Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a

SaPIN2a, a proteinase inhibitor II from American black nightshade (Solanum americanum Mill.) is highly expressed in the phloem and could be involved in regulating proteolysis in the sieve elements. To further investigate the physiological role of SaPIN2a, we have produced transgenic lettuce (Lactuca sativa L.) expressing SaPIN2a from the CaMV35S promoter by Agrobacterium-mediated transformation. Stable integration of the SaPIN2a cDNA and its inheritance in transgenic lines were confirmed by Southern blot analysis and segregation analysis of the R₁ progeny. SaPIN2a mRNA was detected in both the R₀ and R₁ transformants on northern blot analysis but the SaPIN2a protein was not detected on western blot analysis using anti-peptide antibodies against SaPIN2a. Despite an absence of significant inhibitory activity against bovine trypsin and chymotrypsin in extracts of transgenic lettuce, the endogenous trypsin-like activity in each transgenic line was almost completely inhibited, and the endogenous chymotrypsin-like activity moderately inhibited. Our finding that heterogeneously expressed SaPIN2a in transgenic lettuce inhibits plant endogenous protease activity further indicates that SaPIN2a regulates proteolysis, and could be potentially exploited for the protection of foreign protein production in transgenic plants.Abbreviations CaMV cauliflower mosaic virus - cDNA complementary DNA - NOS nopaline synthase - PAGE polyacrylamide gel electrophoresis - PI proteinase inhibitor - SaPIN2a Solanum americanum proteinase inhibitor IIa - SDS sodium dodecyl sulphate - T-DNA transferred DNA     

地衣芽孢杆菌对养殖水体氨氮、残饵降解特性研究

试验对从健康养殖大黄鱼肠道中分离筛选到的一株益生地衣芽孢杆菌X3914的氨氮、残饵降解特性进行了研究。结果表明,X3914菌株降解氨氮的最适温度、盐度和pH分别为30℃、2.0%和7.0,在氨氮初始浓度为100 mg/L时,24h内的氨氮降解率达到36.2%。通过向添加1%饲料的大黄鱼养殖用水中接种1%和5%(体积比)的菌量(菌浓度约为9.0×108 cfu/L)来研究地衣芽孢杆菌浓度对降解饲料中蛋白质、淀粉的影响。结果表明,接种5%菌量的降解率均高于1%菌量的降解率,其中,48h内接种5%菌量的蛋白质和淀粉的降解率分别为35.2%和52.6%。X3914菌株具有较好的氨氮、饲料蛋白质和淀粉的降解能力,在水产养殖上具有潜在的应用价值。     

猪流行性腹泻病毒经其附属蛋白抑制细胞凋亡

【背景】orf3位于猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV) s基因与e基因之间,是目前发现的PEDV唯一一个附属基因,编码附属蛋白(ORF3蛋白)。我们前期研究初步发现ORF3蛋白对PEDV诱导的细胞凋亡有影响。【目的】研究ORF3蛋白在PEDV侵染复制过程中的毒力作用机制。【方法】实验用3种PEDV:rDR13att-?ORF3 (orf3基因全部敲除)、DR13-ORF3att (携带有C端截短orf3)、rDR13att-ORF3wt(携带全长orf3基因)感染Vero细胞,观察病变情况,再用活细胞成像仪、流式细胞仪、 DNA断裂的原位末端标记法[terminaldeoxynucleotidyltransferase(TDT)-mediated dUTP nick end labeling,TUNEL]等方法检测不同感染时间点的细胞凋亡情况,然后用蛋白质印迹方法分析PEDV感染宿主细胞中主要凋亡相关蛋白(如Caspase-3)的活化或裂解,最后进行转录组测序研究病毒感染细胞中差异基因的表达情况,再用荧光定量PCR验证转录组结果。【结果】rDR13att-?ORF3引起较多的细胞病变,活细胞成像仪的动态观察结果显示,3种病毒侵染的细胞凋亡水平随着时间的延长均高于正常阴性细胞,但敲除orf3的病毒感染细胞后细胞凋亡率比其他两种病毒更高;敲除orf3病毒感染细胞凋亡率显著高于其他两种病毒;病毒rDR13att-?ORF3感染细胞后TUNEL阳性细胞数比DR13-ORF3att和rDR13att-ORF3wt更多;表达ORF3蛋白的重组PEDV可以抑制Caspase-3的活化;ORF3蛋白对受感染细胞Heat shock 70 kD protein 1B (HSP70)基因转录有促进作用,荧光定量PCR结果表明rDR13att-ORF3wt感染细胞的HSP70表达量高于rDR13att-?ORF3感染细胞。【结论】PEDV通过ORF3蛋白抑制细胞凋亡,而且这种作用可能是通过抑制Caspase-3的活化或增加HSP70的产生来完成的。     

与细胞周期G2/M期进程相关的H2AX磷酸化

γH2AX焦点(foci)被普遍当做DNA双链断裂（DSB）损伤的分子标志物.为探 讨细胞周期进程相关的H2AX磷酸化规律特征,采用胸腺嘧啶双阻滞结合噻氨酯哒唑(nocodazole)的后续处理,将HeLa细胞同步于有丝分裂的前中期．然后,用流式细胞仪检测细胞周期、Western印迹和免疫荧光法,观察γH2AX表达和γH2AX焦点的形成．结果显示,细胞进入G2/M期和有丝分裂过程中,γH2AX水平显著增加 ;在无DNA DSB发生的情况下,部分M期细胞中也存在大量的γH2AX焦点．随着细 胞完成有丝分裂从M期退出再进入G1期,γH2AX的表达水平逐渐降低．这种 γH2AX表达变化特征与G2/M期密切关联的PLK1和Cyclin B1的表达规律相类似． 在4 Gy大剂量照射下,HeLa细胞于照后8 到12 h出现明显的G2/M期阻滞．γH2AX 焦点数在照后1 h达高峰,随后降低,照后8 h又上升,出现了第2个峰值．与之不同的是,在1 Gy低剂量照射下,细胞的G2/M期阻滞微弱,γH2AX焦点数在照后 0.5 h最高,随后下降,且无反弹,符合DNA DSB的修复动力学特征．因此,将γ H2AX当做DNA DSB分子标志物时,还需要考虑细胞周期变化的影响．γH2AX适合 作为1 Gy以下照射的DNA双链断裂损伤的分子标志．     

Using silica particles to isolate total RNA from plant tissues recalcitrant to extraction in guanidine thiocyanate

The most commonly used protocol of the RNA isolation, the guanidine thiocyanate method, was unsuitable for recalcitrant plant tissues containing a large amount of storage proteins and secondary metabolites. We demonstrated that RNA could bind to the silica particles, which have been used successfully in DNA isolation from various sources, under a high concentration of NaCl in the presence of ethanol and sodium acetate. Based on this observation, an efficient, inexpensive, and highly reproducible technique, the acid phenol-silica method, was developed to isolate high-quality RNAs from various plant tissues recalcitrant to extraction in guanidine thiocyanate.     

两株副溶血弧菌烈性噬菌体的分离鉴定

研究旨在筛选烈性噬菌体, 为副溶血弧菌(Vibrio parahaemolyticus, Vp)病害防控增加新的选择。以副溶血弧菌Vp13为宿主菌, 通过二层琼脂平板法筛选, 分离到了2株烈性噬菌体SX-2和SX-F。对其形态结构进行了透射电镜观察, 利用DNase I、 RNase A、Mung Bean Nuclease和Hind Ш酶进行噬菌体核酸类型鉴定, 并对噬菌体的裂解谱、最佳感染复数、一步生长曲线进行了测定。透射电镜观察结果显示: SX-2核衣壳头部长约110 nm, 宽约50 nm, 尾部长约150 nm, 宽约10 nm, 为典型的复合体制; SX-F核衣壳呈正六边形, 长约为56.86 nm,宽约50.74 nm, 未观察到尾部, 推测为正二十面体对称; 核酸测定结果显示两者均为线性双链DNA。依据国际病毒分类委员会第九次报告, SX-2符合肌尾噬菌体科特征, SX-F符合盖噬菌体科特征。噬菌体SX-2和SX-F对85株弧菌裂解结果显示: 噬菌体SX-2能够裂解23株副溶血弧菌和1株溶藻弧菌(Vibrio alginolyticus), 噬菌体SX-F能够裂解19株副溶血弧菌和1株溶藻弧菌。SX-2和SX-F的最佳感染复数均为0.0001。一步生长曲线结果显示: SX-F的潜伏期约10min, 裂解期约70min, 裂解量为116.2; 噬菌体SX-2的潜伏期小于10min, 裂解期大约70min, 裂解量为209.3。两株噬菌体生物学特性表明SX-2与SX-F均为烈性噬菌体, 这为进一步探讨噬菌体防治技术奠定了基础。     

Production and characterization of soluble human lysosomal enzyme α-iduronidase with high activity from culture media of transgenic tobacco BY-2 cells

Lysosomal storage diseases (LSDs), that collectively represent over 50 disorders, are amenable to enzyme replacement therapies. However, the current methods used to commercially produce recombinant lysosomal enzymes for this purpose, most commonly Chinese Hamster Ovary cells and human fibroblasts, are prohibitively costly. Plant bioreactors hold great promise for economic production of functional human α-l-iduronidase (hIDUA; glycosaminoglycan α-l-iduronohydrolase; EC 3.2.1.76), the enzyme deficient in the human LSD, Mucopolysaccharidosis I. We have developed and tested an expression system using transgenic tobacco BY-2 cells to produce high amounts of active hIDUA. A plant signal peptide was essential for proper expression and secretion of the 78 kDa glycosylated hIDUA into the cultured media of transgenic BY-2 cells. The yield and activity of the secreted hIDUA from long-term cultures of transgenic BY-2 cell lines were as high as 10 μg/mL media and 53,000 pmol/min/mg proteins, respectively. Thus, this transgenic BY-2 cell line presents an attractive platform for economic production and easy downstream purification of hIDUA for enzyme replacement therapy. Furthermore, this system can be used for the production and purification of other human lysosomal enzymes or pharmaceuticals.     

Analysis of expressed sequence tags from biodiesel plant Jatropha curcas embryos at different developmental stages

Jatropha curcas is considered a potential biodiesel feedstock plant whose seeds contain up to 40% oil. However, little is currently known about the seed biology of Jatropha. Therefore, it would be valuable to understand the mechanisms of development and lipid metabolism in Jatropha seeds. In the present study, three cDNA libraries were constructed with mRNA from Jatropha embryos at different stages of seed development. A total of 9844 expressed sequence tags (ESTs) were produced from these libraries, from which 1070 contigs and 3595 singletons were obtained. One hundred and seven unigenes were found to be differentially expressed in the three cDNA libraries of Jatropha embryos, indicating that these genes may play key roles in seed development. We have identified 59 and 61 unigenes that might be involved in the development and lipid metabolism in Jatropha seeds, respectively. Some of these genes may also play important roles in embryogenesis, morphogenesis, defense response and adaptive mechanisms in plants.     

A proteinase inhibitor II of Solanum americanum is expressed in phloem

Although proteinase inhibitor proteins are known to confer insect resistance in transgenic plants, their endogenous roles remain undefined. Here, we describe the expression of a proteinase inhibitor II (PIN2) protein from Solanum americanum in phloem of stems, roots and leaves suggesting a novel endogenous role for PIN2 in phloem. The phloem consists of parenchyma cells, sieve elements (SE), and companion cells (CC) which are in close association with SE. We isolated two cDNAs encoding PIN2, SaPIN2a and SaPIN2b, from a S. americanum cDNA library using a tomato PIN2 cDNA as hybridization probe. SaPIN2a shows 73.6% identity to SaPIN2b. Southern blot analysis confirmed that two genes occur in S. americanum. Northern blot analysis showed that both are wound-inducible and are expressed in flowers. Unlike SaPIN2b and other previously characterized plant PIN2 proteins, SaPIN2a is abundantly expressed in stems. In situ hybridization studies on stem sections showed that SaPIN2a mRNA is expressed in CC and some SE, likely the immature developing SE, of external and internal phloem. Western blot analysis using SaPIN2a-specific antibodies showed SaPIN2a accumulation in stems, leaf midribs and fruits. Immunohistochemical localization, using these antibodies, revealed SaPIN2a expression in external and internal phloem of stem. Immunoelectron microscopy of stem, root and leaf sections further localized SaPIN2a to the CC and predominantly to the SE, particularly the parietal cytoplasm adjacent to the cell wall, the lumen and the sieve-area pores. These results suggest that, other than a possible role in plant defense, SaPIN2a could be involved in regulating proteolysis in the SE.