桑属ITS,TrnL-F,rps16序列分析及12个特殊桑资源评鉴

 【目的】分析桑属ITS,TrnL-F,rps16序列,探讨系统学价值,建立系统进化树,并对12个特殊桑资源进行评鉴,为开发利用提供理论依据。【方法】用67份桑资源,经DNA提取、PCR扩增、测序,测序结果用软件拼接、比对、除去非序列碱基,计算长度、G+C%含量、变异位点、信息位点、以构树、柘树为外类群,MP分析,根据分析结果,结合桑资源研究积累,对12个特殊桑资源进行评鉴。【结果】桑ITS(包括5.8S)基本序列576 bp ,变异范围576-590 bp,G+C% 59.55-62.25,40个信息位点;TrnL-F（包括tRNA-Leu内含子）基本序列923 bp , 变异范围920-924 bp, G+C%33.69-34.13,23个信息位点;rps16内含子基本序列929 bp ,变异范围923-929 bp,G+C%32.51-32.72,17个信息位点。最适碱基进化模型分别为(GTR+G),(GTR),(GTR+G),可能的分支概率,混合卡方概率值分别为0.000001 ,0.027175,0.000222, 三片段合并最适进化模型(GTR+G),基于模型,MP分析, 2538个位点,80个信息位点。分支图首先将黑桑M. nigra分出,其它桑种分成两支,第一支桑(mulberry),包括长穗桑、瑞穗桑、蒙桑、鬼桑、鸡桑、山桑、白桑、广东桑,第二支乔木桑(arbor),包括华桑、奶桑、川桑。12个特殊桑资源,神农华桑♀（优质木材桑）分在第二支,乔木特性,其它11个特殊桑资源分在第一支,具桑(mulberry)特性。【结论】桑属TrnL-F;rps16序列信息位点有限,单独研究桑属系统学,价值不大,与ITS序列合并,能增加分支图的信息位点,使分支图更具客观性,更近于似然。基于ITS,TrnL-F;rps16序列MP分支图,新疆黑桑为最原始类型,乌克兰等栽培种是进化类型。桑属可根据分支的亲缘关系,结合桑资源研究积累,评鉴特殊桑资源。     

腐烂茎线虫种内群体特异性检测研究

对腐烂茎线虫(Ditylenchus destructor Thorne)7个不同地理群体rDNA 中的ITS区进行PCR-RFLP和序列测定, 发现种内群体间无论在序列长度还是酶切图谱均存在一定的变异.根据其序列特征设计出种特异性引物, 对所供试的腐烂茎线虫群体及属间不同种群体样品的rDNA ITS区相应区段扩增以验证其特异性, 同时对分别由1～5条腐烂茎线虫成虫和幼虫提取的DNA进行特异性扩增以验证其灵敏度. 结果表明: 所有供试的腐烂茎线虫群体均可以特异性扩增一个346 bp的片段, 而所有供试的属外种均没有扩增片段;由1～5条腐烂茎线虫成虫和4～5条幼虫进行的特异性扩增也获得稳定的目的片段. 显示该特异性引物具有较高的稳定性、特异性和灵敏度, 能快速、准确地检测出腐烂茎线虫.     

中草药青蒿(Artemisia annua L.)的实时荧光PCR检测

中草药青蒿,学名黄花蒿(Artem isia annuaL.),属于中国独有的抗疟药用植物资源之一。根据蒿属植物rDNA ITS序列多态性设计TaqM an探针及引物,通过实时荧光PCR方法对黄花蒿进行检测。结果表明:该组探针-引物对黄花蒿有很强的特异性,除黄花蒿外,其余8种对照蒿属材料均未检测到荧光信号。该方法快速、简便、安全、准确,适用于黄花蒿的出入境检验。     

3种鳢属鱼类ITS序列及系统进化分析

[目的]阐明乌鳢、斑鳢、月鳢核基因序列(ITS)的遗传变异情况,并用ITS序列研究鳢属系统进化关系.[方法]利用PCR扩增3种鳢的ITS,经克隆、拼接得到ITS全长后进行分析.[结果]乌鳢、斑鳢和月鳢ITS全序列长度为902、927、902或903bp.3种鳢ITS的G+C含量较高,约占72％.3种鳢之间核苷酸差异明显大于种内,明显的差异性ITS片段可以鉴别3种鳢.采用NJ和ML方法建立的进化树都表明乌鳢和斑鳢的遗传距离最近,而乌鳢和月鳢遗传距离最远.[结论]该研究为鳢属的分类鉴定、系统进化关系以及种间杂交提供基础资料.     

基于Smad 7基因为靶序列的RNA干涉作用研究

为了探讨小干涉RNA(siRNA)对靶基因Smad7mRNA表达的阻断作用,采用体外转录方法制备Smad7基因的小干涉RNAs(siRNAs),用脂质体介导瞬时转染BERP35T 2肺癌细胞系,并采用Northernblot杂交检测靶基因mRNA的表达丰度。结果表明,根据Smad7编码区序列在体外成功地制备了针对2个不同靶序列的siRNAs;Northernblot杂交显示,在转染siRNA的BERP35T 2细胞中,不管是内源性的还是外源性的,Smad7mRNA的表达丰度均明显下降。说明Smad7基因编码区中542563bp及701722bp2个区域均是siRNA作用的有效靶序列,本研究设计并制备的siRNAs能有效抑制Smad7基因的表达。     

凡纳滨对虾微卫星DNA的筛选及其特性的研究

采用小片段克隆法构建凡纳滨对虾的部分基因文库，用放射性同位素[y-^12P]ATP标记的（CA）15（AT）12（AG）12、（AAT）8、（AAG）8做探针筛选阳性克隆。共获得微卫星序列152个，其中二核苷酸为核心的微卫星序列：（AG）a〉（AC）a〉（AT）a，三核苷酸为核心的微卫星序列：（AAT）a〉（CAT）a〉（AAG）a应用引物设计软件primer3．0设计引物105对。选择合成10对理论上易出现影子带的引物，筛选后用31个凡纳滨对虾个体对这些引物进行了评估，结果10对引物中有8对引物能扩增出谱带，其中1对扩增产物出现影子带，1对扩增产物为单态．有6对扩增产物出现多态。     

烟粉虱复合种不同地理种群的遗传分化

烟粉虱Bemisia tabaci(Gennadius) 是由具有遗传分化的不同地理种群组成的复合种。本文介绍了烟粉虱不同地理种群遗传分化的最新研究进展，并在世界各国对烟粉虱核糖体ITS1(rDNA ITS1)、线粒体COI（mtDNA COI）基因大量测序的基础上，进一步分析了烟粉虱不同地理种群的遗传分化。根据mtDNA COI和rDNA ITS1基因序列分析的结果，烟粉虱不同地理种群可分为5组，即亚洲组（Asia group）、美洲组（America group）、非洲组（Africa group）、澳洲组(Australia group)、B型/地中海/中东/北非/Ms型组(Biotype B/Mediterranean/Middle Eastern/Northern Africa/Biotype Ms group)；此外，还包括3个没有特定组的种群，即乌干达（Uganda）、科特迪瓦（Ivory Coast和台湾（Taiwan）种群。地理隔离可能是造成烟粉虱不同地理种群遗传分化的最重要驱动力。许多具有入侵性或生物学优势的烟粉虱种群随着人类活动而传入新的地区，造成了严重的经济损失。有必要加强烟粉虱生物型的监测，遏制已入侵烟粉虱种群的蔓延，防止新的具有潜在危险性的烟粉虱种群传入中国。     

Two- and three-domain bacterial laccase-like genes are present in drained peat soils

Laccases of fungal origin have been intensively studied due to their importance in various biotechnological applications. There is a constant demand for new laccases with improved properties such as stability at higher temperatures or at an alkaline pH. Growing molecular evidence suggests that laccases may also be widespread in bacteria. While only a handful of bacterial laccases have been purified and characterized, several novel traits have already been discovered (e.g. pH-stability and 2-domain organization of the enzyme as opposed to the usual 3-domain structure of fungal laccases). The aim of this study was to examine the diversity of bacterial laccase-like genes in two types of high-organic peat soil using a cloning and sequencing approach. Gene libraries prepared of small fragments (150 base pairs) revealed an amazing diversity of bacterial laccases. The fragments clustered in 11 major lineages, and one third of the 241 sequences resembled laccase-like genes of Acidobacteria. Additionally, a new primer was used to retrieve several larger fragments of the putative bacterial laccase genes that spanned all four copper-binding sites. Both “conventional” 3-domain laccases and the recently described 2-domain small laccases have been obtained using this approach, demonstrating the potential of the primer. The present study thus contributes to the understanding of the diversity of bacterial laccases and provides a new tool for finding laccase-like sequences in bacterial strains and soil samples.     

利用ITS序列和trnL-F序列探讨赖草属6个物种的系统发育关系

本研究克隆了赖草属6个物种Leymus karelinii,L.angustus,L.racemosus,L.arenarius,L.triticoides和L.ambiguus的ITS序列和trnL-F序列,并选用3个新麦属物种的ITS序列和27个小麦族二倍体物种的trnL-F序列,均以Bromus catharticus为外类群,用最大简约法构建系统发育树。结果发现:不同组的赖草属物种分别聚在一起;赖草属植物与新麦草属植物亲缘关系较近;新麦草属植物是这6个赖草属物种的母本来源。     

RNA- and DNA-based profiling of soil fungal communities in a native Australian eucalypt forest and adjacent Pinus elliotti plantation

Total and active soil fungal communities in a native eucalypt forest and first rotation Pinus elliotti plantation were investigated by direct extraction of DNA and RNA from soil. Terminal restriction fragment length polymorphism (T-RFLP) analysis of internal transcribed spacer (ITS) and 18S rRNA profiles indicated that total and active fungal communities differed significantly in both forest types. This was supported by DGGE profile analysis on an individual plot basis for both forest types and when groups in the canonical analysis were redefined to allow comparison between forest types. Analyses of both ITS and 18S T-RFLP profiles indicated that conversion from native eucalypt forest to P. elliottii plantation may significantly alter total and active soil fungal communities. ITS DGGE (DNA) and 18S (RNA) profiles also suggested differences in fungal communities in the two forest types. No significant separation of the fungal communities in the two forest types was observed, however, when ITS DGGE (RNA) profiles were compared. Overall, the data suggest that conversion from native eucalypt forest to P. elliottii plantation at the Beerburrum State Forest in subtropical Australia has significantly altered soil fungal communities.