梨不同DNA提取方法的效果研究

以7个梨品种为实验材料,比较分析了SDS法、CTAB法、SDSCTAB法、改良的CTAB法、高盐低pH值法、分步离心法对梨总DNA提取的效果。结果表明:利用以上6种方法提取的梨总DNA在纯度和量上有很大的差别。所得到的平均DNA量从大到小依次为:分步离心法、SDS法、SDSCTAB法、改良的CTAB法、CTAB法、高盐低pH值法。DNA提取纯度依次为分步离心法、SDSCTAB法、改良的CTAB法、高盐低pH值法、CTAB法、SDS法。RAPD和自交不亲和基因(S基因)特异性引物扩增实验结果都比较理想,但分步离心法和SDSCTAB法提取的DNA双酶切效果较好。分步离心法提取的梨总DNA更适用于后续的分子生物学实验操作。     

Effect of ammonium-N on malic enzyme and lipid production in Rhodotorula glutinis grown on monosodium glutamate wastewater

Rhodotorula glutinis, an oil producing strain, can utilize monosodium glutamate (MSG) wastewater as a raw material for lipid production. The effects of ammonium-N in the MSG wastewater (ammonium 15,000–25,000?mg/L, COD 30,000–50,000?mg/L) on cell growth, lipid accumulation and malic enzyme activity of R. glutinis have been studied. Four initial ammonium sulfate concentrations in the medium were set, which were 20, 60, 100, and 140?g/L. With an increase in the ammonium sulfate concentration, the uptake of ammonia nitrogen and lipid accumulation increased while the biomass decreased at 72?h. The maximum value of ammonia nitrogen consumption reached 5.77?g/L for an initial ammonium sulfate concentration of 140?g/L at 72?h. In addition, 60?g/L ammonium sulfate concentration may be an appropriate concentration for R. glutinis cultivation. The activity of the malic enzyme was measured and the results showed that there was a linear relationship between the intracellular lipid content and the total malic enzyme activity.     

Homo- and hetero-dimerization of LPA/S1P receptors, OGR1 and GPR4

G protein coupled receptors (GPCRs) form homo- and hetero-dimers or -oligomers, which are functionally important. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive lysophopholipids involved in diverse biological processes. We have examined homo- and hetero-dimerization among three major LPA receptors (LPA(1-3)), three major S1P receptors (S1P(1-3)), as well as OGR1 and GPR4. Using LacZ complementation assays, we have shown that LPA receptors form homo- and hetero-dimers within the LPA receptor subgroup and hetero-dimers with other receptors (S1P(1-3) and GPR4). In addition, we have found that although GPR4 and OGR1 share more than 50% homology, GPR4 forms strong homo- and hetero-dimers with LPA and S1P receptors, but OGR1 forms very weak homo-dimer and relatively weak hetero-dimers with other receptors. Using chimeric receptors between GPR4 and OGR1, we have shown that different domains of GPR4 receptor are involved in its dimerization with different GPCRs and more than one domain may be involved in some of the complex formation. Our results suggest that when studying a signal transduction induced by a stimulus, not only is the expression and activation of its own receptor(s), but also the status of the interacting receptors should be taken into consideration.     

Transformation of Soybean (Glycine max) by Infecting Germinating Seeds with Agrobacterium tumefaciens

The transfer of genetic material into soybean tissue was accomplished by using an avirulent strain of Agrobacterium tumefaciens which contained the binary vector pGA482. The method used for transformation requires no tissue culture steps as it involves the inoculation of the plumule, cotyledonary node, and adjacent cotyledon tissues of germinating seeds. The identification of neomycin phosphotransferase (NPT) II enzyme activity in the tissues of 16 (R₀) soybean plants indicated that the plant expressible Nos-NPT II gene, contained within the T-DNA region from pGA482, had been transferred at least into somatic tissues. Putative transformed R₀ soybean plants were advanced to produce R₁ plants which were also assayed for the presence of the transferred Nos-NPT II gene. The combined results of these assays indicated that about 0.7% of the surviving inoculated seeds yielded transformed tissues in the R₀ plant, and that about 1/10 of these plants yielded transformed R₁ plants. The presence of the Nos-NPT II gene in DNAs isolated from both R₀ and R₁ plant was demonstrated by using genomic blot hybridization and polymerase chain reaction methods. Integration of this gene into the soybean genome was demonstrated for three R₁ soybean plants.     

Characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors

A novel spectrophotometric method to study the kinetics of the guanine nucleotide exchange factors-catalyzed reactions is presented. The method incorporates two coupling enzyme systems: (a). GTPase-activating protein which stimulates the intrinsic GTP hydrolysis reaction of small GTPases and (b). purine nucleotide phosphorylase and its chromophoric substrate, 7-methyl-6-thioguanosine, for quantitation of the resultant inorganic phosphate. The continuous coupled enzyme system was used for characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors, Lbc and Dbl. Kinetic parameters obtained here show that there is no significant difference in kinetic mechanism of these GEFs in interaction with RhoA. The Michaelis-Menten constants were determined to be around 1micro M, and the rate constants k(cat) were around 0.1s(-1).     

Preliminary findings of age and male sexual characteristics andand potential effect to semen characteristics and cryopreservation of the critically endangered Bornean orangutan in Malaysia

Primates - Bornean orangutan is a critically endangered non-human primate; however, the threat of extinction is not merely from poaching and habitat loss. Orangutan survival is also threatened by...     

Phosphatidylinositol Phosphate 5-Kinase Iγi2 in Association with Src Controls Anchorage-independent Growth of Tumor Cells

A fundamental property of tumor cells is to defy anoikis, cell death caused by a lack of cell-matrix interaction, and grow in an anchorage-independent manner. How tumor cells organize signaling molecules at the plasma membrane to sustain oncogenic signals in the absence of cell-matrix interactions remains poorly understood. Here, we describe a role for phosphatidylinositol 4-phosphate 5-kinase (PIPK) Iγi2 in controlling anchorage-independent growth of tumor cells in coordination with the proto-oncogene Src. PIPKIγi2 regulated Src activation downstream of growth factor receptors and integrins. PIPKIγi2 directly interacted with the C-terminal tail of Src and regulated its subcellular localization in concert with talin, a cytoskeletal protein targeted to focal adhesions. Co-expression of PIPKIγi2 and Src synergistically induced the anchorage-independent growth of nonmalignant cells. This study uncovers a novel mechanism where a phosphoinositide-synthesizing enzyme, PIPKIγi2, functions with the proto-oncogene Src, to regulate oncogenic signaling.     

Correlations and comparisons of quantitative trait loci with family per se and testcross performance for grain yield and related traits in maize

Simultaneous improvement in grain yield and related traits in maize hybrids and their parents (inbred lines) requires a better knowledge of genotypic correlations between family per se performance (FP) and testcross performance (TP). Thus, to understand the genetic basis of yield-related traits in both inbred lines and their testcrosses, two F _2:3 populations (including 230 and 235 families, respectively) were evaluated for both FP and TP of eight yield-related traits in three diverse environments. Genotypic correlations between FP and TP, $ \hat{r}_{\text{g}} $ (FP, TP), were low (0–0.16) for grain yield per plant (GYPP) and kernel number per plant (KNPP) in the two populations, but relatively higher (0.32–0.69) for the other six traits with additive effects as the primary gene action. Similar results were demonstrated by the genotypic correlations between observed and predicted TP values based on quantitative trait loci positions and effects for FP, $ \hat{r}_{\text{g}} $ (M _FP, Y _TP). A total of 88 and 35 QTL were detected with FP and TP, respectively, across all eight traits in the two populations. However, the genotypic variances explained by the QTL detected in the cross-validation analysis were much lower than those in the whole data set for all traits. Several common QTL between FP and TP that accounted for large phenotypic variances were clustered in four genomic regions (bin 1.10, 4.05–4.06, 9.02, and 10.04), which are promising candidate loci for further map-based cloning and improvement in grain yield in maize. Compared with publicly available QTL data, these QTL were also detected in a wide range of genetic backgrounds and environments in maize. These results imply that effective selection based on FP to improve TP could be achieved for traits with prevailing additive effects.     

Phosphorylation of eIF2α signaling pathway attenuates obesity-induced non-alcoholic fatty liver disease in an ER stress and autophagy-dependent manner

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder and frequently exacerbates in postmenopausal women. In NAFLD, the endoplasmic reticulum (ER) plays an important role in lipid metabolism, in which salubrinal is a selective inhibitor of eIF2α de-phosphorylation in response to ER stress. To determine the potential mechanism of obesity-induced NAFLD, we employed salubrinal and evaluated the effect of ER stress and autophagy on lipid metabolism. Ninety-five female C57BL/6 mice were randomly divided into five groups: standard chow diet, high-fat (HF) diet, HF with salubrinal, HF with ovariectomy, and HF with ovariectomy and salubrinal. All mice except for SC were given HF diet. After the 8-week obesity induction, salubrinal was subcutaneously injected for the next 8 weeks. The expression of ER stress and autophagy markers was evaluated in vivo and in vitro. Compared to the normal mice, the serum lipid level and adipose tissue were increased in obese mice, while salubrinal attenuated obesity by blocking lipid disorder. Also, the histological severity of hepatic steatosis and fibrosis in the liver and lipidosis was suppressed in response to salubrinal. Furthermore, salubrinal inhibited ER stress by increasing the expression of p-eIF2α and ATF4 with a decrease in the level of CHOP. It promoted autophagy by increasing LC3II/I and inhibiting p62. Correlation analysis indicated that lipogenesis in the development of NAFLD was associated with ER stress. Collectively, we demonstrated that eIF2α played a key role in obesity-induced NAFLD, and salubrinal alleviated hepatic steatosis and lipid metabolism by altering ER stress and autophagy through eIF2α signaling.Subject terms: Obesity, Metabolic syndrome, Outcomes research