The effect of salt stress on Arabidopsis thaliana and Phelipanche ramosa interaction

Salinity and Orobanche or Phelipanche spp. infection are important crop stress factors in agricultural areas. In this study, we investigated the effect of salt stress on Phelipanche ramosa seed germination and its attachment onto Arabidopsis thaliana roots. We also evaluated the effect of both stresses on the expression of genes regulated by abiotic and biotic stresses. According to our results, high concentration of NaCl delayed P. ramosa seed germination in the presence of a strigolactone analogue (GR24). A similar pattern was observed in the presence of A. thaliana plants. Furthermore, we found that salt‐treated A. thaliana seedlings were more sensitive to P. ramosa attachment compared with the untreated plants, indicating that there was a positive correlation between salt sensitivity and the ability of P. ramosa to infect A. thaliana plants. At the molecular level, a synergystic effect of both salt and P. ramosa stresses was observed on the cold‐regulated (COR) gene expression profile of treated A. thaliana seedlings. Our data clarify the interaction between parasitic plants and their hosts under abiotic stress conditions.     

不同继代培养次数对苹果八棱海棠离体培养和遗传转化的影响

以苹果八棱海棠离体新梢和幼叶为外植体,研究不同继代次数对其离体繁殖、再生和遗传转化的影响。结果表明,随着继代培养次数的增加,继代10次和48次的八棱海棠离体增殖倍数、生根能力、不定芽的再生频率、每外植体再生芽数和GUS基因瞬时表达率均显著高于继代5次的;继代10次的增殖倍数和GUS阳性率明显多于继代48次的,其它指标相互间差异不显著;说明继代10次的芽苗较适合用于八棱海棠离体培养的外殖体材料,其幼叶较适于遗传转化研究。     

Comprehensive Analysis of Plant Gene Expression in Soybean Root Nodules at Different Growth Stages

Gene expression profiles in early (12 d after sowing, DAS), mature (35 DAS) and senescent (77 DAS) soybean ( Glycine max (L.) Merr. cv. Enrei) root nodules were analyzed using Lotus japonicus cDNA macroarray. The cDNA macroarray membranes contained 18,144 independent cDNAs of L. japonicus isolated from various organs. The membranes were hybridized to ³³P-labeled single-strand cDNAs transcribed from mRNA extracted from the soybean root nodules at three different growth stages. The results showed that over 75% of the cDNA clones gave a relative signal intensity (RSI) lower than 300, irrespective of the soybean growth stage. cDNA with an RSI value over 5,000 accounted for 2.1, 1.2 and 0.5% of the total cDNA clones at 12, 35 and 77 DAS, respectively. The highest RSI value was detected in a clone hybridized to putative pectinesterase (MWM097c10_r) at 12 DAS, while the RSI value of lycopene β-cyclase (MWL025d06_r) was the highest at 35 and 77 DAS. At 77 DAS, the percentage of transferase gene cDNA clones accounted for 20.5% of the total cDNA clones showing an RSI value over 500, a value clearly lower than that at 12 and 35 DAS. On the other hand, the percentage of hydrolase gene cDNA clones accounted for 45.2% at 77 DAS, a value around 1.5 times higher than that at 12 and 35 DAS. The chronological RSI profiles of individual cDNA clones were classified into nine patterns, and the profiles of the cDNA clones belonging to 15 homologous gene groups encoding enzymes of nitrogen and carbon metabolism, nodule specific proteins, enzymes of RNA and protein hydrolysis, and synthesis of jasmonic acid precursor were compared within and between groups. As a result, significant variations both within- and between-groups were revealed.     

水稻OsRFP1基因的原核表达与蛋白纯化

以推导的氨基酸序列分析,水稻OsRFP1基因编码一分子量为34.24kDa锌指蛋白。将OsRFP1基因编码区cDNA序列插入到pGEX4T-3载体中构建OsRFP1-gst融合基因的表达载体,并将质粒转化宿主菌大肠杆菌BL21DE3。经IPTG诱导,融合蛋白在BL21DE3细胞中获得表达。利用亲和层析的方法对融合蛋白进行了纯化,SDS-PAGE蛋白电泳显示获得一60kDa的唯一蛋白条带,即OsRFP1-GST融合蛋白。     

乳腺特异表达载体缺失片段的鉴定与修复

通过对pBC1中的乳腺特异表达启动子成分进行分析,鉴定了位于β酪蛋白启动子上游两个EcoRⅠ位点(-89~-94和-120~-125)之间的缺失片段,31bp的缺失使得该载体中的一个乳控盒元件(-112~-141)和一个乳腺因子识别序列(-92~-102)遭到破坏,并使得位于TATA框上游的所有启动子成分发生了位置改变。通过与山羊β酪蛋白启动子序列进行比较,设计并合成了缺失片段,经一系列的酶切和连接处理,使得该缺失得到了修复,修复后的载体具有所有乳蛋白表达需要的启动子调控原件。本试验结果为利用修复后的乳腺特异高效表达载体进行乳腺生物反应器的研究奠定了分子生物学基础。     

利用PBR322质粒监控MSAP甲基化检测过程

[目的]监控MSAP甲基化检测过程,以控制MSAP每一反应步骤。[方法]利用PBR322质粒作为阳性对照,全程监控MSAP分析中酶切、连接、预扩与选扩体系。[结果]PBR322质粒作为阳性对照电泳时条带有限,可以根据条带有无和位置对MSAP甲基化检测过程进行全程监控,有助于发现问题的具有所在。[结论]该研究可以为MSAP技术提供可靠保障。     

哺乳动物性腺特异表达基因研究进展

在哺乳动物配子发生过程中,需要一个严格的阶段性和细胞特异性的基因表达程序。近年来,随着基因克隆、表达及功能研究技术的发展与应用,发现了许多配子发生过程中发挥特定作用的阶段特异和细胞特异表达的基因,有的已被证明在配子发生中起重要作用,但生殖细胞在分化及发育时受其调节的分子机制尚未了解,需要进行更加广泛深入的研究。文章综述了动物性腺特异表达基因研究的最新进展,旨在为从事该领域研究的工作者提供参考信息。     

Expression of esterase gene in yeast for organophosphates biodegradation

Organophosphates are esters of phosphoric acid and can be hydrolyzed and detoxified by carboxylesterase and phosphotriesterase. In this work esterase enzyme (Est5S) was expressed in yeast to demonstrate the organophosphorus hydrolytic activity from a metagenomic library of cow rumen bacteria. The esterase gene (est5S) is 1098 bp in length, encoding a protein of 366 amino acid residues with a molecular weight of 40 kDa. Est5S enzyme was successfully produced by Pichia pastoris at a high expression level of approximately 4.0 g L⁻¹. With p-nitrophenol butyrate as the substrate, the optimal temperature and pH for enzyme activity were determined to be 40 °C and pH 7.0, respectively. The esterase enzyme was tested for degradation of chlorpyrifos (CP). TLC results obtained inferred that CP could be degraded by esterase enzyme (Est5S) and HPLC results revealed that CP could be efficiently degraded up to 100 ppm. Cadusafos (CS), coumaphos (CM), diazinon (DZ) dyfonate (DF), ethoprophos (EP), fenamiphos (FM), methylparathion (MPT), and parathion (PT) were also degraded up to 68, 60, 80, 40, 45, 60, 95, and 100%, respectively, when used as a substrate with Est5S protein. The results highlight the potential use of this enzyme in the cleanup of contaminated insecticides.     

芜菁花叶病毒长春分离物p3基因的克隆、序列分析及P3蛋白抗血清的制备

 用RT-PCR方法从长春感染芜菁花叶病毒( Turnip mosaic virus,TuMV)的十字花科蔬菜中扩增获得该病毒的p3基因,并对其序列进行了比较分析。结果表明,本研究所获得的10个TuMV分离物p3基因含1 065个核苷酸,其序列一致率为98.8%~99.6%,与GenBank中其他15个TuMV分离物核苷酸一致率为80.9%~99.4%。根据p3基因核苷酸序列构建的系统进化树显示:25个TuMV分离物可分为4个组,本研究得到的10个TuMV分离物均属于basal-BR组。将TuMV JCR06分离物p3基因N端663 bp片段克隆至原核表达载体pET-28a(+),并在大肠杆菌BL21(DE3) pLysS中表达出分子量约为28 kDa的融合蛋白。以纯化的融合蛋白为抗原免疫家兔,制备了P3蛋白的特异性抗血清。以TuMV侵染的萝卜为抗原,间接ELISA测定抗血清的效价为1∶2 048。Western blotting分析表明,制备的抗血清能与诱导表达的融合蛋白发生特异性反应。