Cloning of resistance gene analogs located on the alien chromosome in an addition line of wheat-Thinopyrum intermedium

Homology-based gene/gene-analog cloning method has been extensively applied in isolation of RGAs (resistance gene analogs) in various plant species. However, serious interference of sequences on homoeologous chromosomes in polyploidy species usually occurred when cloning RGAs in a specific chromosome. In this research, the techniques of chromosome microdissection combined with homology-based cloning were used to clone RGAs from a specific chromosome of Wheat-Thinopyrum alien addition line TAi-27, which was derived from common wheat and Thinopyrum intermedium with a pair of chromosomes from Th. intermedium. The alien chromosomes carry genes for resistance to BYDV. The alien chromosome in TAi-27 was isolated by a glass needle and digested with proteinase K. The DNA of the alien chromosome was amplified by two rounds of Sau3A linker adaptor-mediated PCR. RGAs were amplified by PCR with the degenerated primers designed based on conserved domains of published resistance genes (R genes) by using the alien chromosome DNA, genomic DNA and cDNA of Th. intermedium, TAi-27 and 3B-2 (a parent of TAi-27) as templates. A total of seven RGAs were obtained and sequenced. Of which, a constitutively expressed single-copy NBS-LRR type RGA ACR3 was amplified from the dissected alien chromosome of TAi-27, TcDR2 and TcDR3 were from cDNA of Th. intermedium, AcDR3 was from cDNA of TAi-27, FcDR2 was from cDNA of 3B-2, AR2 was from genomic DNA of TAi-27 and TR2 was from genomic DNA of Th. intermedium. Sequence homology analyses showed that the above RGAs were highly homologous with known resistance genes or resistance gene analogs and belonged to NBS-LRR type of R genes. ACR3 was recovered by PCR from genomic DNA and cDNA of Th. intermedium and TAi-27, but not from 3B-2. Southern hybridization using the digested genomic DNA of Th. intermedium, TAi-27 and 3B-2 as the template and ACR3 as the probe showed that there is only one copy of ACR3 in the genome of Th. intermedium and TAi-27, but it is absent in 3B-2. The ACR3 could be used as a specific probe of the R gene on the alien chromosome of TAi-27. Results of Northern hybridization suggested that ACR3 was constitutively expressed in Th. intermedium and TAi-27, but not 3B-2, and expressed higher in leaves than in roots. This research demonstrated a new way to clone RGAs located on a specific chromosome. The information reported here should be useful to understand the resistance mechanism of, and to clone resistant genes from, the alien chromosome in TAi-27.     

Excision of a selectable marker in transgenic rice (Oryza sativa L.) using a chemically regulated Cre/loxP system

Removal of a selectable marker gene from genetically modified (GM) crops alleviates the risk of its release into the environment and hastens the public acceptance of GM crops. Here we report the production of marker-free transgenic rice by using a chemically regulated, Cre/loxP-mediated site-specific DNA recombination in a single transformation. Among 86 independent transgenic lines, ten were found to be marker-free in the T₀ generation and an additional 17 lines segregated marker-free transgenic plants in the T₁ generation. Molecular and genetic analyses indicated that the DNA recombination and excision in transgenic rice were precise and the marker-free recombinant T-DNA was stable and heritable.The first two authors contributed equally to the work     

Characterisation of recombinant epithiospecifier protein and its over-expression in Arabidopsis thaliana

Epithiospecifier protein (ESP) is a protein that catalyses formation of epithionitriles during glucosinolate hydrolysis. In vitro assays with a recombinant ESP showed that the formation of epithionitriles from alkenylglucosinolates is ESP and ferrous ion dependent. Nitrile formation in vitro however does not require ESP but only the presence of Fe(II) and myrosinase. Ectopic expression of ESP in Arabidopsis thaliana Col-5 under control of the strong viral CaMV 35S promoter altered the glucosinolate product profile from isothiocyanates towards the corresponding nitriles.     

中小负荷运动对冷应激大鼠白细胞介素2和β-内啡肽的影响

目的:探讨中小负荷运动对IL-2和β-EP的影响及其调节机制.方法:对SD大鼠进行为期4周中小负荷运动,并在运动后期施加冷应激,测定大鼠外周血液IL-2和β-EP的含量.结果:①应激组IL-2显著低于对照组,但β-EP含量显著高于对照组.②经过4周运动,30 mm运动组和60 min运动组,β-EP含量显著低于对照组,而IL-2水平显著高于应激组.同时30 min运动 应激组和60min运动 应激组血清IL-2显著高于应激组,而β-EP含量显著低于应激组.结论:中小负荷运动降低冷应激反应程度,减少内源性β-EP释放,使IL-2含量升高,维持机体在应激状态下免疫功能稳定.     

低氧及低氧预处理时心肌细胞HIF-1α与凋亡相关蛋白表达的变化

目的:观察低氧时心肌细胞HIF-1α表达变化与凋亡相关蛋白表达关系.方法:采用体外心肌细胞培养的方法,将原代培养4～6 d的大鼠乳鼠心肌细胞随机分为对照组、低氧组与低氧预处理组.低氧预处理组在低氧培养箱中通入1%O2、5%CO2、94%N2的低氧混合气体,每天低氧12 h,低氧5 d,第6 d与急性低氧组一同放入0%O2、5%CO2、95%N2的低氧培养箱中进行低氧暴露.低氧48 h后,通过Western blot方法分别检测心肌细胞中HIF-1α、Bcl-2、P53及Bax的表达变化.结果:常氧时细胞不表达HIF-1α,低氧可增加HIF-1的表达,低氧预处理后,能降低HIF-1α的表达.低氧时,Bax的表达变化大致与此相同.p53在低氧时的变化也与其相同,但低氧预处理后似乎没有明显的改变.Bcl-2在低氧时表达下降,低氧预处理后可增加其表达.结论:HIF-1α的表达可协同Bcl-2家族凋亡相关蛋白的表达,在低氧导致的心肌细胞凋亡中发挥重要作用.     

Theoretical and practical advances in genome halving

MOTIVATION: Duplication of an organism's entire genome is a rare but spectacular event, enabling the rapid emergence of multiple new gene functions. Over time, the parallel linkage of duplicated genes across chromosomes may be disrupted by reciprocal translocations, while the intra-chromosomal order of genes may be shuffled by inversions and transpositions. Some duplicate genes may evolve unrecognizably or be deleted. As a consequence, the only detectable signature of an ancient duplication event in a modern genome may be the presence of various chromosomal segments containing parallel paralogous genes, with each segment appearing exactly twice in the genome. The problem of reconstructing the linkage structure of an ancestral genome before duplication is known as genome halving with unordered chromosomes. RESULTS: In this paper, we derive a new upper bound on the genome halving distance that is tighter than the best known, and a new lower bound that is almost always tighter than the best known. We also define the notion of genome halving diameter, and obtain both upper and lower bounds for it. Our tighter bounds on genome halving distance yield a new algorithm for reconstructing an ancestral duplicated genome. We create a software package GenomeHalving based on this new algorithm and test it on the yeast genome, identifying a sequence of translocations for halving the yeast genome that is shorter than previously conjectured possible.     

Application of response surface methodology in medium optimization for spore production of <Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in solid-state fermentation

Summary Spore production of Coniothyrium minitans was optimized by using response surface methodology (RSM), which is a powerful mathematical approach widely applied in the optimization of fermentation process. In the first step of optimization, with Plackett–Burman design, soluble starch, urea and KH²PO⁴ were found to be the important factors affecting C. minitans spore production significantly. In the second step, a 2³ full factorial central composite design and RSM were applied to determine the optimal concentration of each significant variable. A second-order polynomial was determined by the multiple regression analysis of the experimental data. The optimum values for the critical components for the maximum were obtained as follows: soluble starch 0.643 (36.43 g. l⁻¹), urea −0.544 (3.91 g l⁻¹) and KH²PO⁴ 0.049 (1.02 g l⁻¹) with a predicted value of maximum spore production of 9.94 × 10⁹ spores/g IDM. Under the optimal conditions, the practical spore production was 1.04 × 10¹⁰ spores/g IDM. The determination coefficient (R²) was 0.923, which ensure an adequate credibility of the model.     

Discovery and SAR studies of a novel series of noncovalent cathepsin S inhibitors

A novel series of competitive, reversible cathepsin S (CatS) inhibitors was discovered and optimized. The 4-(2-keto-1-benzimidazolinyl)-piperidin-1-yl moiety was found to be an effective replacement for the 4-arylpiperazin-1-yl group found in our earlier series of CatS inhibitors. This replacement imparted improved PK properties as well as decreased off-target activity. Optimization of the ketobenzimidazole moiety led to the discovery of the lead compound JNJ 10329670, which represents a novel class of selective, noncovalent, reversible, and orally bioavailable inhibitors of cathepsin S.     

Membrane transformation during malaria parasite release from human red blood cells

Three opposing pathways are proposed for the release of malaria parasites from infected erythrocytes: coordinated rupture of the two membranes surrounding mature parasites; fusion of erythrocyte and parasitophorus vacuolar membranes (PVM); and liberation of parasites enclosed within the vacuole from the erythrocyte followed by PVM disintegration. Rupture by cell swelling should yield erythrocyte ghosts; membrane fusion is inhibited by inner-leaflet amphiphiles of positive intrinsic curvature, which contrariwise promote membrane rupture; and without protease inhibitors, parasites would leave erythrocytes packed within the vacuole. Therefore, we visualized erythrocytes releasing P. falciparum using fluorescent microscopy of differentially labeled membranes. Release did not yield erythrocyte ghosts, positive-curvature amphiphiles did not inhibit release but promoted it, and release of packed merozoites was shown to be an artifact. Instead, two sequential morphological stages preceded a convulsive rupture of membranes and rapid radial discharge of separated merozoites, leaving segregated internal membrane fragments and plasma membrane vesicles or blebs at the sites of parasite egress. These results, together with the modulation of release by osmotic stress, suggest a pathway of parasite release that features a biochemically altered erythrocyte membrane that folds after pressure-driven rupture of membranes.