Molecular cloning and characterization of a Brassica juncea yellow stripe-like gene, BjYSL7, whose overexpression increases heavy metal tolerance of tobacco

Key message

BjYSL7 encodes a plasma-localized metal–NA transporter and has transport Fe(II)–NA complexes activity. BjYSL7 is involved in the transport of Cd and Ni from roots to shoots.

Abstract

Heavy metal transporters play a key role in regulating metal accumulation and transport in plants. In this study, we isolated a novel member of the yellow stripe-like (YSL) gene family BjYSL7 from the hyperaccumulator Brassica juncea. BjYSL7 is composed of 688 amino acids with 12 putative transmembrane domains and is over 90 % identical to TcYSL7 and AtYSL7. Real-time PCR analysis revealed that BjYSL7 mRNA was mainly expressed in the stem under normal condition. The expression of BjYSL7 was found to be up-regulated by 127.1-, 12.7-, and 3.4-fold in roots and 6.5-, 4.3-, and 2.8-fold in shoots under FeSO₄, NiCl₂, and CdCl₂ stresses, respectively. We have demonstrated that BjYSL7 is a Fe(II)–NA influx transporter by yeast functional complementation. Moreover, a BjYSL7::enhanced green fluorescent protein (EGFP) fusion localized to the plasma membrane of onion epidermal cells. The BjYSL7-overexpressing transgenic tobacco plants exhibited longer root lengths, lower relative inhibition rate of lengths and superior root hair development compared to that of wild-type (WT) plants in the presence of CdCl₂ and NiCl₂. Furthermore, the concentrations of Cd and Ni in shoots of BjYSL7-overexpressing plants are significantly higher than that of WT plants. Compared with WT plants, BjYSL7-overexpressing plants exhibited Fe concentrations that were higher in the shoots and seeds and lower in the roots. Taken together, these results suggest that BjYSL7 might be involved in the transport of Fe, Cd and Ni to the shoot and improving heavy metal resistance in plants.     

中国鸟类亚种新记录——黑冠黄鹎

2010年12月和2011年4月,分别在广西西北部百色市右江区和靖西县古龙山自然保护区拍摄和观察到黑冠黄鹎(Pycnonotus flaviventris)的johnsoni亚种.其喉部为红色,与中国其他记录亚种区别明显,属中国鸟类亚种的新记录.该文亦初步讨论了该亚种的分类和分布.     

一种新型蛋白类药物口服载体的海藻酸钠/巯基化果糖-壳聚糖复合水凝胶

壳聚糖经果糖修饰以改善其水溶性,再经半胱氨酸修饰得到巯基化果糖-壳聚糖。海藻酸钠与巯基化果糖-壳聚糖混合溶液经氯化钙及硫酸钠双重交联制得复合水凝胶珠。溶胀试验结果表明:该凝胶珠在pH值6.8及7.4时的溶胀率分别是pH值1.2时的7倍和10倍左右。牛血清白蛋白(BSA)包载试验结果表明:BSA重量为凝胶珠质量的20%时,包载率可达94%以上,随着巯基化果糖-壳聚糖在凝胶珠中比例增加,BSA包载率上升。BSA释放试验表明:pH值1.2时BSA的释放率很低,只有6%～10%的BSA从凝胶珠中释放出来,随后累积释放量基本不变;pH值6.8和pH值7.4时BSA的释放率迅速提高,因此这种复合水凝胶珠可作为一种潜在的口服蛋白类药物载体。     

Immunosensor based on magnetic relaxation switch and biotin-streptavidin system for the detection of Kanamycin in milk

A rapid, sensitive, and simple immunosensor was developed for the detection of Kanamycin (KM) in milk. This immunosensor is based on magnetic relaxation switch (MRS) assay and biotin-streptavidin system (B-SA system). The target analyte (KM) competed with those on the surface of the superparamagnetic iron oxide (SPIO) nanoparticles and hence affected the formation of SPIO aggregates. The dispersed and aggregated states of SPIO can modulate the spin-spin relaxation time (T(2)) of the neighboring water molecule. T(2) was then changed as an effect of the target analyte. The B-SA system was used to amplify the SPIO binding, thus enhance the sensitivity. The detection working was 1.5 to 25.2ngmL(-1) and limit of detection (LOD) was determined to be 0.1ngmL(-1). The LOD of the immunosensor decreased tenfold, and its analysis time (45min) was much shorter than that of enzyme-linked immunosorbent assay (6h to 8h). The average recoveries of the KM at various spiking levels ranged from 80.2% to 85.6% with a relative standard deviation (RSD) below 4.0%. The results showed that the MRS immunosensor was a promising platform for the determination of small molecular residues because of its high sensitivity, specificity, homogeneity, and speed.     

Efficient secretion of lipase r27RCL in Pichia pastoris by enhancing the disulfide bond formation pathway in the endoplasmic reticulum

The lipase r27RCL from Rhizopus chinensis CCTCC M201021 was heterologously expressed in Pichia pastoris GS115 by simultaneous co-expression with two secretion factors ERO1p and PDI involved in the endoplasmic reticulum (ER). Compared to the expression of the lipase alone (12,500 U/ml), co-expression with these two proteins resulted in the production of larger total quantities of enzymes. The largest increase was seen when the combined ERO1p/PDI system was co-expressed, resulting in approximately 30 % higher enzyme yields (16,200 U/ml) than in the absence of co-expressed secretion factors. The extracellular protein concentration of the recombinant strain Co XY RCL-5 reached 9.39 g/l in the 7-l fermentor. Simultaneously, the fermentation time was also shortened by about 8 h compared to that of the control. The substrate-specific consumption rate (Qs) and the product-specific production rate (Qp) were both investigated in this research. In conclusion, the space–time yield was improved by co-expression with ERO1p and PDI. This is a potential strategy for high level expression of other heterologous proteins in P. pastoris.     

Computational studies on polynitropurines as potential high energy density materials

As part of a search for high energy density materials (HEDMs), a series of purine derivatives with nitro groups were designed computationally. The relationship between the structures and the performances of these polynitropurines was studied. Density functional theory (DFT) at the B3LYP/6-311G** level was employed to evaluate the heats of formation (HOFs) of the polynitropurines by designing an isodesmic reaction method. Results indicated that the HOFs were influenced by the number and positions of substituent groups. Detonation properties were evaluated using the Kamlet–Jacobs equations, based on the theoretical densities and heats of formation of the polynitropurines. The relative stabilities of the polynitropurines were studied via the pyrolysis mechanism and the UB3LYP/6-311G** method. Homolysis of the ring–NO₂ bond is predicted to be the initial step in the thermal decomposition of these purine derivatives. Considering their detonation properties and relative stabilities, the tetranitropurine (D1) derivatives may be regarded as potential candidates for practical HEDCs. These results may provide useful information for further investigations.     

Characterization of two novel family 12 xyloglucanases from the thermophilic Rhizomucor miehei

Two novel glycoside hydrolase (GH) family 12 xyloglucanase genes (designated RmXEG12A and RmXEG12B) were cloned from the thermophilic fungus Rhizomucor miehei. Both genes contained open reading frames of 729 bp encoding 242 amino acids. Their deduced amino acid sequences shared 68 % identity with each other and less than 60 % with other xyloglucanases. The two genes, without the sequences for the signal peptides, were cloned and successfully expressed in Escherichia coli as active xyloglucanases, designated RmXEG12A and RmXEG12B, with similar molecular masses—25.6 and 25.9 kDa, respectively. RmXEG12A showed optimal activity at pH?6.5 and 65 °C, RmXEG12B at pH?5.0 and 60 °C. Both recombinant xyloglucanases displayed very high specific activities, 6,681.4 and 3,092.2 U?mg^?1, respectively, toward tamarind xyloglucan, but no activity toward carboxymethylcellulose, Avicel, or p-nitrophenyl derivatives. The main products of tamarind xyloglucan hydrolysis by the two xyloglucanases were XXXG, XXLG/XLXG, and XLLG (where G is an unsubstituted β-d-Glc residue, X is a xylosylated β-d-Glc residue, and L is a β-d-Glc residue substituted by xylosyl-galactose).     

斑马鱼整胚原位杂交和显微注射转基因技术的建立(简报)

随着人类基因组框架图的完成,解读大量基因的功能和表达调控成为亟待解决的问题。因此通过模式生物从个体水平、细胞水平和分子水平研究基因功能及其表达调控日益成为一个重要的研究领域。斑马鱼(Brachydanio rerio)是近年来崛起的一种模式生物。它的显著优势在于:个体小,便于大批量饲养;产卵量大,胚胎透明,易于观察和操作;体外发育,发育迅速,2—3天即可完成主要器官的建成;可以方便的进行大规模的诱变和突变型筛选等研究。它已成为脊椎动物胚胎发育遗传学和基因功能研究中理想的模式生物。整胚原位杂交技术是     

烟草乙烯转录因子ERF基因NtTOE3的克隆、载体构建及表达分析

ERF（Ethylene-responsive factor）是一类具有AP2特征结构域的乙烯应答转录因子,在响应植物的逆境胁迫应答中起关键作用。为明确ERF家族成员TOE3在烟草中的生物学功能和调控机制,同源克隆普通烟草K326中NtTOE3基因cDNA全长,利用实时荧光定量PCR（qRT-PCR）表征基因表达模式,结合生物信息学分析,对基因功能和调控机制进行初步预测。结果表明,该基因全长1 356 bp,编码451个氨基酸残基,预测分子量为49.84 ku。生物信息学分析表明,该蛋白是一个亲水蛋白,可能定位在细胞核,且与美花烟草（Nicotiana sylvestris）NsTOE3有96%的同源性,故命名为NtTOE3。组织表达分析发现,该基因在烟草根、茎、叶、花中均有表达,其中茎中表达量最高。逆境胁迫试验表明,该基因能快速响应低钾、高盐、干旱、H₂O₂、ABA和4℃等逆境处理。表明NtTOE3转录因子在烟草非生物胁迫中起着重要调节作用。     

Inhibitory Effect of Vaginal Lactobacillus Supernatants on Cervical Cancer Cells

Lactobacilli have been shown to inhibit the proliferation of several types of cancer cells, but the effects of vaginal Lactobacilli on cervical cancer cells have seldom been reported. We incubated Caski cells with supernatants of predominant strains in the vagina and investigated their effects on cell growth and the possible mechanisms. Cell-free supernatants of Lactobacillus crispatus, L. jensenii, and L. gasseri were prepared and purified. Caski cells were treated with various concentrations of Lactobacillus supernatants (LS). The effect of LS on cell growth was investigated using MTT assays. The influence of LS on the cell cycle and expression of human papillomavirus (HPV) E6 and E7 oncogenes was determined by flow cytometry and RT-PCR, respectively. LS-inhibited Caski cell proliferation caused morphological changes in a pH-independent manner. Flow cytometric analysis revealed that cells exposed to LS exhibited a significant increase of cell number in S phase and a strong decrease of cell number in G2/M phase. Expression of HPV E6 and E7 oncogenes, as well as CDK2 and cyclin A was decreased after treatment with LS, while expression of p21 was increased. Supernatants of L. crispatus, L. jensenii, and L. gasseri have inhibitory effects on the viability of cervical cancer cells via regulation of HPV oncogenes and cell cycle-related genes. Lactobacillus, as a promising treatment for cancer, is being assessed for its effect, and these results provide further evidence in this respect.