桃PGIP蛋白基因片段的克隆、序列分析及在大肠杆菌中的表达

【目的】克隆桃（Prunus persica）多聚半乳糖醛酸酶抑制蛋白（polygalacturonase-inhibiting protein,PGIP）基因,并进行原核表达研究。【方法】根据李属植物pgip保守区域设计1对特异引物,以桃叶片总RNA为模板,通过RT-PCR获得了约1 kb的cDNA片段,T/A克隆后进行序列测定,并对该序列进行分析。随后将该蛋白成熟肽cDNA片段克隆到原核表达载体pET-32a(+)中,构建融合表达质粒,转化到Escherichia coli Rosetta-gami（DE3）pLysS中进行表达。【结果】测序结果显示,桃多聚半乳糖醛酸酶抑制蛋白cDNA编码区长993 bp,编码330个氨基酸残基,命名为Pppgip。PpPGIP分子量为36.4 kD,等电点为7.42,信号肽为N端24个氨基酸残基,具有7个潜在的N-糖基化位点。Pppgip与李属其它pgip核苷酸和氨基酸序列的同源性分别为93.2%～94.6%和89.7%～92.7%。同源树分析表明,该基因明显区别于其它pgip,与马哈利樱桃pgip的关系较近,与寿星桃、桃栽培品种‘久保’等pgip的关系都较远。该基因所编码的蛋白质的三维结构含11个α-螺旋和21个β-折叠,中心LRR结构域由10个串联的LRRs基序组成。原核表达产物经SDS-PAGE分析表明,重组蛋白主要以包涵体形式出现。【结论】克隆了桃PGIP基因,并可在大肠杆菌中表达。     

冷激对玉米幼苗可溶性蛋白质含量与组分的影响

以胚芽期与3叶期玉米幼苗叶片为试材,进行0、2、4、6℃温度下冷激处理,分析不同冷激强度与可溶性蛋白质含量及组分的关系。结果表明,冷激处理下可溶性蛋白质含量先增加后减少;胚芽期增加幅度大于3叶期,3叶期幼苗在较低温度下(0℃、2℃)可溶性蛋白质含量先降低后升高。冷激蛋白的诱导在不同苗龄表现不一致,胚芽期有部分蛋白质带(40KD、26KD)表达增强,但没有发现新的蛋白质。3叶期幼苗有分子量在80KD、45KD左右的两条新蛋白带。温度越低出现的新蛋白越少,随着温度的升高新蛋白出现的时间越迟。     

Polymorphism of waxy proteins in Spanish durum wheats

A collection of 547 durum wheats (103 cultivars and 444 landraces) from Spain was analysed for waxy protein composition. The electrophoretic patterns showed low polymorphism. At the Wx‐A1 locus, 99.8% of the wheats had the Wx‐Ala allele and only one had the null Wx‐Alb allele. The Wx‐Bl locus was more polymorphic and four different alleles were detected: Wx‐Bla (41.3%), Wx‐Blc (42.6%), a new allele, not detected before in bread wheat and named Wx‐Blf (16.0%), and the null Wx‐Blb allele, found for the first time in one durum wheat. Eleven durum wheats with different allelic composition at the Wx‐l loci were analysed for amylose content. Wheats with the Wx‐Bla allele had a lower amylose content than those with Wx‐Blc or Wx‐Blf. The lowest amylase content was found in the only durum wheat having the null Wx‐Blb allele.     

Allelic variants of granule-bound starch synthase proteins in European bread wheat varieties

The composition of 324 European wheat cultivars were analysed at the three granule‐bound starch synthase (GBSS I) loci. Protein separation was first made by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE. A specific two‐dimensional (2D) electrophoresis (immobilized pH gradient × SDS‐PAGE) using an Immobiline dry strip in the first dimension was developed to resolve the GBSS I proteins more clearly and to confirm some results. Very low polymorphism was found. Among the 324 cultivars analysed, only one carried a Wx‐A1 null allele (Wx‐A1b) and none was found to have the Wx‐2D null allele. As described in the literature the Wx‐B1 locus was more polymorphic and the null allele was encountered in 11 cultivars. The use of 2D electrophoresis allowed us to find another type of variant which presented as having thicker band with same mobility as the Wx‐D1 protein in SDS‐PAGE. Twelve per cent of the cultivars analysed presented this band and could have been previously mistaken for cultivars carrying the Wx‐B1 null allele. Indeed this band probably corresponded to the Wx‐B1? or Wx‐B1e allele overlapping with the Wx‐D1a allele in SDS‐PAGE.     

Determination of the outcrossing rate of Phaseolus vulgaris L. using seed protein markers

The outcrossing rates of four varieties of Phaseolus vulgaris in Asturias (Northern Spain) were studied using seed protein polymorphisms as genetic markers. No evidence of outcrossing was obtained, and the outcrossing rate of this species at Asturias was estimated, with a confidence of 95%, as being less than 0.74%. The usefulness of seed proteins as genetic markers for obtaining estimates of outcrossing is also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Absence of albumin in the plasma of the common carp Cyprinus carpio: binding of fatty acids to high density lipoprotein

It was investigated whether an albumin-like protein, active in the transport of free fatty acids, is present in the blood of the common carp, Cyprinus carpio. In contrast with the brown trout Salmo trutta, no free fatty acid-binding protein could be found with a molecular mass and isoelectric point similar to human serum albumin. On the other hand, free fatty acids bound in this fish species to the apolipoproteins of high density lipoprotein (HDL), namely apolipoprotein A-I and apolipoprotein A-II. In addition to the binding of free fatty acids, carp HDL was also capable of interacting with the lipophilic human serum albumin binding dye, Cibracon Blue. The results obtained with plasma of carp, trout and man suggest that HDL acts as an alternative carrier for the transport of free fatty acids in the blood when albumin concentrations are low or absent.     

Genome-wide analysis of OVATE family proteins in cucumber (Cucumissativus L.)

OVATE family proteins (OFPs) are plant-specific proteins with a conserved OVATE domain that regulate plant growth and development. Although OFPs have been studied in several species, their biological functions remain largely unknown in cucumber (Cucumis sativus L.). This study identified 19 CsOFPs distributed on seven chromosomes in cucumber. Most CsOFP genes were expressed in reproductive organs, but with different expression patterns. Ectopic expression of CsOFP12-16c in Arabidopsis resulted in shorter and blunt siliques. The overall results indicated that CsOFP12-16c regulates silique development in Arabidopsis and may have a similar function in cucumber.     

Mucosal responses of brown-marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) following intraperitoneal infection with Vibrio harveyi

Groupers are popular aquaculture species in South-East Asia, but their cultivation is affected by infectious disease outbreaks. Mucosa-associated lymphoid tissues provide a first-line defence against pathogens; however, few studies are available relating to cellular or proteomic responses of mucosal immunity in grouper. Skin, gill and intestine were sampled from brown-marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) at 4 and 96 hr post-infection (hpi) and 7 days post-infection (dpi) following intraperitoneal infection with Vibrio harveyi, and stained with haematoxylin/eosin and Alcian Blue/periodic acid–Schiff. Skin mucus was analysed by 2D-gel electrophoresis, and proteins modulated by the bacterial infection identified. In the infected fish, significant increases in sacciform cells in skin and increased levels of nucleoside diphosphate kinase in mucus were detected at 4 hpi. At 96 hpi, goblet cells containing acidic mucins significantly increased in the intestine, while those containing mixed mucins increased in skin and gills of infected fish. Proteasome subunit alpha type-I and extracellular Cu/Zn superoxide dismutase levels also increased in mucus. Rodlet and mast cells did not appear to respond to the infection. Mucosal tissues of grouper appeared actively involved in response to Vibrio infection. This information may help future research on improving grouper health, production and vaccine development.     

3株水产动物病原弧菌主要分泌性蛋白的鉴定及其分泌性序列的分析

从3株水产动物病原菌鳗弧菌MN、鳗弧菌3101、费氏弧菌培养液中分别获取了分泌性蛋白,进行SDS-PAGE,对表达量较高的5条蛋白区带进行MALDI-TOF/TOF质谱鉴定表明,它们分别是鳗弧菌MN金属蛋白酶(empA-MN)、鳗弧菌3101金属蛋白酶(empA-3101)、锌金属蛋白酶(zincempA)、费氏弧菌VFMJ11_1094蛋白(VFMJ11_1094)和费氏弧菌ES114的外膜蛋白(OMP).根据所鉴定的序列设计引物,分别从鳗弧菌MN、鳗弧菌3101和费氏弧菌中扩增出相应基因,克隆后测定emp-MN、empA-3101、zinc empA、VFMJ11-1094和OMP相应基因的序列,经在线软件SignaIP 3.0分析,确定emp-MN、empA-3101、zinc empA、VFMJ11-1094和OMP均存在不同的分泌性信号肽序列angMN-35、ang3101-35、ang3101-25、vf-38和vf-23,通过在线软件PSORT分析表明,所有信号肽均定位在细胞的周质空间或细胞外膜上.