凡纳滨对虾感染致急性肝胰腺坏死病副溶血弧菌(VpAHPND)的定量分析

对虾急性肝胰腺坏死病(Acute hepatopancreatic necrosis disease, AHPND)是由致AHPND副溶血弧菌(AHPND-causing Vibrio parahaemolyticus, VpAHPND)携带的pVA1-like质粒所表达的PirAVp和PirBVp毒力蛋白对对虾肝胰腺的急性毒性所致。本研究用2.19×105 CFU/ml VpAHPND分离株20130629002S01对凡纳滨对虾(Litopenaeus vannamei)进行浸泡感染,于感染后2~9 d采集对虾的肝胰腺、鳃、肠道、肌肉组织,采用实时荧光定量PCR方法,检测各组织中的pirAVp拷贝数。结果显示,感染后凡纳滨对虾各组织均能检测到pirAVp,其中,肝胰腺在感染后第4天达到峰值, 为8.71×104 copies/mg,而鳃、肌肉、肠道分别在第3、4、5天达到峰值,分别为9.08×103、2.59×104、5.76×104 copies/mg。早期感染鳃组织中先出现VpAHPND的富集,在高死亡发生期,VpAHPND数量在肝胰腺和肠道出现高峰,在死亡数量逐渐下降的后期,各组织的VpAHPND均快速下降,肠道、肝胰腺和肌肉中的VpAHPND水平趋于接近。对虾肝胰腺组织病理切片显示,同一时间有临床症状的病虾和濒死对虾相比,濒死对虾表现出更严重的AHPND病理特征,且二者的组织病理特征均随着感染时间的延长变得更为严重,但检测到的VpAHPND数量呈下降趋势。研究表明,在VpAHPND感染过程中,组织中的pirAVp基因数量不能代表对虾的发病程度,发病程度及组织病理严重的AHPND样品中VpAHPND的数量不一定处于高水平状态。     

合浦珠母贝实时定量PCR内参基因的稳定性比较

以合浦珠母贝（Pinctada fucata）为研究对象,应用实时荧光定量PCR技术检测了甘油醛-3-磷酸脱氢酶（GAPDH）、β-肌动蛋白（β-actin）和18S核糖体rRNA（18S rRNA）3个管家基因在合浦珠母贝不同组织、性腺发育时期、胚胎发育时期mRNA水平的表达情况。同时比较了BestKeeper、geNorm和NormFinde软件对试验数据处理的差异,从中选出合适的内参基因。结果表明β-actin在不同组织和胚胎发育不同阶段表达最稳定,18S rRNA在性腺发育不同时期表达最稳定。     

Rhizosphere microbial communities associated with Rhizoctonia damage at the field and disease patch scale

Rhizoctonia solani AG-8 is a major root pathogen in wheat (Triticum aestivum L.) systems worldwide and while natural disease suppression can develop under continuous cropping, this is not always the case. The main aim of our work was to elucidate the rhizosphere microbial community underlying a Rhizoctonia suppressive soil (Avon, South Australia) and to investigate how this community may develop in agricultural soils conducive to disease and of different soil type (Galong and Harden, New South Wales). The Avon suppressive soil community included Asaia spp. and Paenibacillus borealis, which were absent from a paired non-suppressive site. At Galong, soil taken from inside and outside disease patches showed no evidence of suppression, and disease suppression could not be transferred from the suppressive soil to the conducive soil from a different soil type and climatic area. 16S rRNA microarray analysis revealed Pseudomonas spp. were significantly more abundant inside than outside three disease patches at Galong. However, a survey of 32 patches across a range of stubble and tillage treatments at a nearby site showed no correlation between Pseudomonas and disease incidence. R. solani levels were significantly lower when stubble was retained rather than burnt or when nutrients (N, P and S) were incorporated with stubble during the non-crop period. Our results suggest soil type is an important factor for suppressive capability and that where specific disease suppression is absent, agronomic practice to increase soil carbon can encourage a non-specific microbial response that limits disease severity.     

一种基于TaqMan探针法的阪崎肠杆菌qPCR全自动检测技术体系的建立

[目的]建立快速准确地检测食物样品中食源性致病菌的体系。[方法]配套湖州市微生物制剂与农产品安全重点实验室自主研发的微生物分子检测仪,建立一种基于Taq Man探针法的阪崎肠杆菌q PCR检测体系。[结果]体系建立成功,标准曲线斜率为-3.44,R~2=0.995 5,扩增效率为95.3%。[结论]该q PCR体系特异性强,灵敏度高,明显缩短了检测时间,并具有良好的稳定性与重复性,为食品中阪崎肠杆菌的高通量和自动化检测提供了一个新的途径。     

龙眼体胚发生过程中tRNA怀丁苷合成蛋白基因(Dltyw1)的克隆及表达分析

根据龙眼胚性愈伤组织转录组数据库信息,进行龙眼胚性愈伤组织tRNA怀丁苷合成蛋白基因(Dltyw1)cDNA全长克隆及其在体胚发生过程中的定量表达分析.结果表明:Dltyw1基因全长2273 bp,包含1920 bp ORF,编码640个氨基酸,将此基因登录GenBank,登录号为JF733783;Dltyw1含有flavodoxin 1、Radical SAM及Wyosine form功能域,进化树分析显示,Dltyw1与其他物种twy1基因具有较高同源性.荧光定量PCR技术分析表明,Dltyw1基因在龙眼体细胞心形胚时期呈现表达高峰,并在子叶形胚阶段迅速下降至最低点,由于心形胚是体细胞胚胎发生由球形胚向各组织器官发育的关键阶段,表明twy1基因在体胚发生过程中的组织器官发育及形态建成阶段的转录后调控方面起重要作用.     

龙眼胚性愈伤组织AGO6基因克隆及其在体胚发生过程中的表达分析

依据龙眼胚性愈伤组织转录组数据库Unigene序列,利用RT-PCR结合RACE技术,以龙眼胚性愈伤组织c DNA为模板,获得Dl AGO6基因的c DNA全长序列,共3 168 bp(登录号为KF819529),完整开放阅读框2 700 bp,编码900个氨基酸。生物信息学分析表明:Dl AGO6的c DNA所编码的氨基酸序列含有2个高度保守的PAZ和PIWI结构域,具有典型的AGO类蛋白的结构特征;与拟南芥AGO6蛋白序列有较高的同源性。龙眼体胚发生过程中Dl AGO6表达量的q PCR分析表明:Dl AGO6在龙眼松散型胚性愈伤组织时期的表达量最高,在鱼雷形胚时期表达量最低,推测DLAGO6在龙眼松散型胚性愈伤组织阶段的高表达量,可能与细胞的旺盛分裂有关。     

Microbial communities in rice rhizosphere altered by intermittent and continuous flooding in fields with long-term arsenic application

Rice cultivated on arsenic (As)-contaminated soils can, under some conditions, accumulate high concentrations of As in grain, mostly as a result of the continuous flooding practices commonly used for rice cultivation. Intermittent flooding, as opposed to continuous flooding, might reduce soluble As concentrations in the rice rhizosphere, but it might also alter soil microbial populations that may impact As chemistry. A field-scale study was conducted to analyze As concentrations and microbial populations in the rice rhizosphere, in response to intermittent and continuous flooding in plots that were historically amended with “As-containing” pesticide and unamended soil. Rhizosphere, pore-water and grain As concentrations were quantified, and microbial populations in the rhizosphere were characterized using community quantitative-PCR and 16S rRNA gene sequencing. Pore-water As concentrations decreased by 41-81% and grain As by 31-48% in the intermittently flooded plots relative to the continuously flooded plots. The relative abundance of bacteria increased over the course of the growing season, while archaeal and fungal gene abundances decreased. Bacterial community structure and composition were significantly different between As amended and unamended plots, as well as between the flooding treatments. Proteobacteria was the predominant phylum detected in most treatments with relative abundance of 24-29%. The relative abundance of iron-reducing bacteria was higher with the continuous flood compared to the intermittent-flood treatment, implying greater relative iron reduction and possibly As release from the iron oxides under the continuously flooded conditions. These differences in rhizosphere-microbial communities may have contributed to the lower pore-water arsenic concentrations in the intermittently flooded conditions.     

Expression study on the porcine PTHLH gene and its relationship with sow teat number

Teat number is an important trait in sows that should accompany the increase in litter size that has been achieved in the last decades through selection. We have previously identified a genome-wide significant QTL for teat number in porcine chromosome SSC5 by means of an experimental Meishan by Iberian F(2) intercross population. In the present report, we have studied the porcine parathyroid hormone-like hormone (PTHLH) gene, which maps to SSC5, as a candidate gene for this trait, as PTHLH is involved in nipple formation during embryogenesis and nipple development during pregnancy and lactation. We have found that porcine PTHLH gene is transcribed into three mRNA species differing in the 5'UTR region. Two of these variants are reported in pigs here for the first time: one was similar to variant 1 described in humans while the other, which was generated by the retention of two small introns, has not been identified before in any other species. In addition, mRNA expression profile for two of the mRNA variants was assessed in 19 pig tissues. Porcine PTHLH showed a widespread expression as it was present in all tested tissues and relative expression of each variant was tissue dependent. Finally, we have performed an association study between a non-synonymous mutation in the coding region of this gene and sow teat number. The PTHLH polymorphism was segregating in our Meishan by Iberian F(2) population at intermediate allelic frequencies. We compared here six different statistical models to choose the one with a better fit and a lower degree of complexity. However, despite the potential negative effect of the PTHLH mutation in the signal peptide of this protein, we did not detect any association between the PTHLH genotype and the sow teat number phenotype, concluding that the causal mutation of the observed QTL is very likely not related to this gene.     

Molecular detection and quantification of Colletotrichum abscissum in sweet orange propagative material

Postbloom fruit drop disease (PFD) has caused serious impacts on citrus production in the Americas, occurring sporadically and suddenly during rain in the flowering period. In São Paulo State, Brazil, Colletotrichum abscissum is the causal agent responsible for over 80% of the disease incidence. Pathogen dispersal over long distances and the origin of primary inoculum are still unclear for PFD epidemics. We tested the hypothesis that citrus propagation material can harbour C. abscissum DNA by quantifying it in leaves of budwood increase block trees (BIB) and young citrus plants (YP) using multiplex quantitative PCR (qPCR). C. abscissum DNA was detected in all citrus nurseries, regardless of the type of propagative material, the sweet orange variety, or the nursery location. Overall, 73.4% of all samples from citrus nurseries have DNA from the pathogen, with a detection limit of 10 conidia. The average of 155 conidia found in YP was higher than the conidia observed on leaf samples from BIB (p = 0.03), although leaf samples from cultivars Valencia and Pera did not differ significantly, with means around 127 and 118 conidia, respectively (p = 0.75). This is the first molecular detection of C. abscissum in citrus propagative material. The multiplex qPCR assay may be used as a protocol for an accurate diagnosis of C. abscissum in citrus propagative material, may assist a better understanding of the pathogen dispersal over long distances, and may be used for further studies involving the quantification of C. abscissum in citrus orchards.