar-type H+-ATPase(V-ATPase) β-subunit gene in the gill of Litopenaeus vannamei. Semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) assay showed that the expression of NKA α-subunit and V-ATPase β-subunit gene was significantly influenced by salinity. It was found that the NKA activity significantly varied with salinity in time and dose dependent manner; whereas the V-ATPase activity did not. The abundance of NKA α-subunit gene transcript increased rapidly when the salinity decreased from 26 b to 21, and slowly when the salinity decreased from 26 to 31 within the first 24 h. When the salinity decreased from 26 to 21, the transcription of NKA α-subunit gene in gill epithelium was higher at 12 h than that at 0 h, which was consistent with the result of immunoblotting assay of NKA α-subunit. In addition, salinity had a significant time- and dose-dependent effect on the concentration of biogenic amines in both hemolymph and gill. As compared to other parameters, the concentration of dopamine(DA) and 5-hydroxytryptamine(5-HT) varied in different patterns when the salinity decreased from 26 to 21 or increased from 26 to 31, suggesting that DA and 5-HT played different regulatory roles in osmotic adaption and modulation of shrimp when salinity varies.

Systems perspectives on mRNA processing

The application of genomic technologies to the study of mRNA processing is increasingly conducted in metazoan organisms in order to understand the complex events that occur during and after transcription. Large-scale systems analyses ofmRNA-protein interactions and mRNA dynamics have revealed specificity in mRNA transcription, splicing, transport, translation, and turnover, and have begun to make connections between the different layers ofmRNA processing. Here, we review global studies of post-transcriptional processes and discuss the challenges facing our understanding of mRNA regulation in metazoan organisms. In parallel, we examine genome-scale investigations that have expanded our knowledge of RNA-binding proteins and the networks of mRNAs that they regulate.

Genome-wide identification and transcriptome-based expression analysis of sox gene family in the Japanese flounder Paralichthys olivaceus

Sox genes are transcription factors that ubiquitously exist in animal species, and share a conserved high mobility group(HMG) box. They play important biological roles in diverse developmental processes, such as sex determination and differentiation, chondrogenesis, neurogenesis, and early embryonic development. In this study, we identified 25 sox genes from genome and transcriptome of Japanese flounder Paralichthys olivaceus. These s ox genes could be mainly classified into seven subfamilies(B1, B2, C, D, E, F, and K), and each subfamily exhibited a relatively conserved gene structure. Besides, subfamilies A and G were found exclusively in human and mouse, and sox 32 in subfamily K only existed in teleosts. Compared with other mammals, some s ox genes in teleosts had two duplicates. The loss, duplication, and divergence of sox genes during evolution provided an evidence for whole-genome duplication that occurred in the radiation of teleosts. The expression of Japanese flounder sox genes was also analyzed by FPKM value. Our results showed that certain s ox genes exhibited obviously tissue-specific and spatio-temproal expression. Especially, gonal-basied expression analysis uncovered that s ox7 and s ox2 were ovary-biased, and s ox8 b was testis-biased. Moreover, sox10 a was expressed specifically in ovary, and sox8 a in testis. Therefore this study provide a solid foundation for future functional and evolutionary analysis of sox genes in Japanese flounder.

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.

Applicability of Cytoplasmic Male Sterility （CMS） as a Reliable Biological Confinement Method for the Cultivation of Genetically Modified Maize in Germany

The cultivation of genetically modified （GM） plants requires the reduction of an unwanted spread of genes （biological confinement）. Cytoplasmic male sterility （CMS） inhibits the development of functional pollen, but nuclear restorer （RJ） genes and environmental impacts can restore the fertility. The aim of this study was to verify whether CMS in maize hybrids is a reliable confinement method for the prospective cultivation of GM maize in Germany. Two-year field experiments in three different environments were conducted with three CMS maize hybrids which vary in the CMS stability, one conventional maize variety （all yellow kernels） and white maize as pollen recipient. Tassel characteristics, pollen vitality and cross-pollination rates were investigated. The CMS stability was dependent on the genotype and the specific weather conditions per year and location. In all maize hybrids CMS was unstable. One CMS maize hybrid showed a high level of CMS stability and very low cross-pollination rates in any case （〈 1%）. The two other CMS maize hybrids developed more fluctuant and fertile tassels with few or many pollen, respectively. Compared with a conventional and fully fertile maize variety, cross-pollination of all CMS maize hybrids was strongly reduced （84%-99%）. In conclusion, the CMS trait can be proposed as a useful biological confinement method to reduce pollen-mediated gene flow from GM maize.

Bioinformatics Analysis of VOZ Gene Family in Populus trichocarpa

To explore the biological characteristics of Vascular plant One-Zinc finger(VOZ)gene family in Populus trichocarpa,this paper used bioinformatics to analyze the nucleotide sequences and protein sequences of four members of VOZ gene family of P.trichocarpa.The results showed that the four PtVOZ genes of P.trichocarpa were evenly distributed on four chromosomes.The length and molecular weight of the encoded protein were almost the same,and the subcellular localization was located in the nucleus,belonging to the unstable acidic hydrophilic non-aliphatic soluble protein.The gene structures were all in the patterns of 4 exons and 3 introns.The proportion order of PtVOZ transcription factor secondary structure components was random coil>αhelix>extended strand>βsheets,and the tertiary structure was very similar in spatial conformation.The phylogenetic tree analysis showed that P.trichocarpa was more closely related to VOZ transcription factors of dicotyledons.The four PtVOZ genes of P.trichocarpa were expressed in seedlings and different tissues,but there were differences in the expression intensity.This study provided a necessary theoretical basis for further exploring the molecular biological function of PtVOZ genes.

Risk Assessment and Biosafety Studies of Transgenic Bt Rice （Oryza sativa L.）

Transgenic crops having alien genes from different sources are getting popularity. A Rice （Oryza sativa L.） containing gene from Bacillus thuringiensis, a soil bacterium, was assessed to study the potential risks of transgenic plants on environment. The crop was found resistant to target insect pests. Rice variety Basmati-370 transformed with two insecticidal genes, crylAc and cry2A, was grown under field conditions for several years. Data were collected at different stages of the plant growth. Fate of Cry protein in soil, effect of Bt protein on non-target insects, risks of vertical and horizontal gene flow were evaluated. No potential hazard was found at all levels. Bt protein was unstable and degraded significantly in soil within 30 days after harvesting the crop. No harmful effects were found on non-target insects （insects other than order lepidoptera）. Maximum gene flow of 0.02% was observed at close spacing and no evidence of horizontal gene transfer to Rhizobium spp. was found. In conclusion, the transgenic rice plants transformed with Bt genes have no harmful effects on the environment.