超表达OsPR1A增强了Xa21介导的水稻对白叶枯病的抗性反应

【背景】前期研究发现,水稻病程相关蛋白质OsPR1A的表达受上游抗病基因Xa21调控,接菌后早期启动Xa21介导的OsPR1A较高水平表达对水稻抵抗白叶枯病菌至关重要。同时OsPR1A也受到水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae,Xoo）的诱导表达。对于OsPR1A的研究绝大部分是作为抗性反应发生的标志基因佐证其他基因或途径在抗性中的作用,缺乏直接的证据证实OsPR1A本身的生物学功能。【目的】通过获得OsPR1a-OX超表达转基因植株,调查其表型及农艺性状,并明确OsPR1A蛋白质表达与抗性的关系,为鉴定OsPR1A功能提供依据。【方法】通过农杆菌介导法,将构建的OsPR1a-OX转化载体转入到水稻受体4021中,利用PCR和免疫印迹（western blot,WB）技术分别在基因水平和蛋白质水平上筛选并鉴定OsPR1A超表达阳性纯合株系。在成熟期,调查OsPR1A超表达转基因植株的表型及农艺性状（株高、穗长、分蘖数、结实率和籽粒大小等）。在31℃条件下,将生长2周的水稻幼苗TP309、4021和OsPR1A超表达转基因植株接种水稻白叶枯病菌,并在接菌0、2、4、6、8、10和12 d时测量病斑长度。在接菌0、4和6 d时,收集TP309、4021和OsPR1A超表达转基因植株的水稻叶片,提取蛋白质,利用WB技术检测OsPR1A的表达特征。【结果】构建了OsPR1a-OX转化载体,并转入到受体4021中,筛选并鉴定到2个OsPR1A超表达转基因纯合株系（#704和#709）。调查了OsPR1A超表达转基因植株在成熟期的表型及农艺性状,与对照4021相比,#704和#709的株高较矮、穗长较短、分蘖数减少、结实率降低,但籽粒稍大,可能与结实率低有关。在31℃条件下,OsPR1A超表达转基因植株的病斑长度与对照4021相比明显缩短,结果具有显著性差异（P<0.05）。在接菌0、4和6 d的材料中,超表达转基因植株#704和#709中OsPR1A始终有较高水平的表达丰度,从而提高了对白叶枯病菌的抗性。【结论】采用农杆菌介导法,获得OsPR1A超表达转基因植株;超表达OsPR1A影响到水稻的正常发育过程;超表达OsPR1A后增强了Xa21介导的水稻对白叶枯病的抗性。     

以CP23重组蛋白为抗原建立检测微小隐孢子虫抗体间接ELISA方法的研究

摘要：以在E.coli高效表达的微小隐孢子虫子孢子表面抗原CP23为抗原，以辣根过氧化物酶(HRP)标记的山羊抗鼠IgG为二抗，建立了检测微小隐孢子虫抗体的间接ELISA方法。经检测筛选出最佳反应条件为1μg／孔纯化的E.coli表达的CP23抗原包被酶标板，用10％免血清进行封闭，以正常E.coli裂解上清液稀释待检血清。实验表明应用CP23重组蛋白作为诊断C.parvum抗原具有特异性高、抗原易纯化和成本低等特点。     

Epidemiology of beet necrotic yellow vein virus in sugar beet at different initial inoculum levels in the presence or absence of irrigation

A field experiment was set up in 1988 to study the development of rhizomania disease of sugar beet at different inoculum levels of beet necrotic yellow vein virus (BNYVV) in soil. Five, tenfold different, inoculum levels were created by addition of the approximate amounts of 0, 0.5, 5, 50 and 500 kg infested soil per ha (the latter corresponding to 0.01% v/v calculated to the tillage layer). A drip irrigation treatment was applied to study the influence of soil moisture on disease. Susceptible sugar beet, cv. Regina, was grown for three consecutive years.In the first year, root symptoms were not observed, but BNYVV-infected plants were detected by ELISA in low numbers at all inoculum levels at harvest. After late drilling in 1989, high numbers of infected plants, up to 90–100% in plots with the highest inoculum level, were detected already in June. Root symptoms were also observed from June onwards. In both these years disease incidence increased in time and was significantly influenced by the initial inoculum level. In the third year, the whole field was heavily diseased, and only for the non-irrigated plots incidence differed for different initial inoculum levels. The expression of symptoms by BNYVV-infected plants was influenced by initial inoculum level, thus by the amount and timing of primary infection.Root weight at harvest was not affected, but sugar content decreased with increasing inoculum level already in 1988, leading to a reduction in sugar yield of 10% at the highest inoculum level. In 1989, both root weight and sugar content decreased progressively with increasing inoculum level, resulting in sugar yield reductions of 11–66% (down to approximately 3000 kg ha^–1) for low to high inoculum levels compared to the control. As the control plots became contaminated, all yields were low in 1990, still showing a decrease with increasing inoculum level in the non-irrigated plots, but an overall mean sugar yield of 3323 kg ha^–1 for the irrigated ones.Sodium and -amino nitrogen content of the root, additional quality parameters determining extractability of sucrose, showed an increase and decrease, respectively, with increasing initial inoculum level already in the first year. The relative differences in contents compared to those from the control were largest for Na content. A significant negative correlation was found between Na (mmol kg^–1 root) and sugar content (% of fresh weight); linear for 1988, exponential for 1989 and 1990.In spring 1989, the infestation of individual plots was assessed using a quantitative bioassay estimating most probable numbers (MPNs) of infective units of BNYVV per 100 g dry soil. The relationship between the MPns determined and root weight, sugar content and sugar yield at harvest could be described by Gompertz curves. The increase in disease incidence with increasing MPN in 1989 was adequately fitted with a logistic equation.     

Molecular Identification of a New Member of the Clover Proliferation Phytoplasma Group (16SrVI) Associated with Centaurea Solstitialis Virescence in Italy

In the United States, yellow starthistle (Centaurea solstitialis) is an annual invasive weed with Mediterranean origins. Malformed plants displaying witches' broom, fasciations, abortion of buds and flower virescence symptoms were observed in central Italy. Attempts to transmit the causal agent from the natural yellow starthistle host to periwinkle by grafting, resulted in typical symptoms of a phytoplasma, i.e. yellowing and shortening of internodes. The detection of phytoplasmas was obtained from both symptomatic yellow starthistle and periwinkle by the specific amplification of their 16S-23S rRNA genes. PCR amplification of extracted DNA from symptomatic plant samples gave a product of expected size. Asymptomatic plants did not give positive results. An amplicon obtained by direct PCR with universal primers P1/P7 was cloned and sequenced. The homology search using CLUSTALW program showed more than 99% similarity with Illinois elm yellows (ILEY) phytoplasma from Illinois (United States) and 97% with Brinjal little leaf (BLL) phytoplasma from India. Digestion of the nested-PCR products with restriction enzymes led to restriction fragment length polymorphism patterns referable to those described for phytoplasmas belonging to the clover proliferation (16S-VI) group. Since this is a previously undescribed disease, the name Centaurea solstitialis virescence has been tentatively assigned to it. This is a new phytoplasma with closest relationships to ILEY and BLL, but distinguishable from them on the basis of 16S rDNA homology, the different associated plant hosts and their geographical origin.     

Comparison of phenolic profiles and antioxidant activities in skins and pulps of eleven grape cultivars(Vitis vinifera L.)

Eleven grape cultivars were analysed to explore the variety differences of fresh grape phenolic profiles. The results showed that free phenolics were predominant in grape skins and pulps, and showed the higher antioxidant activities than bound. In 11 cultivars, Muscat Kyoho extracts had the highest total phenolic content in skins(10.525 mg GAE g~(–1) FW) and pulps(1.134 mg GAE g~(–1) FW), and exhibited the highest DPPH radical scavening capacity(EC_(50)=11.7 μg mL~(–1)) and oxygen radical absorbance capacity(ORAC) value(190.57 μmol TE g~(–1) FW) of free phenolic in skin. In addition, the most abundant phenolics in grape skins were found to be flavonoids such as kaempferol in Kyoho skin(541.2 μg g~(–1) FW), rutin, catechin and epicatechin in Muscat Kyoho skin(262.3, 86.3 and 70.0 μg g~(–1) FW, respectively). Furthermore, the principal component analysis showed a strong difference of phenolic profiles with the cultivars, existing forms and distributions. Pearson correlation coefficient analysis showed a significant linear correlation between total phenolic content and antioxidant activity(P0.05). Therefore, both skins and pulps were rich sources of bioactive phenolic compounds, and Muscat Kyoho was the ideal source among all samples.     

土耳其斯坦裂体吸虫23kDa蛋白基因的序列分析及抗原表位预测

为研究土耳其斯坦裂体吸虫23 kDa蛋白的生物学特性,以土耳其斯坦裂体吸虫cDNA为模版,利用PCR对23 kDa基因进行扩增,并将其克隆到T-easy载体后进行序列测定。利用生物信息学对其结构、抗原指数进行分析,并对其抗原表位进行预测。序列分析结果表明,该虫体的23 kDa基因长度为657 bp,A+T含量为57.38%,与曼氏血吸虫、埃及血吸虫和日本血吸虫的23 kDa基因的相似性分别为85.24%、83.71%和81.89%。蛋白二级结构分析表明,23 kDa蛋白经过4次跨膜,主要由6个α-螺旋、3个β-折叠、7个β转角和若干个无规则卷曲构成。抗原表位预测结果表明,该蛋白有3个B细胞抗原表位。综合分析土耳其斯坦裂体吸虫23 kDa蛋白是一种较好的抗原分子,是土耳其斯坦裂体吸虫疫苗的重要候选分子。     

松辽黑猪METTL23基因多态性及其与繁殖性状的关联分析

为探究松辽黑猪METTL23基因多态性及其与繁殖性状的关联性,试验选取178头松辽黑猪母猪为研究对象,利用Sanger直接测序法查找METTL23基因外显子1~5的单核苷酸多态性(SNP),使用SPSS 19.0软件分析METTL23基因SNP位点与松辽黑猪繁殖性状(总产仔数、产活仔数、断奶仔猪数、初生重、3周龄重、断奶重、乳头数)的关联性。结果显示,仅在松辽黑猪METTL23基因外显子4发现1个SNP位点,命名为A62G,在A62G位点上检测到3种基因型,分别是AA、AG和GG。GG和G分别为优势基因型和优势等位基因,且A62G位点处于Hardy-Weinberg平衡状态(P>0.05)。该位点的遗传纯合度(Ho)为0.6516,高于遗传杂合度(He,0.3484),说明其在松辽黑猪群体中的变异较小;多态信息含量(PIC)为0.2877,属于中度多态位点(0.25<PIC<0.5),说明该遗传标记能够提供一定量的遗传信息。关联分析结果表明,GG基因型个体总产仔数、产活仔数和断奶仔猪数均显著或极显著高于AA基因型个体(P<0.05;P<0.01),总产仔数还显著高于AG基因型个体(P<0.05);AG基因型个体断奶仔猪数显著高于AA基因型个体(P<0.05);初生重、3周龄重、断奶重、乳头数各基因型间差异均不显著(P>0.05)。综上所述,METTL23基因外显子4存在多态性位点A62G,该位点与松辽黑猪产仔数存在显著关联,可作为松辽黑猪产仔数的遗传标记,用于分子育种。     

Genetic, phenotypic and pathogenic diversity among xanthomonads isolated from pistachio (Pistacia vera) in Australia

Repetitive extragenic palindromic polymerase chain reaction (rep-PCR), sequencing of the 16S−23S rDNA internal transcribed spacer (ITS), biochemical and physiological tests, the Biolog microplate system, polyacrylamide gel electrophoresis (PAGE) of whole-cell proteins, and pathogenicity tests were used to characterize variability among xanthomonads isolated from pistachio trees suffering from bacterial dieback in four regions of Australia. ITS sequencing and rep-PCR revealed two distinct genotypes among the strains. The ITS sequencing suggested that the pistachio strains were closely related to Xanthomonas translucens pathovars, in particular X. translucens pv . poae . Results of physiological and biochemical tests, as well as Biolog microplate analysis and protein profiling, confirmed the existence of two groups. Furthermore, pathogenicity and host-range studies indicated that the two groups were biologically different. There was an association between the two groups and the geographical origin of the strains.     

Relationship between genetic instability of nm23H1 gene and clinical pathological characteristic in hepatocellular carcinoma

AIM: The aim of this study was to examine the microstatellite instability (MSI) and loss of heterozygosity(LOH) of locus D17S396 on chromosome 17 and their influence on the expression of nm23H1 in hepatocellular carcinoma (HCC),which may provide experimental evidence for the mechanism of nm23H1 gene and tumor metastasis.METHODS: Techniques such as DNA extraction from formalin-fixed paraffin-embedded tissues,PCR-SSCP,ordinary silver stain were used to study MSI and LOH of locus D17S396.Envision immunohistochemistry and Leica-Qwin computer imaging techniques were used to assess the expression of nm23H1.RESULTS: ① The frequency of heredity instability of HCCs was 35.42%.The frequency of LOH in the cases with lymph node or distant organs metastasis or not and with intrahepatic metastasis or embolus of portal vein or not was significantly different (P<0.01),it was higher in stage TNM Ⅲ than that in stageⅠ and Ⅱ.Moreover,it was higher in high tendency to invasion or metastasis cases than that in the low tendency cases (P<0.01).② The expression of nm23H1 was 56.25%.It was significantly different in Edmondson grade,TNM stage and in lymph node or distant organ metastasis cases (P<0.01).The cases with high tendency of invasion or metastasis exhibited lower nm23H1 expression compared with low tendency cases (P<0.01).③ The positive rate of nm23H1 protein in LOH positive group was lower than that in LOH negative group (P<0.05).CONCLUSION: The results indicate that both MSI and LOH of nm23H1 gene control the development of HCC independently in different pathways.LOH inhibits the expression of nm23H1,which endows it with high aggressive and poor prognosis.Increase in the amount of nm23H1 protein expression effectively restrains the tendency to invasion or metastasis of HCCs and improves prognosis of patients.