檀香SaRbohA基因的克隆与吸器诱导因子响应分析

植物Rboh基因家族编码产生活性氧(ROS)的NADPH氧化酶。了解半寄生植物檀香(Santalum album Linn.)基因Rboh相关信息和表达特性,可为研究檀香SaRbohA基因通过活性氧信号(ROS)调控吸器发育的响应提供理论依据。该研究以全长转录组测序为基础,通过序列拼接设计合成基因特异引物,从根中克隆获得了1个檀香respiratory burst oxidase homolog(Rboh)基因cDNA全长,命名为SaRbohA。序列分析表明,该基因cDNA全长2 790 bp,编码929个氨基酸,分子量105.37 kD,理论等电点9.13,预测亚细胞定位于细胞膜。结构预测表明,SaRbohA具有6个跨膜结构域,在膜内侧的C端包含典型的NADPH结合结构域和FAD结合结构域, N端含有两个EF手性结构。序列比对分析表明,檀香SaRbohA与苹果MdRboh同源进化关系较近,相似度为63.65%。组织特异性表达分析表明,SaRbohA基因在茎中表达量最低,幼叶和茎尖中表达量较高,而在根中表达量最高。采用2, 6-二甲氧基对苯醌(寄生植物吸器诱导因子)处理,可以强烈诱导檀香SaRbohA基因的响应并伴随大量活性氧信号。研究推测,SaRbohA基因的在ROS信号介导的檀香吸器发育过程中起重要作用,且受化学诱导因子调控表达。     

Characteristics of the microbial community in rhizosphere of Camptotheca acuminata cultured with exotic invasive plant Eupatorium adenophorum

The traditional culture-dependent plate counting and culture-independent small-subunit-ribosomal RNA gene-targeted molecular techniques, Single-Strand Conformation Polymorphism (SSCP) and ter-minal Restriction Fragment Length Polymorphism (tRFLP) combined with 16S rDNA clone library were adopted to investigate the impacts of secretion from Camptotheca acuminata (abbreviated to Ca) roots on the quantities and structure of eukaryotic microbes and bacteria in the rhizosphere, and the possi-bility that Ca controls exotic invasive plant Eupatorium adenophorum (Ea). The counting results indi-cated that the number of bacteria increased in turn in rhizospheres of Ea, Ca-Ea mixed culture and Ca, while that of eukaryotic microbes decreased. PCR-SSCP profiles showed eukaryotic microbial bands (corresponding to biodiversity) in rhizosphere of Ea were more complex than those of Ca and CE. Meristolohmannia sp., Termitomyces sp. and Rhodophyllus sp. were the dominant populations in the rhizosphere of Ca. Bacterial terminal restriction fragments (TRFs) profiles showed no difference among three kinds of rhizospheres, and the sequences of the 16S rDNA clone library from Ca rhizospheres were distributed in 10 known phyla, in which phylum Proteobacteria were the absolute dominant group and accounted for 24.71% of the cloned sequences (δ-Proteobacteria accounted for up to 17.65%), and phyla Acidobacteria and Bacteroidetes accounted for 16.47% and 10.59% of the cloned sequences, respectively. In addition, high performance liquid chromatography detected a trace amount of camp-tothecin and hydroxycamptothecin in the rhizospheric soil of Ca and CE, but examined neither camp-tothecin nor hydroxycamptothecin in rhizospheric soil of Ea. Therefore, invasion and diffusion of Ea evidently depended on distinguishing the eukaryotic community structure, but not on that of the bac-terial pattern. Ca was able to alter the eukaryotic community structure of invasive Ea by secreting camptothecin and hydroxycamptothecin into rhizospheres, and may benefit the control of overspread of Ea. This study provided theoretical evidence for rhizospheric microbial aspects on substituting Ca for Ea.     

楸树CbuATX1，CbuATX1-like和CbuATX2基因克隆及生物信息学分析

开花是高等植物内部遗传因素和外界环境因素共同调节完成的复杂生命过程,是植物进入生殖生长从而具备遗传能力和繁殖能力的象征。使用PCR技术,以楸树（Catalpa bungei）混合芽cDNA为模板分别克隆CbuATX1,CbuATX1-like和CbuATX2 3个基因,并利用相关生物信息学软件对这3个基因编码的蛋白质结构进行预测,同时对基因的启动子序列进行顺式作用元件分析,通过qRT-PCR技术检测3个基因在楸树混合芽不同发育时期的表达量。结果表明：CbuATX1的CDS全长为726 bp,编码241个氨基酸,存在跨膜运输结构,属于HMA蛋白家族,与芝麻（Sesamum indicum）、甜菜（Beta vulgaris）亲缘关系较近。CbuATX1-like的CDS全长为801 bp,编码266个氨基酸,存在跨膜运输结构,属于HMA蛋白家族,与胡萝卜（Daucus carota）、欧洲橄榄（Olea europaea）亲缘关系较近。CbuATX2的CDS全长为1 554 bp,编码517个氨基酸,不存在跨膜运输结构,属于PWWP蛋白家族,与马尾草（Erythranthe guttatus）亲缘关系较近。这3个基因启动子均含有多个真核生物启动子的基本元件,如CAAT-box和TATA-box等,此外,还含有光响应、低温响应、生长素响应,以及与干旱诱导相关的MYB结合位点等3个基因与光响应密切相关,还可能参与外界环境胁迫响应等过程。qRT-PCR结果显示,上述3个基因在1年生普通楸树无性系9-1和突变株系-百日花楸树不同发育时期的表达量呈现显著差异。通过以上研究以期能够进一步阐述百日花楸树开花的性状,为楸树开花机制的研究和楸树的定向遗传改良提供理论支持。     

桃Pp4CL2基因的克隆及抗寒功能验证

基于‘丁家坝李光桃’抗寒转录组数据,采用RT PCR技术克隆桃4 香豆酸辅酶A连接酶基因(Pp4CL2),并对其进行生物信息学及转化模式植物拟南芥和烟草的抗寒分析,以解析‘丁家坝李光桃’的抗寒机制。结果显示：(1)成功克隆获得桃Pp4CL2基因(登录号：LOC18792923),其cDNA序列为1 635 bp,编码544个氨基酸残基,具有4CL基因家族保守结构域。(2)二级结构分析显示,Pp4CL2蛋白由4种状态的二级结构组成,其中α螺旋占30.51%、β 折叠占7.35%、不规则卷曲及延伸链分别占41.54%和20.59%。(3)顺式作用元件分析发现,Pp4CL2基因上游启动子区含有光、低温以及多种激素响应元件。(4)序列系统进化分析显示,桃Pp4CL2与杏(Prunus armeniaca)、欧洲甜樱桃(Prunus avium)和梅(Prunus mume)的蛋白相似性最高,分别为99.08%、97.98%和96.14%。(5)成功构建转化载体Pp4CL2 pRI101,对拟南芥和烟草进行遗传转化并通过PCR鉴定获得转基因拟南芥和烟草。(6)与野生型相比,低温胁迫下转基因拟南芥和烟草的Pp4CL2基因相对表达量高,受冷害程度轻,具有更高的渗透调节物质含量和抗氧化酶活性,对低温具有更强的耐受性。研究表明,过表达Pp4CL2基因可增强植物对低温的耐受性,推测Pp4CL2基因在‘丁家坝李光桃’响应低温胁迫过程中具有重要作用。     

Real‐time PCR detection of Enterococcus faecalis associated with amyloid arthropathy

Aims: To develop a RT‐PCR method for detection of the multilocus sequence type 82 of Enterococcus faecalis associated with amyloid arthropathy (AA) in layers. Methods and Results: Bacteria were selected from lesions including AA in layers. The primers were designed based on the phosphate ATP binding cassette transporter (pstS) and xanthine phosphoribosyltransferase (xpt) genes and first tested against three isolates with known base pairs at the specific sites. Subsequently, 12 isolates were selected from our collection by one researcher, and RT‐PCR was performed blinded. The sequence type (ST) was then confirmed by multilocus sequence analysis. Two single‐nucleotide polymorphisms in the pstS and xpt genes allowed an unambiguous identification of ST82. As an alternative to DNA extraction, a boiling method for release of DNA from cells was used. Conclusions: The real‐time PCR targeting ST82 enables rapid screening of Ent. faecalis cultured from suspect cases with results available after a few hours, much faster than multilocus sequence typing and pulse field gel electrophoresis. Significance and Impact of the Study: The new method allows a rapid screening of isolates with results available after only few hours. This RT‐PCR method could be a useful tool for molecular epidemiological studies on the spread of arthropathic and amyloidogenic Ent. faecalis within and between birds more efficiently.     

Detection of sequence polymorphisms in red junglefowl and White Leghorn ESTs

Over 16,000 high quality expressed sequence tags (ESTs) from red junglefowl (RJ) and White Leghorn (WL) brain and testis cDNA libraries were generated. Here, we have used this resource for detection of single nucleotide polymorphisms (SNPs), and also completed full-length sequencing of 46 pairs of clones, representing the same gene from both the RJ and WL libraries. From the main set of ESTs, which were assembled using Phrap, 746 putative SNPs were identified, of which 76% were transitions and 24% were transversions. A subset of SNPs was evaluated by sequence analysis of five RJ and five WL birds. Nine of 12 SNPs were verified in this limited sample, suggesting that a majority of the putative polymorphisms documented in this study represent real SNPs. During full-length sequencing of the 46 RJ/WL clones 100 SNPs were identified, which translated to a frequency of 1.90 SNPs/1000 bp. The number of transitions and transversions were 77% and 23%, respectively, and the proportion of non-synonymous vs. synonymous SNPs was 20% and 80%, respectively. Four large insertions/deletions were identified between the RJ and WL full-length sequences, and they appear to represent different splice variants.     

Variations in levels of clonality among Populus nigra L. stands of different ages

The evolution of genotypic diversity with population age remains poorly explored in clonal plant populations despite the potential for important shifts to occur through the course of time. Woody sprouting species are particularly under-represented in studies investigating intra-specific variations in levels of clonality from one locality to the next and through time. In this study we sought to determine the incidence and frequency of replicate genotypes in natural Populus nigra L. (Salicaceae) stands of different ages. Ten stands of this woody riparian sprouting species were selected in each of three distinct age groups (young, middle-aged and old) along a 30 km stretch of the River Garonne (south-west France). Leaf samples were collected from 15 neighbouring trees in each stand (450 samples in total) and replicate genotypes were identified using five SSR markers. Replicate genotypes were identified in two-thirds of all stands sampled (i.e. 50 of young stands, 100 of middle-aged stands and 50% of old stands). Young stands had significantly fewer replicated genotypes than middle-aged or old stands, while middle-aged stands had the greatest number of replicated genotypes. Replicate genotypes were most often found to occur as nearest neighbours and formed relatively small, discrete units (i.e. 2–4 trees growing in close proximity to one another). This suggests that asexual regeneration frequently occurs through flood-training in this species, although asexual regeneration from translocated fragments also evidently occurs as evidenced by 11 cases of replicate genotypes occurring in widely separated stands (up to 19 km apart). The results of this study highlight the need for a hierarchical sampling strategy in space and across age groups for an accureate understanding of the genotypic structure of woody sprouting species populations. Conservation and management of effective population sizes will benefit from better insight into not only spatial, but also temporal variations in levels of genotypic diversity.Co-ordinating editor: J. Tuomi     

Cloning and tissue distribution of the human B3GALT7 gene, a member of the β1,3-Glycosyltransferase family

We report here the cloning and tissue distribution of the human B3GALT7 gene, a member of the beta1,3-Glycosyltransferase family, structurally related to the beta1,3-Galactosyltransferase family and beta1,3- N -acetylglucosaminyltransferase family, isolated from a human lung cDNA library. B3GALT7 is mapped to chromosome 19q13.2 by browsing the UCSC genomic database. It contains an ORF with length of 1191bp, encoding a protein with a signal peptide sequence and galactosyl-T domain, and its molecular weight and isoelectric point is predicted to be 43.3 kDa and 8.67 respectively. The molecular weight of the protein when expressed in E. coli corresponded to that expected. Northern blotting showed that B3GALT7 was highly expressed in lung, throat and ileum, whereas the expression level was low in tongue, breast, uteri, testis. In addition, it was also demonstrated that B3GALT7 is differentially transcribed in human tumor cell lines.     

水葫芦苗(Halerpestes cymbalaris)的生长特征研究

以调查统计的方法在中国科学院海北定位站研究了高寒湿地植物水葫芦苗无性系的生长特征、形态特征以及能量分配规律。结果表明：匍匐茎只有1条的水葫芦苗最多,占35．29％,匍匐茎有4条的水葫芦苗只占8．82％。同一水葫芦苗无性系中,随着匍匐茎数目的增多,分株数、间隔子数、茎总长和匍匐茎比节问重变小。分株一般在第一级最高,末级较低;第1条匍匐茎的间隔子较长。随水葫芦苗匍匐茎数目的增多,用于无性繁殖的分株干重比例逐渐增加,用于有性繁殖的花的干重比例下降。水葫芦苗无性系这种生长特征、形态特征以及能量分配规律是其生物—生态学特性和所处高寒湿地生境共同决定的。     

Mitochondrial DNA heteroplasmy in calves cloned by using adult somatic cell

Adult somatic cell cloned calves were produced by somatic cell nuclear transfer prepared by fusion of cultured ear fibroblast from a Holstein cow into enucleated oocytes of Luxi Yellow cow. In order to determinate the source of mitochondrial DNA of cloned calves, we designed the breed-specific PCR primers by aligning the known D-loop sequences of Bos taurus and analyzed the displacement loop sequences of five live cloned calves by breed-specific primers PCR. The results demonstrated that mtDNA originated from Holstein breed and that from Luxi breed co-exist in all five live calves.