草鱼肠系膜脂肪组织高质量总RNA提取方法的研究

基于传统的酸性酚—异硫氰酸胍—氯仿一步提取法,比较分析多种优化操作步骤,摸索出一种高质量提取体质量为80~150 g草鱼肠系膜脂肪组织总RNA的改良方法。试验结果显示,相较于肝脏、脾脏、肠道等脏器组织,草鱼肠系膜脂肪组织RNA丰度低,且极易在样品前处理阶段出现顽固性降解问题。探索发现,将取样量增至约30 mg,可提升RNA产量以满足常规试验需求。针对降解难题,改良常规的样品前处理技术流程,采用鲜样液氮速冻,冻样直接放入TRIzol试剂中裂解,并即刻进行长时间机械匀浆,时长约3 min等核心操作步骤,可显著降低脂肪组织样品RNA的降解。Agilent生物分析仪检测结果显示,改良方法提取的草鱼肠系膜脂肪组织RNA完整度高,关键RIN值为8.7~9.0。研究推测,长时间机械匀浆所形成的持续剪切冲击力或许有助于TRIzol试剂中的异硫氰酸胍等成分突破油滴阻碍而有效抑制内源性RNA酶。本方法提取的草鱼脂肪组织总RNA质量可满足高通量转录组测序要求。     

马尾松叶表微生物多样性

为发掘对林木生长发育有利的优良微生物资源,并筛选适合叶表微生物的收集方法,以马尾松针叶为试验材料,分别用悬摇法和超声波法收集马尾松叶表微生物,用扩增子高通量测序技术、MUSCLE和Qiime软件研究马尾松叶表微生物的多样性。结果表明:扫描电镜观测结果显示,马尾松针叶表面定殖有大量微生物,包括真菌(菌丝及孢子)和细菌。扩增子高通量测序结果表明,马尾松叶表微生物物种丰富,包含细菌运算分类单位(OTUs)490个,真菌OTUs 1273个。马尾松叶表细菌以未分类的蓝细菌属(unidentified_Cyanobacteria)(36.53%)、未分类的拜叶林克氏菌属(unidentified_Beijerinckia)(28.60%)、鞘氨醇单胞菌属(Sphingomonas)(2.35%)为优势属;叶表真菌以枝孢属(Cladosporium)(2.45%)、拟盘多毛孢属(Pestalotiopsis)(0.92%)、无头孢菌属(Capnobotryella)(0.91%)为优势属。针对叶表细菌多样性的研究表明,悬摇法和超声波法均有较高的物种检出度;在叶表真菌多样性的研究中超声波法优于悬摇法,但超声波法样品间数据变异性较大,测定结果不稳定。     

间歇式双循环工厂化养殖系统构建及其养殖效果

为改善工厂化循环水养殖系统水质净化效果,提高养殖密度和成活率,构建了间歇式双循环工厂化养殖系统。通过间歇运行生物膜反应器增加水力停留时间,充分降解含氮污染物;连续运行弧形筛及时去除固体颗粒物。考察了该系统的启动过程及石斑鱼高密度养殖效果。启动初期,将硝化型生物絮团与海绵填料混合培养,生物膜22 d即可挂膜成功。以30.03 kg/m~3为初始养殖密度开展石斑鱼养殖试验,经66 d养殖,石斑鱼平均质量从(273.00±12.22)增至(552.52±107.04) g,最终养殖密度达到60.78 kg/m~3,成活率为100%。养殖过程中,生物膜逐渐适应养殖环境,氨氮、亚硝酸盐氮去除率从13.33%、14.84%增至93.73%、93.50%。此外,在弧形筛进水槽增加曝气形成曝气式弧形筛,可进一步除去细小颗粒物,有效控制养殖水体浊度。     

Identification,characterization and full-length sequence analysis of a novel endornavirus in common sunflower (Helianthus annuus L.)

To identify the possible quarantine viruses in seven common sunflower varieties imported from the United States of America and the Netherlands, we tested total RNAs extracted from the leaf tissues using next-generation sequencing of small RNAs. After analysis of small RNA sequencing data, no any quarantine virus was found, but a double-stranded RNA(dsRNA) molecule showing typical genomic features of endornavirus was detected in two varieties, X3939 and SH1108. Full-length sequence and phylogenetic analysis showed that it is a novel endornavirus, temporarily named as Helianthus annuus alphaendornavirus(HaEV). Its full genome corresponds to a 14 662-bp dsRNA segment, including a 21-nt 5′ untranslated region(UTR), 3' UTR ending with the unique sequence CCCCCCCC and lacking a poly(A) tail. An open reading frame(ORF) that encodes a deduced 4 867 amino acids(aa) polyprotein with three domains: RdRP, Hel and UGT(UDP-glycosyltransferase). HaEV mainly distributed in the cytoplasm but less in the nucleus of leaf cells by fluorescence in situ hybridization(FISH) experiment. This virus has a high seed infection rate in the five varieties, X3907, X3939, A231, SH1108 and SR1320. To our knowledge, this is the first report about the virus of the family Endornaviridae in the common sunflower.     

A genetic linkage map in southern‐by‐spring oat identifies multiple quantitative trait loci for adaptation and rust resistance

We developed 178 recombinant inbred lines from a southern‐by‐spring oat population designated as “TxH.” These lines were genotyped to generate a high‐quality linkage map that resolved 6,902 markers into 21 linkage groups that matched closely with the latest hexaploid oat consensus map. Three major quantitative trait loci (QTLs) affecting heading date were found in locations that are consistent with known QTLs and candidate genes, and two other QTLs affecting heading date were found in novel locations. Five QTLs affecting plant height were found. Both sets of QTLs are responsible for transgressive segregation observed for these two traits. Four QTLs affecting resistance to crown rust, caused by the pathogen Puccinia coronata f. sp. avenae, were identified. Two of these QTLs are consistent with known clusters of rust resistance genes, while two may represent new locations of novel rust resistance genes. A complete set of SNP sequences suitable for generating markers for molecular selection is provided.     

鸭瘟病毒的PCR检测及其产物分析

根据基因库中已有的鸭瘟 Ul6的序列 ,合成一对引物 ,对 3株鸭瘟病毒进行 PCR扩增 ,均扩增出大小约 42 0 bp的特异性片段。进行测序 ,结果表明 3个毒株扩增后的片段均为 42 1bp。经重复实验后确定最佳反应条件 ,按该条件对两例临床病料进行检测 ,结果均出现大小约为 42 0 bp的特异片段。PCR产物与 L ake Andes株的相关序列比较 ,广东毒株与 L ake Andes株同源性较高     

T7 RNA多聚酶基因的克隆、序列测定及其在E.coli中的表达

从BL21(DE3)E．coli菌株中以PCR的方法扩增得到了与T7RNA多聚酶(T7RNApolymerase，T7pol)基因大小一致的DNA片断。将PCR产物纯化后直接克隆到pGEM—T载体中，经酶切鉴定和DNA序列分析表明克隆得到了正确的T7pol基因。将T7pol基因亚克隆入pET-28b( )中，构建得到原核表达质粒pET28T7。该质粒的BL21(DE3)pLysS转化菌在IPTG的诱导下可表达约98800的蛋白，这与T7pol的相对分子质量一致。将该质粒转化DH5α、JMl09、HBl01、BL21(DE3)和BL21(DE3)pLysS等5种不同的宿主菌，仅有转化T7pol酶活性受到抑制的宿主菌BL21(DE3)pLysS才能得到转化子，而其余4种T7T7pol酶活性不受抑制的E．coli宿主菌不能得到转化子。pET28T7原核表达质粒这种仅能在T7pol酶活性受到抑制的宿主菌中才能存活的现象说明本试验所克隆的T7pol基因能正确表达出具有RNA转录酶活性的蛋白。     

The RNA interference

The phenomenon of RNA interference (RNAi) is highly conserved mechanism in the organism evolution. As a immune system, RNAi is a ubiquitous mechanism against invading microorganism in plant and animal cells. Recently, it has been found that RNAi is the process by which double-strand RNA(dsRNA) directs sequence-specific degradation of messenger RNA and the mediations of sequence specific messenger RNA degradation are 21-and 23-nucleotide small interfering RNAs that generate by ribonuclease from endogenous longer dsRNA or by transfectious technics from heterologous dsRNA. Over the past few years, the way in which cells respond to dsRNA by silencing homologous genes has revealed a new regulating paradigm in biology.     

VSV的RT-PCR快速检测方法的建立

选取水泡性口炎病毒 N基因序列 ,设计 1对引物 ,建立检测水泡性口炎病毒的 RT- PCR方法。对VSV各毒株进行检测 ,结果均为阳性 ,而对反刍动物病毒性疾病相关病毒进行检测 ,结果均为阴性。结果表明所建立的 RT- PCR技术可用于水泡性口炎的诊断和流行病学调查