养殖废弃物堆肥中抗生素和抗性基因的降解研究

抗生素的滥用及排放会造成细菌产生耐药性以及抗生素抗性基因(Antibiotic resistance genes, ARGs)的传播和扩散。畜禽粪便是导致环境中抗生素污染的主要来源之一。本文综述了四环素类、大环内酯类、喹诺酮类、β-内酰胺类、磺胺类和氨基糖苷类等在水土环境中广泛存在的抗生素及其环境残留水平和对动植物、微生物的影响,分析了当前利用堆肥技术降解畜禽粪便中抗生素和ARGs效果及机制的研究情况。总结得出,猪粪中抗生素残留量最高,其中四环素类残留量为1390~354 000 mg·kg^-1,磺胺类170.6~89 000 mg·kg^-1,氟喹诺酮类411.3~1 516.2 mg·kg^-1,硝基呋喃类85.1~158.1 mg·kg^-1,大环内酯类1.4~4.8 mg·kg^-1。堆肥对大部分抗生素具有好的降解效果,其中四环素类抗生素降解率为62.7%~99%,磺胺类为0~99.99%,对大环内酯类几乎可以完全降解,但是,堆肥无法降解喹诺酮类抗生素。养殖废弃物堆肥过程中,ARGs的降解情况同样因抗生素种类和堆肥方式而不同。已有的研究表明,除大环内酯类ARGs外,堆肥对其他ARGs均具有有效的降解效果,降解率为50.03%~100%。堆肥初期的优势菌门是厚壁菌门、放线菌门、变形菌门和拟杆菌门;堆肥结束后放线菌门成为最优势菌门。初始抗生素的浓度不影响堆肥结束时微生物的群落组成。温度和pH是影响抗生素降解的最主要因素,而ARGs的降解效果主要受温度影响。     

捻转血矛线虫GST耐药基因的克隆、表达及生物信息学分析

为探究捻转血矛线虫GST基因的原核表达及其生物信息学特征,以捻转血矛线虫cDNA为模板,设计特异性引物扩增GST基因,构建原核表达载体pET30a(+)-GST转化至E.coil BL21(DE3)感受态细胞,将鉴定正确的重组菌进行表达条件优化后,再通过SDS-PAGE分析蛋白表达的特征,并利用在线软件对GST蛋白进行生物信息学分析。结果表明:捻转血矛线虫GST基因大小为618 bp;诱导后的pET30a(+)-GST重组菌最佳诱导条件为0.05 mmol/L的IPTG在37℃时诱导6 h,以包涵体的形式存在,大小约为29 ku。生物信息学分析表明:GST分子式为C_(1083)H_(1657)N_(277)O_(294)S_6,属于不含信号肽和跨膜区的亲水性蛋白;二级结构主要由α螺旋形成;潜在的抗原表位主要位于25～48、55～63、92～106、113～124、139～147、170～189和195～205位氨基酸附近。本试验成功构建GST基因原核表达系统,优化表达条件下能稳定纯化GST重组蛋白,为进一步探索捻转血矛线虫丙硫咪唑耐药机制及GST蛋白的生物学特性提供研究基础。     

牦牛瘤胃微生物抗生素抗性基因对3种外源性刺激因子的响应

通过给牦牛投喂硫酸头孢喹肟(CEF)、盐酸二氟沙星(DIF)和黄曲霉毒素B1(AFB1),并进行瘤胃微生物宏基因组测序,旨在揭示这3种外源性刺激因子对抗生素抗性基因(ARGs)种类、抗性类型、抗性机制等的影响,对于深入研究微生物抗性组特征和抗性机制具有重要价值。选取15头牦牛,随机分5组。Cef组和Dif组分别根据说明书推荐剂量按体重计算、灌服CEF 1 mg·kg^-1和DIF 1 mL·kg^-1;E1组和E2组分别按采食量投喂AFB120、60μg·kg^-1;C组为对照组。处理7 d后,采集瘤胃液,提取DNA,Illumina HiSeq测序,对reads counts进行标准化得到TPM值,并进行方差分析。结果显示,对照组共获得132种ARGs,分属30种抗性类型,其中,四环素类tetQ和tetW基因丰度较高;Cef组tetW基因丰度增加(P<0.05),Dif组tetQ丰度增加(P<0.05);Cef组四环素类和头孢菌素类抗性基因丰度增加(P<0.05),Dif组四环素类和氨基香豆素类抗性基因丰度增加(P<0.05),E1组氨基香豆素和青霉烯类抗性基因丰度增加(P<0.05),E2组青霉烯类、头孢菌素类等9类抗性基因丰度均增多(P<0.05);Dif组Erm基因23S核糖体RNA甲基转移酶丰度增加(P<0.05),E2组中ATP结合盒超家族等3种抗性机制相关基因的丰度增加(P<0.05);3种因子均显著增加四环素类ARGs宿主的种类。结论:瘤胃是蕴含丰富ARGs的储藏库,其中,四环素类抗生素抗性基因tetQ和tetW是主要的ARGs。不仅CEF和DIF使部分ARGs的种类、抗性类型以及耐药机制相关酶等的丰度升高,增加瘤胃微生物的耐药性,而且AFB1也具有类似作用,且高剂量AFB1对抗性类型的影响范围较抗生素大。这3种因子还导致携带四环素类ARGs宿主微生物的种类数量增加,从而强化横向转移机制,加快ARGs传播,增强微生物对四环素类的耐药性。     

Regional and temporal differentiation of virulence phenotypes of Puccinia triticina from common wheat in Russia during the period 2001–2018

Leaf rust, caused by the fungus Puccinia triticina, is one of the most damaging rust diseases of wheat in Russia. Populations of P. triticina were monitored in seven regions of Russia from 2001 to 2018, with a total of 5,191 single urediniospore isolates from bread wheat (Triticum aestivum) being analysed. Populations have changed significantly in all regions since 2012, after 2 years of drought (2010–2011). Regional collections of P. triticina were also significantly different between the two periods 2001–2009 and 2012–2018, with changes along two geographic gradients from West Siberia to the north-west and south-west (North Caucasia) of the European part of Russia. All tested isolates were avirulent to resistance gene Lr9 in 2001–2009 but, since 2010, virulence to Lr9 has occurred and annually increased in the Asian part of Russia (Ural and West Siberia) due to deployment of cultivars with the Lr9 gene. Virulence to Lr2a and Lr15 was considerably lower in Dagestan (6%–33%) and all European regions (35%–67%) than in Asian regions (84%–96%). During 2001–2009, virulence on Lr1 was also lower in Dagestan (33%) and the European regions (50%–77%) than in Asia (91%–96%); however, by 2012–2018, nearly all isolates were virulent on Lr1. Remarkable changes were observed in frequencies of P. triticina races defined by their virulence/avirulence to Lr1 and Lr2a genes. We postulate the P. triticina population in Dagestan is specific to that area and pathogen populations in European and Asian parts of Russia are distinct.     

旱垂柳转录组测序及赤霉素在垂枝形成中的作用

垂枝作为特殊的林木形态结构,因形态优美在全世界被广泛用于绿化和园林景观而成为研究热点。国内外对垂枝植物的研究表明赤霉素在垂枝形成中起作用,但是垂枝形成的分子背景研究仍比较缺乏。本研究以旱垂柳(Salix matsudana var. pseudo-matsudana)的垂枝为研究对象,通过对旱垂柳在不同生长发育时期的叶片、茎尖和茎基部进行了转录组和数字表达谱测序研究,共获得71 474个Unigenes序列。将Unigenes与COG和KEEG数据库比对,与转录和信号转导机制相关的基因在旱垂柳中高度富集,最终确定8 359个差异表达的基因,其中427个基因与激素相关。进一步分析赤霉素相关基因得出,主要参与2条赤霉素合成途径,具有生物活性的GA4是合成途径中的主要产物,而且GA4在枝条中的含量显著高于GA1和GA3。参与赤霉素信号转导途径中的GID1B在旱垂柳中上调表达,以及在G1和G4生长期的表达量高于其他时期,而DELLA蛋白GAI在G2生长期以后下调表达,GID1B和GAI在旱垂柳的垂枝生长过程中可能起非常重要的作用。这些研究结果将有助于探索赤霉素在旱垂柳垂枝形成中的分子作用,可为林木形态的遗传改良提供基础理论依据。     

碱胁迫下榆叶梅差异基因表达分析

为了解榆叶梅(Prumus triloba Lindl.)在短期碱胁迫下的分子响应机制,本研究采用Illumina HiSeq^TM2500高通量测序技术对其在0、1、3、6、12 h碱胁迫下的叶片进行了de novo转录组测序,以此鉴定与碱胁迫相关的差异表达基因(DEGs)。结果表明,从5 960万条raw reads中获得了约5 300万条高质量clean reads,经从头组装后获得了124 786条榆叶梅unigenes。基于8 948个 unigenes差异表达(DEGs), GO富集分析发现,其中有28个基因可能在早期碱胁迫响应机制中起着重要作用; KEGG富集分析发现,不同碱处理时间下的KEGG途径(pathways)具有显著差异。对7个碱性胁迫反应相关基因进行RT-qPCR验证,所得基因的表达模式与RNA-Seq数据结果一致,证实了RNA-Seq结果的可靠性。本研究首次对碱胁迫下的榆叶梅叶片进行了转录组分析,获得的序列数据极大地丰富了榆叶梅可利用的基因资源,深入了解了榆叶梅碱胁迫的分子响应机制,同时为研究以榆叶梅为砧木的中国李(Prunus salicina)在碱胁迫下的适应机制奠定了分子研究基础。     

温度胁迫对幼体大海马基因转录表达的影响

为探究大海马幼体在高温和低温胁迫条件下基因表达水平的变化规律,采用Illumina平台的PE150测序功能对大海马幼体肝脏进行了转录组测序。对照组、高温胁迫组和低温胁迫组测序数据组装后获得22 513个单基因簇(Unigene),N50为2 223 bp,平均长度为1 122.71 bp。与对照组相比,高温胁迫组共获得14 009个差异表达基因,其中5 543个差异表达基因上调,8 466个差异表达基因下调;低温胁迫组共获得20 030个差异基因,14 016个差异表达基因上调,6 014个差异表达基因下调。差异表达基因经KEGG数据库富集发现,高/低温胁迫均导致大海马体内抗氧化途径相关基因表达(HSP70、HSP90、SOD等)发生显著改变;另外,高温胁迫还造成大海马免疫系统相关基因(PIK3R、Akt、IL-10、TLR等)以及细胞凋亡相关基因(CYCS、CASP9、CASP3等)表达显著改变;低温胁迫造成DNA损伤修复(MSH、DDB2、XRCC2、RAD52、Ogg1、PMS2等)、脂肪酸代谢(StARD7、ApoA4、CYP51、Fadsd6等)和细胞凋亡(P21、P53、BAX等)相关基因显著上调。选择HSP70、Fadsd 6与IL-10等13个差异基因验证,RT-qPCR结果与RNA-Seq测序结果基本一致。本研究结果为深入探究大海马温度应激的调控机制奠定了一定的理论基础,而且有助于预防极端温度对大海马造成损伤。     

Stability comparison of three candidate internal reference genes in Chinese giant salamander (Andrias davidianus)

The Chinese giant salamander (Andrias davidianus) as food and medicinal product has been an important aquaculture object in China. Study of gene function in the Chinese giant salamander requires accurate normalization though the use of appropriate reference genes. In this study, the expression levels of three candidate reference genes including β‐actin, GAPDH and cytb of different tissues, different developmental stages and different challenges in Chinese giant salamander were evaluated by qPCR. The stabilities of these three reference genes were analysed by geNorm, NormFinder and BestKeeper software. The results showed that the expression of GAPDH was more stable than that of β‐actin and cytb in four tissues and at two developmental stages of Chinese giant salamander. Compared with GAPDH and cytb, β‐actin was the most stable in spleen of Chinese giant salamander treated with LPS or GSIV. Therefore, the result showed that GAPDH was the suitable reference gene in different tissues and at different developmental stages of Chinese giant salamander. The β‐actin could be used as a reference gene in spleen of Chinese giant salamander challenged with LPS and GSIV. This study provides convincing information for the GAPDH and β‐actin as suitable reference gene in Chinese giant salamander of different tissues, different developmental stages and different challenges respectively.