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Objective: The main objective of the present study is to evaluate the psychometric properties of the Chinese version of Beliefs about Medicines Questionnaire-specific among asthma patients; and to assess the association between patients’ belief and adherence to inhaled corticosteroid therapy. Methods: A cross-sectional survey was carried out in the asthma clinic of Zhongshan Hospital, to Fudan University (Shanghai, China) between April 2016 and March 2018. The Beliefs about Medicines Questionnaire-specific was translated into Chinese according to international guidelines. Internal consistency, test–retest reliability, and confirmatory factor analysis were calculated to validate the Beliefs about Medicines Questionnaire-specific. The relationship between the adherence and the belief subscale were assessed using Kruskal–Wallis test. Results: Two hundred and seventeen patients were recruited in this study. The Beliefs about Medicines Questionnaire-specific was deemed reliable based on the results of Cronbach’s alpha coefficient and test–retest intraclass correlation coefficient (ICC, ICC= 0.759). Confirmatory factor analysis showed acceptable model fit for the two-factor model. Patients’ compliance was closely related to their belief about inhaled corticosteroid. The adherence rates were highest for the accepting groups, and lowest for the skeptical groups. Higher adherence was significantly associated with higher necessity-concerns differential (p = .001) and lower concern (p = .004). Conclusions: The Chinese version of the Beliefs about Medicines Questionnaire-specific can be used as a reliable tool by the clinicians to identify beliefs and behaviors of individual to improve adherence in Chinese patients.

Increasing evidence has demonstrated that circulating microRNAs (miRNAs) can be utilized as potential biomarkers for the diagnosis of cancer, as well as a prognostic tool for the management of the disease. Therefore, the present study aimed to evaluate the predictive ability of miRNA (miR)-155, miR-96 and miR-99a for the diagnosis and prognosis of hepatocellular carcinoma (HCC). Tissues were collected from 30 patients with HCC and their matched adjacent normal liver tissues, as well as from serum samples from 30 patients with HCC and 30 healthy controls. Reverse transcription-quantitative PCR was used to measure the expression levels of miR-155, miR-96 and miR-99a. The expression levels of miR-155 and miR-96 were upregulated in the tissues and serum of patients with HCC, whereas miR-99a expression levels were decreased. Receiver operating characteristics (ROC) curve analysis revealed that circulating miR-155, miR-96, miR-99a and a combination of these three miRNAs could serve as diagnostic biomarkers for HCC with areas under the curve (AUC) of 0.84, 0.824, 0.799 and 0.931, respectively. Serum α-fetoprotein (AFP) was detected using electrochemiluminescence immunoassay analyzer. The addition of AFP with the combination of these three miRNAs offered a higher accuracy of HCC diagnosis (AUC, 0.979; sensitivity, 90.0%; specificity, 100.0%). In addition, elevated expression levels of miR-155 and miR-96 were associated with poor survival time of patients with HCC. The panel of miR-155, miR-96, miR-99a and AFP had a higher sensitivity and specificity for the diagnosis of HCC when compared with a single marker. Furthermore, the present data suggested that miR-155 and miR-96 may be potential prognostic markers for the clinical management of patients with HCC.

Phosphate (Pi) deficiency has become a significant challenge to worldwide agriculture due to the depletion of accessible rock phosphate that is the major source of cheap Pi fertilizers. Previous research has identified a number of diverse adaptive responses to Pi starvation in the roots of higher plants. In this study, we found that accelerated axile root elongation of Pi-deprived maize plants resulted from enhanced cell proliferation. Comparative phosphoproteome and proteome profiles of maize axile roots were conducted in four stages in response to Pi deficiency by multiplex staining of high-resolution two dimensional gel separated proteins. Pro-Q DPS stained gels revealed that 6% of phosphoprotein spots displayed changes in phosphorylation state following low-Pi treatment. These proteins were involved in a large number of metabolic and cellular pathways including carbon metabolism and signal transduction. Changes in protein abundance of a number of enzymes indicated that low-Pi induced a number of carbon flux modifications in metabolic processes including sucrose breakdown and other downstream sugar metabolic pathways. A few key metabolic enzymes, including sucrose synthase (EC 2.4.1.13) and malate dehydrogenase (EC 1.1.1.37), and several signaling components involved in protein kinase or phosphatase cascades, auxin signaling and 14-3-3 proteins displayed low-Pi responsive changes in phosphorylation state or protein abundance. A variety of key enzymes and signaling components identified as potential targets for phosphorylation provide novel clues for comprehensive understanding of Pi regulation in plants. Protein phosphorylation, coordinating with changes in protein abundance, is required for maize root metabolic regulation and developmental acclimation to Pi starvation.

Bear bile is a traditional Chinese medicine that has been used for millennia. Several arguments support and oppose the use of bear farming in terms of conservation and nonhuman animal welfare. This study involved designing a questionnaire and surveying a random sample of general citizens and college students in Beijing to elicit their attitudes on bile extraction from living bears. Older people and people with lower education levels used more bear bile medicines. In total, 29.47% (n = 204) of citizens and 23.14% (n = 81) of students surveyed used bear bile medicine since 1990. Students were less willing to use bear bile medicines than citizens (p < .05). The level the respondents agreed with the blue side (against the extraction of bile from living bears; anti for short) was significantly higher than that for the red side (support the extraction of bile from living bears; pro for short; p < .05). Additionally, college students had a more distinct attitude toward the opposing views, which indicates they were more inclined to oppose bile extraction from living bears.

Purpose This study aimed to validate a Chinese version of the Morisky Medication Adherence Scale (MMAS-8) in patients with epilepsy. The relationships between adherence, seizure frequency, and adverse effects were assessed using this method. Methods Data from patients diagnosed with epilepsy at the Department of Neurology of Huashan Hospital were collected between January and June 2013. To validate the MMAS-8, internal consistency, test–retest reliability, and factor analysis were calculated. Relationships between adherence, seizure frequency, and adverse effects were assessed using Pearson's correlation. Results One hundred and eleven patients were recruited. The MMAS-8 had moderate internal consistency (Cronbach's α =0.556) and good test–retest reliability (intraclass correlation coefficient=0.729). The MMAS-8 adherence rate was 79.2%. MMAS-8 adherence was negatively correlated with seizure frequency and adverse effects ( r =–0.708, p r =–0.484, p Conclusion The MMAS-8 scale can be used as a tool to assess medication adherence in Chinese patients with epilepsy. Better seizure control and lower rates of adverse effects were significantly correlated with higher adherence scores.

Phosphatidylinositol (PtdIns) synthase is a key enzyme in the phospholipid pathway and catalyses the formation of PtdIns. PtdIns is not only a structural component of cell membranes, but also the precursor of the phospholipid signal molecules that regulate plant response to environment stresses. Here, we obtained transgenic maize constitutively overexpressing or underexpressing PIS from maize (ZmPIS) under the control of a maize ubiquitin promoter. Transgenic plants were confirmed by PCR, Southern blotting analysis and real-time RT-PCR assay. The electrospray ionization tandem mass spectrometry (ESI-MS/MS)-based lipid profiling analysis showed that, under drought stress conditions, the overexpression of ZmPIS in maize resulted in significantly elevated levels of most phospholipids and galactolipids in leaves compared with those in wild type (WT). At the same time, the expression of some genes involved in the phospholipid metabolism pathway and the abscisic acid (ABA) biosynthesis pathway including ZmPLC, ZmPLD, ZmDGK1, ZmDGK3, ZmPIP5K9, ZmABA1, ZmNCED, ZmAAO1, ZmAAO2 and ZmSCA1 was markedly up-regulated in the overexpression lines after drought stress. Consistent with these results, the drought stress tolerance of the ZmPIS sense transgenic plants was enhanced significantly at the pre-flowering stages compared with WT maize plants. These results imply that ZmPIS regulates the plant response to drought stress through altering membrane lipid composition and increasing ABA synthesis in maize.

The human CD14, a high affinity receptor for lipopolysaccharides (LPS), is involved in the innate immunity system and the inflammatory response. There is increasing interest in using recombinant approaches to produce purified CD14 protein for therapeutic uses. Plants provide ideal expression systems for the production of recombinant proteins, but the levels of expression of recombinant proteins produced in planta are still not high. To improve expression levels of CD14 the 22-kDa alpha-zein signal peptide (ZSP) from maize was fused to the human CD14 cDNA so that recombinant CD14 could stably accumulate in plant cells. The human CD14 gene and the modified human CD14 cDNA with the 22-kDa ZSP were respectively transformed into tobacco to produce transgenic plants. Western blot analysis confirmed human CD14 accumulation in the transgenic tobacco. The concentration of the recombinant protein in the tobacco leaves was measured by ELISA, and the results suggested that fusion with the 22-kDa alpha-ZSP effectively increased the accumulation of the recombinant protein (rCD14). The concentration of rCD14 in some of the transgenic lines was 19.54 μg g−1 tobacco leaf (fw), which was about 0.6 % of the total soluble protein. The rCD14 protein showed natural LPS-binding bioactivity by using U937 cells mensuration. Our results suggested that the maize 22-kDa alpha-zein signal peptide could be used to increase the accumulation of recombinant protein in tobacco leaves so that proteins can be produced in abundant biomass.

Drought is a major agricultural menace reducing crop productivity and limiting the successful realization of land potential throughout the world. Therefore, breeding common wheat with improved drought-tolerance via genetic manipulation is of great importance. We have introduced the betA gene encoding choline dehydrogenase from Escherichia coli into common wheat (Triticum aestivum L.) by Agrobacterium-mediated transformation. Various levels of expression of the betA gene were confirmed by RT-PCR among the transgenic lines and different levels of glycine betaine accumulation were detected in these lines. Several wheat transgenic lines with different betA expression levels in the T3 generation and wild-type (WT) were selected to test their performance under drought stress conditions. Water deficit in plants caused a reduction in photosynthesis and activity of the PSII complex and resulted in increased accumulation of osmolytes. Drought stress also led to lower membrane stability along with much higher activities of superoxide dismutase and peroxidase in all wheat lines. However, wheat lines that were transgenic for the betA gene were less injured and exhibited greater root length and growth compared with the WT. It was concluded that the amount of injury to the wheat plants was negatively correlated with the level of accumulation of glycine betaine, and the glycine betaine acted as an important osmoprotectant in transgenic plants to improve root growth, and enhance the resistance of transgenic plants to drought stress.

The DREB/CBF transcription factors play important roles during low temperature, drought, and high-salt stress in higher plants. In this paper, we transferred TsCBF1 gene from a dicotyledonous halophyte Thellungiella halophila into the monocotyledonous crop maize (Zea mays L.). PCR, Southern blot, and RT-PCR analysis indicated that the TsCBF1 gene had been integrated into the genome of transgenic plants and was expressed in their progeny. After 14 days of drought stress treatments, transgenic plants showed improved drought tolerance with higher Relative Water Content (RWC), higher solute accumulation, and less cell damage compared with wild-type (WT) plants. Most importantly, they showed shorter anthesis-silking interval (ASI) and produced much higher grain yield than WT under drought stress. The results indicate that the TsCBF1 gene conferred enhanced drought tolerance to maize plants and may have utility for improving tolerance to other abiotic stresses as well.

A betA gene encoding choline dehydrogenase from Escherichia coli (E. coli) was transformed into wheat (Triticum aestivum L.) via Agrobacterium-mediated transformation. PCR amplification and Southern blotting confirmed the existence of transgene in transformed plants and their progeny. Levels of expression of the betA gene varied among the different transgenic lines based on RT-PCR analysis. Under salt stress conditions, transgenic lines L2 and L3 had higher levels of glycine betaine and chlorophyll, lower Na+/K+ ratios and solute potential, and less cell membrane damage. These lines also retained moderately higher photosynthesis rates and more vigorous growth than the wild-type line at 200 mM NaCl. In a field trial in a high salt field, transgenic lines L2 and L3 had higher germination rates, more tillers and higher grain yields in comparison to the wild-type plants. This suggested that the transgenic plants were more tolerant to salt stress and have potential for breeding salt-tolerant wheat.

The possible release of selectable marker genes from genetically modified transgenic plants, or of gut microbes, to the environment, has raised worldwide public concerns. In this study, we showed the generation of marker-free transgenic maize plants constitutively expressing AtNHX1, a Na(+)/H(+) antiporter gene from Arabidopsis that conferred salt tolerance on plants, using the FLP/FRT site-specific recombination system. Transgenic plant expressing a modified FLP recombinase gene was crossed with transgenic plant harboring AtNHX1 and mutant als, a selectable marker gene flanked by two directed FRT sites. The sexual crossing led to precise and complete excision of the FRT-surrounded als marker gene in the F1 progenies. Further salt tolerance examinations indicated that marker-free AtNHX1 transgenic plants accumulated more Na(+) and K(+), and produced greater biomass and yields than did the wild-type plants when grown in high saline fields. These results demonstrate the feasibility of using this FLP/FRT-based marker elimination system to generate marker-free transgenic important cereal crops with improved salt tolerance.

Previous studies have shown that the overexpression of betA (encoding choline dehydrogenase from Escherichia coli) or AtNHX1 (a vacuolar Na(+)/H(+) antiport from Arabidopsis thaliana) gene can improve the salt tolerance of transgenic plants. However, little is known about the effects of the transgene pyramiding of betA and AtNHX1. Here, betA + AtNHX1 transgene pyramiding tobacco was produced by sexual crossing, and the salt tolerance was evaluated at the cellular and plant levels. In NaCl stress, the Na(+) concentration in vacuoles and vacuolar membrane potential of transgene pyramiding cells were similar to those of AtNHX1-transgenics, and much higher than those of betA-transgenics when detected using fluorescent dye staining; transgene pyramiding cells showed a higher protoplast viability and comparable mitochondrial activity as compared with single transgenics; and transgene pyramiding plants showed comparable Na(+) content in leaves as compared with AtNHX1-transgenics and remarkably higher than betA-transgenics; and transgene pyramiding lines exhibited higher percentage of seed germination, better seedling growth and higher fresh weight than lines that had betA or AtNHX1 alone. Based on the integrative analysis of salt tolerance, the consistency between the cellular level and the whole plant level was confirmed and the transgene pyramiding plants exhibited improved salt tolerance, but compared with the plants with betA or AtNHX1 alone, the differences were relatively small. Other mechanisms involved in salt tolerance should be considered to further enhance transgene pyramiding plants salt tolerance.

Low phosphorus (P) availability is a major limitation for plant growth. To better understand the molecular mechanism of P efficiency in maize, comparative proteome analyses were performed on the roots of the low-P-tolerant mutant 99038 and wild-type Qi-319 grown under P-sufficient (+P) or P-deficient (-P) conditions. Over 10% of proteins detected on two-dimensional electrophoresis (2-DE) gels showed expression that was altered twofold or more between the genotypes under +P or -P conditions. We identified 73 (+P) and 95 (-P) differentially expressed proteins in response to phosphate (Pi) starvation. These proteins were involved in a large number of cellular and metabolic processes, with an obvious functional skew toward carbon metabolism and regulation of cell proliferation. Further analysis of proteome data, physiological measurements and cell morphological observations showed that, compared to the wild-type, the low-P-tolerant mutant could accumulate and secrete more citrate under Pi starvation, which facilitates solubilization of soil Pi and enhances Pi absorption. The proportion of sucrose in the total soluble sugars of the low-P-tolerant mutant was significantly higher, and cell proliferation in root meristem was accelerated. This resulted in better developed roots and more advantageous root morphology for Pi uptake. These results indicate that differences in citrate secretion, sugar metabolism and root-cell proliferation are the main reasons for higher tolerance to low-P conditions in the mutant compared to the wild-type. Thus, the mutant displayed specialized P-efficient root systems with a higher capacity for mobilization of external Pi and increased cell division in the root meristem under Pi starvation.

The vacuolar H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) uses pyrophosphate as substrate to generate the proton electrochemical gradient across the vacuolar membrane to acidify vacuoles in plant cells. The heterologous expression of H(+)-PPase genes (TsVP from Thellungiella halophila and AVP1 from Arabidopsis thaliana) improved the salt tolerance of tobacco plants. Under salt stress, the transgenic seedlings showed much better growth and greater fresh weight than wild-type plants, and their protoplasts had a normal appearance and greater vigor. The cytoplasmic and vacuolar pH in transgenic and wild-type cells were measured with a pH-sensitive fluorescence indicator. The results showed that heterologous expression of H(+)-PPase produced an enhanced proton electrochemical gradient across the vacuolar membrane, which accelerated the sequestration of sodium ions into the vacuole. More Na(+) accumulated in the vacuoles of transgenic cells under salt (NaCl) stress, revealed by staining with the fluorescent indicator Sodium Green. It was concluded that the tonoplast-resident H(+)-PPase plays important roles in the maintenance of the proton gradient across the vacuolar membrane and the compartmentation of Na(+) within vacuoles, and heterologous expression of this protein enhanced the electrochemical gradient across the vacuolar membrane, thereby improving the salt tolerance of tobacco cells.

The tolerance to drought stress of the homozygous transgenic cotton (Gossypium hirsutum L.) plants with enhanced glycinebetaine (GB) accumulation was investigated at three development stages. Among the five transgenic lines investigated, lines 1, 3, 4, and 5 accumulated significantly higher levels of GB than the wild-type (WT) plants either before or after drought stress, and the transgenic plants were more tolerant to drought stress than the wild-type counterparts from young seedlings to flowering plants. Under drought stress conditions, transgenic lines 1, 3, 4, and 5 had higher relative water content, increased photosynthesis, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation than WT plants. The GB levels in transgenic plants were positively correlated with drought tolerance under water stress. The results suggested that GB may not only protect the integrity of the cell membrane from drought stress damage, but also be involved in OA in transgenic cotton plants. Most importantly, the seedcotton yield of transgenic line 4 was significantly greater than that of WT plants after drought stress, which is of great value in cotton production.

Sucrose: sucrose 1-fructosyltransferase (1-SST) cDNA from Lactuca sativa, coding the enzyme responsible for lower degree polymers fructan biosynthesis, was cloned by RT-PCR and RACE methods. The 1-SST cDNA under the control of CaMV 35S promoter was introduced into tobacco by Agrobacterium-mediated leaf disc transformation protocol. Fructan synthesis in vitro and carbohydrate analysis showed that sense transgenic tobacco plant displayed sucrose: sucrose 1-fructosyltransferse activity. After freezing stress, significant increases in electrolyte leakage and malondialdehyde were found in the wild type and anti-sense transgenic plants, while no apparent differences were observed in sense transgenic plants. Meanwhile, water soluble carbohydrate, fructan and fructose of sense transgenic plants remarkably increased, compared with those of wild type and anti-sense plants. No significant difference was detected in superoxide dismutase activity between transgenic and wild type plants. The above results demonstrated that the expression of 1-SST gene improved the freezing resistance of transgenic tobacco plants.

Embryogenic calli derived from the young embryo of maize inbred line Qi-319, were subcultured for half a year and screened continuously on the medium without phosphate. Low-P tolerant cell lines were obtained and the regenerated plants (R0) were self-pollinated to harvest seeds. The progeny (R1) of 21% regenerated plants showed low-P tolerance traits. Low-P tolerant R1 plants and their progeny were self-pollinated for 5 generations, and a number of low-P tolerant inbred lines including 99038 were obtained. Comparison of response of 99038 and Qi-319 to P nutrition indicated that under P deficiency the growth of 99038 plants were less influenced, especially roots, indicating that low-P tolerance was improved significantly. Inbred line 99038 had larger root systems with modification and capability of P acquisition improved significantly compared to Qi-319, and a larger proportion of P was retained in roots of 99038, so P concentration in roots was obviously higher, which improved root growth under P deficiency. Comparison of 2-DE gel maps of proteins isolated from roots treated with P deficiency suggested that more than 13% of the proteins were different in 99038 and Qi-319, which supported the general viewpoint that the P-efficiency of plant was a trait controlled by multi-gene. We concluded that maize mutants with low-P tolerance could be obtained efficiently by cellular engineering and the low-P tolerance of maize inbred lines 99038 appeared to be a complicated metabolism process controlled by major genes.

An H(+)-pyrophosphatase (PPase) gene named TsVP involved in basic biochemical and physiological mechanisms was cloned from Thellungiella halophila. The deduced translation product has similar characteristics to H(+)-PPases from other species, such as Arabidopsis and rice, in terms of bioinformation. The heterologous expression of TsVP in the yeast mutant ena1 suppressed Na(+) hypersensitivity and demonstrated the function of TsVP as an H(+)-PPase. Transgenic tobacco overexpressing TsVP had 60% greater dry weight than wild-type tobacco at 300 mM NaCl and higher viability of mesophyll protoplasts under salt shock stress conditions. TsVP and AVP1, another H(+)-PPase from Arabidopsis, were heterologously expressed separately in both the yeast mutant ena1 and tobacco. The salt tolerance of TsVP or AVP1 yeast transformants and transgenic tobacco were improved to almost the same level. The TsVP transgenic tobacco lines TL3 and TL5 with the highest H(+)-PPase hydrolytic activity were studied further. These transgenic tobacco plants accumulated 25% more solutes than wild-type plants without NaCl stress and 20-32% more Na(+) under salt stress conditions. Although transgenic tobacco lines TL3 and TL5 accumulated more Na(+) in leaf tissues, the malondialdehyde content and cell membrane damage were less than those of the wild type under salt stress conditions. Presumably, compartmentalization of Na(+) in vacuoles reduces its toxic effects on plant cells. This result supports the hypothesis that overexpression of H(+)-PPase causes the accumulation of Na(+) in vacuoles instead of in the cytoplasm and avoids the toxicity of excessive Na(+) in plant cells.

This study was carried out to evaluate the effects of purine synthesis inhibitor mizoribine, purine and pyrimidine synthesis inhibitors azaserine and acivicin, and surfactant Silwet L-77 on Agrobacterium-mediated transformation efficiency of embryogenic calluses from maize elite inbred lines Qi 319 and Ye 515. After transformation and three rounds of selection on 2.8 μM chlorsulfuron, resistant calluses were obtained subsequently, and morphologically normal plantlets were regenerated from 80 to 90% of the resistant calluses treated with the compounds. There were no obvious discrepancies between the frequencies of plantlet regeneration and the ratio of PCR positive plantlets of calluses treated with different compounds. Results of PCR assay with primers for betA showed that 40.2% (103/256) of the regenerated plantlets were positive. The percentage of resistant calluses was 2–3-fold higher than the control after being treated with 0.19–0.27 mM mizoribine. The most suitable concentration of azaserin was 0.36 mM, which gave a 4-fold increase in the percentage of resistant calluses. Acivicin at 0.28–0.84 mM yielded a 3–5-fold increase in the percentage of resistant calluses, which is significantly better than the control. When the calluses were treated with 0.01 or 0.02% Silwet L-77, the percentages of resistant calluses were 34.89 and 25.60%, respectively. We concluded that purine synthesis inhibitors, purine and pyrimidine synthesis inhibitor and the surfactant Silwet L-77 at optimal concentrations significantly improved the Agrobacterium-mediated transformation of maize calluses.

Small bud tips of 1–3 mm in length were taken from multiple shoot clumps that derived from immature inflorescence cultures of beet as recipient for the Agrobacterium-mediated transformation and transgenic plants were obtained from eight genotypes. The optimal genetic transformation protocol was established as followed: the buds were immersed in Agrobacterium suspension of OD600 =0.3–0.5 for 5–10 min, with vacuum infiltration (0.3–0.5 × 105 Pa) or supplemented with 0.01% Silwet L-77, co-cultured for 2–4 days and followed by 10-day culture on medium containing 100 mg l−1 cefotaxime, then the buds were selected on medium containing 10 mg l−1 hygromycin B for three consecutive generations. The percentage of hygromycin-resistant buds after three selections varied from 13.3 to 30.6% with genotypes. The results of PCR and further Southern blotting of genomic DNA of hygromycin-resistant buds or plants showed that the exogenous hpt and AtNHX1 gene had been integrated into the genomes of some transformed buds or plants. The transgenic buds or plants with AtNHX1 gene encoding Na+/H+ antiport on the vacuole membrane of Arabidopsis showed improved salt-tolerance than the controls. AtNHX1gene inherited in some transgenic lines as Mendelian segregation. This result revealed that it was feasible to improve salt-tolerance of beets by the introduction of AtNHX1 gene into cultured buds.

An efficient protocol for Kentucky bluegrass (Poa pratensis L.) in vitro culture was established using shoot apices of seedlings as explants. The optimal procedure of this protocol for majority of the genotypes was that meristematic cell clumps and small calluses were firstly induced from the bases of explants on initial culture medium supplemented with 0.9 μM 2,4-d and 8.9 μM 6-BA for 20 d, then were separated and transferred to shoot clumps induction medium containing 8.9 μM 6-BA for the formation of multiple shoot clumps. The percentage of multiple shoot clumps and numbers of shoots per clump were deeply related with the combinations of different plant growth regulators, duration of initial culture, the intensity of illumination and genotypes. Histological observation of the induced explants revealed that the meristematic cell clumps were produced from repeated division of the cortical cells and original meristematic primodium cells of explants, and the multiple shoots were formed via organogenesis pathway in the meristematic cell regions of cultures on shoot clumps induction medium. In this study, plantlets were efficiently regenerated on large scale from seven cultivars of Kentucky bluegrass. Hence the meristematic cell clumps and small calluses in this protocol could be considered good targets for genetic transformation of Kentucky bluegrass.

The inheritance and stability of the acetolactate synthase (als) transgene were compared in transgenic maize plants, generated using the pollen-tube pathway, particle bombardment, or Agrobacterium-mediated methods of transformation. Progeny populations generated by successive selfing or backcrossing of primary transformants were analyzed over three generations, using PCR and herbicide screening, to examine segregation and als activity, respectively, and transgenic homozygous plants were selected. The pollen-tube method resulted in a higher rate of primary normal transgenic plants and a less-stable transmission of the als locus than did the other two methods. When transferred by the particle bombardment and Agrobacterium-mediated methods, the als gene was in a much higher proportion of Mendelian transmission than transferred by the pollen-tube method. Compared to the Agrobacterium-mediated transformation, the particle bombardment method tends to create multiple copies and insert sites of the als gene in maize genome, which delaying the homogenization of the als locus with advancing generations. Agrobacterium-mediated transformation resulted in a greater proportion of stable, low copy number (in general 1–2) transgenic events, facilitating the stable inheritance of the als gene, and producing multiple desirable transgenic plants.

A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) has been isolated from Zea mays by screening a cDNA library. The cDNA, designated ZmPLC, encodes a polypeptide of 586 amino acids, containing the catalytic X, Y and C2 domains found in all PI-PLCs from plants. Northern blot analysis showed that the expression of the ZmPLC gene in roots is up-regulated under conditions of high salt, dehydration, cold or low osmotic stress conditions. Recombinant ZmPLC protein was expressed in Esch- erichia coli, purified and used to produce polyclonal antibody, this polyclonal antibody is important for further studies to assess the ultimate function of the ZmPLC gene in plants.

We developed an efficient method for sugar beet multiplication in vitro from excised immature inflorescence tips. On Murashige & Skoog medium supplemented with 4.4 μM 6-benzylamino- purine and 1.3 μM naphthaleneacetic acid, multiple bud clumps were induced from segments of inflorescence tips. The clumps proliferated rapidly. By radiation of small bud clumps at an appropriate dose and by directional selection for NaCl tolerance, we obtained salt-tolerant bud clumps and regenerated plantlets. The plants were vernalized and self-pollinated. The seeds of the regenerated plants were sown in pots of sand and irrigated every day with a solution of 342 mM NaCl. Some of the seeds germinated and grew normally in the 342 mM NaCl solution, exhibiting higher salt-tolerance than the control ones; such seedlings after the saline selection were transplanted to soil and the plants grew normally and produced plump root tuber similar to controls. The seeds from two selected lines germinated and grew for a few weeks in 513 mM NaCl solution before the seedlings withered. In saline soil where the salt concentration was about 154 mM, the yields of tuber from the plants of three salt-tolerant lines were about 45–50 tons ha−1, approximately 2.6–2.9 times of the controls. It is concluded that we have got salt-tolerant materials with good agronomic traits for sugar beet breeding via selection in vitro.

The study aims to conduct a meta-analysis of randomized controlled trials to compare the efficacy of tacrolimus (TAC)/mycophenolate mofetil (MMF)/corticosteroids (CSs) with sirolimus (SRL)/MMF/CSs in renal transplant recipients: Research 2 databases, PubMed, and Web of Science, selecting relevant articles. Data were selected for acute rejection and patient and graft survival. Statistical value relative risk (RR) and 95% confidence intervals (CIs) were recorded. Six randomized controlled trials involving 885 patients were included. There was a significant difference in acute rejection (P = 0.001, RR = 1.69, 95% CI, 1.23-2.34). Two groups, patient survival (P = 0.96, RR = 1.02, 95% CI, 0.54-1.91) and graft survival (P = 0.09, RR = 1.56, 95% CI, 0.93-2.60), had no statistical difference. Acute rejection by those taking SRL/MMF/CSs is worse than those taking TAC/MMF/CSs. Patient and graft survival in TAC/MMF/CSs is similar to that in SRL/MMF/CSs.

Low phosphate (Pi) availability is a major constraint on maize growth and productivity. MicroRNAs (miRNAs) are known to play critical roles in plant responses to various environmental conditions. The identification of low Pi tolerance-associated miRNAs will accelerate the development of Pi starvation tolerant maize plants. However, miRNAs associated with low Pi tolerance have not been identified. In this study, we compared deep sequencing small RNA reads from two maize genotypes, the wild type, Qi319, and the low Pi tolerant mutant, 99038, under normal and low Pi conditions. Six known miRNA families and seven novel miRNAs were found differently expressed by the two genotypes. All these miRNAs were confirmed by sequencing a second batch of small RNA libraries constructed in the same way as those used in the first sequencing. The expression profiles of some of these miRNAs were further confirmed by real-time PCR. The predicted target genes of the low Pi tolerance-associated miRNAs were involved in root development or stress responses. Expression levels of some of target genes were significantly different between Qi319 and 99038. These findings suggested that miRNAs may play important roles in low Pi tolerance in maize and may be a key factor in determining the level of low Pi tolerance in different maize genotypes. This study provides an approach for identifying low Pi tolerance-associated miRNAs and can help in the selection and manipulation of high performing maize genotypes under low Pi conditions.

Phosphatidylinositol (PtdIns) is an important lipid because it serves as a key membrane constituent and is the precursor of the inositol-containing lipids that are found in all plants and animals. It is synthesized from cytidine-diphosphodiacylglycerol (CDP-DG) and myo-inositol by PtdIns synthase (PIS). We have previously reported that two putative PIS genes from maize (Zea mays L.), ZmPIS and ZmPIS2, are transcriptionally up-regulated in response to drought (Sui et al., Gene, 426:47-56, 2008). In this work, we report on the characterization of ZmPIS in vitro and in vivo. The ZmPIS gene successfully complemented the yeast pis mutant BY4743, and the determination of PIS activity in the yeast strain further confirmed the enzymatic function of ZmPIS. An ESI-MS/MS-based lipid profiling approach was used to identify and quantify the lipid species in transgenic and wild-type tobacco plants before and after drought treatment. The results show that the overexpression of ZmPIS significantly increases lipid levels in tobacco leaves under drought stress compared to those of wild-type tobacco, which correlated well with the increased drought tolerance of the transgenic plants. Further analysis showed that, under drought stress conditions, ZmPIS overexpressors were found to exhibit increased membrane integrity, thereby enabling the retention of more solutes and water compared with the wild-type and the vector control transgenic lines. Our findings give us new insights into the role of the ZmPIS gene in the response of maize to drought/osmotic stress and the mechanisms by which plants adapt to drought stress.

The herbicide-resistant geneals ofArabidopsis thaliana has been transferred into embryonic calli of maize by microprojectile bombardment. We have obtained chlorsulfuron-resistant calli and regenerated plantlets through selection by herbicide chlorsulfuron. The results of PCR analysis and Southern blotting indicate that geneals has been transferred into some plantlets. The test of spraying chlorsulfuron indicated that the transgenic plantlets had favorable herbicide-resistant trait. The purpose of the research was to obtain chlorsulfuron-resistant transgenic maize and hope that this kind of high efficient herbicide could be widely used in rotation soil of wheat and maize. In addition, through spraying herbicide, we could eliminate the hybrid plants and thereby increase the purity of F1 seeds.

Two full-length cDNAs, designated as ZmFtsH2A and ZmFtsH2B, were isolated from maize (Zea mays L.) by suppression subtractive hybridization coupled with in silico cloning approach. The predicted proteins of ZmFtsH2A and ZmFtsH2B both consisted of 677 amino acid residues and displayed high similarity to FtsH2 protease of Arabidopsis thaliana. DNA gel blotting analysis indicated that AtFtsH2-like genes exist as two copies in maize genome. The genomic sequences of ZmFtsH2A and ZmFtsH2B were cloned and the main difference was that the first intron of ZmFtsH2B was much longer than that of ZmFtsH2A. RT-PCR analysis revealed that both genes were constitutively expressed in all examined tissues and the expression level of ZmFtsH2B transcripts was higher than that of ZmFtsH2A. The responses of the two genes in maize seedlings to PEG, cold, high salt, and ABA treatments were compared, and the results showed that ZmFtsH2B transcription in leaves was markedly up-regulated by water deficit stress and ABA treatments while ZmFtsH2A constitutively expressed both in leaves and roots under all tested stressful conditions. Drought tolerance of transgenic tobaccos overexpressing ZmFtsH2A and ZmFtsH2B weren't improved compared to wild-type controls, which indicated that two genes might not be directly involved in plant drought tolerance or the number of functional FtsH heterocomplex might not be increased in this condition. Our current study provides fundamental information for the further investigation of the maize FtsH proteins.

Phytohormones play critical roles in regulating plant responses to stress. We investigated the effects of water stress induced by adding 12% (w/v) polyethylene glycol to the root medium on the levels of abscisic acid (ABA), indole-3-acid (IAA), zeatin (ZT), and gibberellin(3) (GA(3)) in maize leaves. The results suggested that water stress had significant effects on the four hormone levels. There was a transient increase in the IAA content during the initial stage of adaptation to water stress in maize leaves, but it dropped sharply thereafter in response to water stress. ABA content increased dramatically in maize leaves after 24 h of exposure to water stress, and then the high levels of ABA were maintained to the end. The contents of ZT and GA(3) rapidly declined in maize leaves subjected to water stress. The effects of water stress on chlorophyll content, electrolyte leakage and malondialdehyde levels in maize leaves were also studied. The variation of cell damage was negatively correlated with ZT and GA(3) levels in maize leaves under water stress. Thus, we explored the roles of ZT and GA(3) on the growth of maize seedlings under water stress by exogenous application. It is possible that both ZT and GA(3) were effective in protecting maize seedlings from water stress, which would be of great importance for the improvement of drought tolerance in maize by genetic manipulation.

Previous studies have indicated the phosphoinositide and phospholipid signaling pathways play a key role in plant growth, development and responses to environmental stresses. However, little is known about the phosphoinositide and phospholipid signaling pathways in maize (Zea mays L.). To better understand the function of genes involved in the phosphoinositide and phospholipid signaling pathways in maize, the cDNA sequences of ZmPIS2, ZmPLC2, ZmDGK1, ZmDGK2 and ZmDGK3 were obtained by RACE (rapid amplification of cDNA ends) or in silico cloning combined with PCR. RT-PCR analysis of cDNA from five tissues (roots, stems, leaves, tassels, and ears) indicated that the expression patterns of the five cDNAs we isolated as well as ZmPIS, ZmPLC, ZmPLD varied in different tissues. To determine the effects of different environmental conditions such as cold, drought and various phytohormones (abscisic acid, indole-3-acetic acid and gibberellic acid) on gene expression, we analyzed expression by Real-Time (RT-PCR), and found that the different isoforms of these gene families involved in the phosphoinositide and phospholipid signaling pathways have specific expression patterns. Our results suggested that these genes may be involved in the responses to environmental stresses, but have different functions. The isolation and analysis of expression patterns of genes involved in the phosphoinositide and phospholipid signaling pathways provides a good basis for further research of the phosphoinositide and phospholipid signaling pathways in maize and is a novel supplement to our comprehension of these pathways in plants.

Phosphatidylinositol-specific phospholipase C (PI-PLC) plays an important role in a variety of physiological processes in plants, including drought tolerance. It has been reported that the ZmPLC1 gene cloned from maize (Zea mays L.) encoded a PI-PLC and up-regulated the expression in maize roots under dehydration conditions (Zhai SM, Sui ZH, Yang AF, Zhang JR in Biotechnol Lett 27:799-804, 2005). In this paper, transgenic maize expressing ZmPLC1 transgenes in sense or antisense orientation were generated by Agrobacterium-mediated transformation and confirmed by Southern blot analysis. High-level expression of the transgene was confirmed by real-time RT-PCR and PI-PLC activity assay. The tolerance to drought stress (DS) of the homogenous transgenic maize plants was investigated at two developmental stages. The results demonstrated that, under DS conditions, the sense transgenic plants had higher relative water content, better osmotic adjustment, increased photosynthesis rates, lower percentage of ion leakage and less lipid membrane peroxidation, higher grain yield than the WT; whereas those expressing the antisense transgene exhibited inferior characters compared with the WT. It was concluded that enhanced expression of sense ZmPLC1 improved the drought tolerance of maize.

Phosphorus (P) deficiency is a major limitation for plant growth and development. Plants can respond defensively to this stress, modifying their metabolic pathways and root morphology, and this involves changes in their gene expression. To better understand the low P adaptive mechanism of crops, we conducted the comparative proteome analysis for proteins isolated from maize roots treated with 1000 microM (control) or 5 microM KH2PO4 for 17 days. The results showed that approximately 20% of detected proteins on 2-DE gels were increased or decreased by two-fold or more under phosphate (Pi) stress. We identified 106 differentially expressed proteins by MALDI-TOF MS. Analysis of these P starvation responsive proteins suggested that they were involved in phytohormone biosynthesis, carbon and energy metabolisms, protein synthesis and fate, signal transduction, cell cycle, cellular organization, defense, secondary metabolism, etc. It could be concluded that they may play important roles in sensing the change of external Pi concentration and regulating complex adaptation activities for Pi deprivation to facilitate P homeostasis. Simultaneously, as a basic platform, the results would also be useful for the further characterization of gene function in plant P nutrition.

Low phosphate availability is a major constraint on plant growth and agricultural productivity. Engineering a crop with enhanced low phosphate tolerance by transgenic technique could be one way of alleviating agricultural losses due to phosphate deficiency. In this study, we reported that transgenic maize plants that overexpressed the Thellungiella halophila vacuolar H(+)-pyrophosphatase gene (TsVP) were more tolerant to phosphate deficit stress than the wild type. Under phosphate sufficient conditions, transgenic plants showed more vigorous root growth than the wild type. When phosphate deficit stress was imposed, they also developed more robust root systems than the wild type, this advantage facilitated phosphate uptake, which meant that transgenic plants accumulated more phosphorus. So the growth and development in the transgenic maize plants were not damaged as much as in the wild type plants under phosphate limitation. Overexpression of TsVP increased the expression of genes involved in auxin transport, which indicated that the development of larger root systems in transgenic plants might be due in part to enhanced auxin transport which controls developmental events in plants. Moreover, transgenic plants showed less reproductive development retardation and a higher grain yield per plant than the wild type plants when grown in a low phosphate soil. The phenotypes of transgenic maize plants suggested that the overexpression of TsVP led to larger root systems that allowed transgenic maize plants to take up more phosphate, which led to less injury and better performance than the wild type under phosphate deficiency conditions. This study describes a feasible strategy for improving low phosphate tolerance in maize and reducing agricultural losses caused by phosphate deficit stress.