油菜(Brassica napus)Bna-miR1140基因启动子miR1140Pro的克隆与表达模式研究北大核心CSCD

旨在研究Bna-miR1140的表达模式及其调控机理,依据本实验室前期的油菜miRNA芯片实验结果,从油菜栽培种Westar中克隆了Bna-miR1140前体序列上游1.5 kb的片段,并进行了顺式作用元件分析,然后构建了GUS报告基因植物表达载体,通过农杆菌介导法将miR1140 pro∷GUS转化到甘蓝型油菜Westar品种中,经PCR法鉴定获得5株阳性株。对阳性株油菜T1代各组织的GUS化学染色分析表明,miR1140前体序列上游1.5 kb区域具有启动子的功能,能够驱动GUS在油菜中表达,并且GUS基因仅在叶柄及叶腋中特异性表达,说明油菜miR1140Pro为特异性启动子。     

油菜(Brassica napus)Bna-miR1140基因启动子miR1140Pro的克隆与表达模式研究

旨在研究Bna-miR1140的表达模式及其调控机理,依据本实验室前期的油菜miRNA芯片实验结果,从油菜栽培种Westar中克隆了Bna-miR1140前体序列上游1.5 kb的片段,并进行了顺式作用元件分析,然后构建了GUS报告基因植物表达载体,通过农杆菌介导法将miR1140 pro∷GUS转化到甘蓝型油菜Westar品种中,经PCR法鉴定获得5株阳性株。对阳性株油菜T1代各组织的GUS化学染色分析表明,miR1140前体序列上游1.5 kb区域具有启动子的功能,能够驱动GUS在油菜中表达,并且GUS基因仅在叶柄及叶腋中特异性表达,说明油菜miR1140Pro为特异性启动子。     

沙柳SpsLAS基因超表达对拟南芥营养生长的影响

用沙柳SpsLAS基因构建35S∷SpsLAS超表达载体并转化野生型拟南芥,对转基因拟南芥进行表型观察,利用荧光定量PCR,对分枝、生长素及细胞分裂素相关基因进行表达分析。结果显示:(1)成功构建35S∷SpsLAS超表达载体,并获得9株纯合转基因株系,且转基因株系的萌芽速率快于野生型(对照),生活周期也较长;其中7个株系表现为生长迅速、株高增加、莲座叶叶片增大、分枝增加,2个株系表现为矮化、分枝增加、育性降低等一系列变化。(2)荧光定量PCR显示,与对照相比24h时转基因株系幼苗生长素及细胞分裂素途径关键基因无明显变化,4d时各基因在各转基因株系呈上调趋势;30d时分枝相关基因RAX1、RAX3表达量均上调,而MAX1、MAX3、REV、AXR1无明显变化。研究表明,SpsLAS基因过表达对拟南芥株型、莲座叶有明显影响,该研究结果为进一步研究该基因对分枝调控机制奠定了基础。     

拟南芥株型突变体zpr1的性状特征与基因鉴定分析

对本研究室经T-DNA插入法获得的拟南芥株型突变株系——隐性突变体zpr1植株进行植物学性状调查和遗传分析,并对该突变基因进行鉴定、表达定位和调控元件分析。结果显示:(1)性状分析表明,与野生型拟南芥Ws-2相比,突变体zpr1的茎生叶分枝数量增加,茎生叶分枝发生于拟南芥顶端花序部位;野生型拟南芥茎生叶为披针形,而突变体zpr1没有出现分枝的茎生叶呈倒卵形,出现分枝的茎生叶呈披针型;突变体zpr1的主花序高度、株高、分枝高度和分枝长度都高于野生型,且分枝数多于野生型。(2)利用质粒挽救和反向PCR法(IPCR)确定了ZPR1基因突变发生位置是该基因起始密码子上游426bp处,证明T-DNA插入破坏了ZPR1基因的启动子区域,导致该基因在拟南芥内不能正常表达。(3)基因转录调控区域的顺式作用元件分析发现在ZPR1基因的转录调控区有多个与植物激素相关的调控元件,还有与光周期调节相关的调控元件。(4)亚细胞定位发现,ZPR1基因在所有细胞中的细胞膜中表达,而在部分细胞的细胞膜、细胞质和细胞核中均有表达。研究表明,ZPR1基因的表达对植物株型发育有重要的调控作用,该基因的表达水平受植物激素和光照的调节,最终导致了植物株型的变化。     

适宜河北省旱寒区的冬油菜品种的筛选

筛选适宜河北省低平原地区种植的油菜品种,同时实现生态效益和经济效益,为相似气候区油菜品种的种植选择及种植结构的调整提供理论依据。以16个油菜品种为材料,对品种间农艺性状的变化进行方差分析、相关性分析和聚类分析。各品种间分枝部位存在差异,变异系数为61.82%,品种JR5分枝部位最低,14T×38最高;其次差异较为明显的是产量和二次分枝数,变异系数分别为26.69%和24.89%,14T×38产量最高,20SY-13最低。二次分枝数最大和最小的品种分别为20SY-13和天油142。油菜产量与一次分枝数和主花序角果数呈极显著正相关(P<0.01),株高与分枝部位、主花序长度和主花序角果数呈显著正相关关系,一次分枝数与主花序角果数呈显著正相关(P<0.05),二次分枝数与主花序角果数呈极显著负相关。当欧式距离为5时,16个油菜品种可分为五大类。选择株高较高、一次分枝数和主花序角果数较多、二次分枝数较少且可以在当地正常越冬的油菜品种进行种植,可以获得较高水平产量;20SY-13在当地秋季种植应注意安全越冬。     

烟草microRNA171c的功能分析

MicroRNA是一类长度为20~24碱基的非编码小RNA,调控植物多种生理代谢途径。MicroRNA171(miR171)在拟南芥、大麦和水稻中通过负调控SCL靶基因使植物表现出分枝结构变化和其他一些发育表型,还可调控拟南芥叶绿素的合成代谢。但其他植物miR171的功能仍然未知。为了探明烟草miR171c的功能,本研究根据烟草miR171c序列设计了靶基因模拟物STTM171,通过病毒表达载体在烟草中进行表达,抑制miR171c的活性后观察植物表型变化。结果表明在病毒表达STTM171烟草中,植株出现顶端优势丧失、茎干增多等表型。荧光定量PCR检测到STTM171过表达植株miR171c表达量下降,两个推测的SCL靶基因TC134811和TC127385表达量上升,表明miR171c可能在烟草和拟南芥等植物中的功能比较保守,可以通过调控可能的靶基因SCL来调节植物的生长发育。     

过表达AhAREB1影响GA代谢调控拟南芥的株型

与野生型相比,过表达AhAREB1的拟南芥植株茎节间的生长受抑制,花序轴短和分枝数多。普遍认为植株的矮化可能与植物体内促进生长的激素作用的异常有关。本文测定了过表达AhAREB1植株体内激素GA3、IAA和ABA含量,并通过外施ABA、IAA和GA3观察植株表型变化及检测相关基因表达,研究AhAREB1在植物株型方面的作用。结果表明,与野生型植株相比,过表达AhAREB1拟南芥植株内源ABA、IAA和GA含量均升高,外施GA3可使植株的表型恢复至野生型水平,推测AhAREB1通过影响GA代谢失活及降低其生理活性水平而引起株型矮化。     

反义磷脂酶Dγ基因转化小麦的研究

目的:获得抗寒、耐盐等能力增强的小麦新种质或新品种。方法:利用穗茎注射法将含有反义磷脂酶Dγ基因(PLDγ)的根癌农杆菌菌液注入到春小麦富硒乌麦旗叶鞘内,T1代经田间表型选择筛选到10株变异株,通过特异PCR扩增和Southern杂交技术对获得的变异株进行分子检测,并对转基因植株进行耐盐性鉴定。结果:变异株均扩增出大小为430bp的目的片段,Southern印迹结果与其一致,证实反义PLDγ基因已经整合到小麦的核基因组中;6株转基因植株的抗寒力比对照显著增强,4株的熟期提早3～4d;在室内耐盐性筛选中,2株变异植株耐NaCl的能力比对照有不同程度的提高。结论:反义PLDγ基因在提高小麦的抗寒、耐盐性上具有很大的应用价值。     

肺炎克雷伯菌耐氨基糖苷类抗生素基因aac（3）-I的改构与功能

研究氨基糖苷乙酰转移酶基因aac(3)-I碱基变异与功能的关系。构建表达载体pET28a-aac(3)-I,转化感受态细胞E.coli BL21(DE3),筛选到阳性克隆送测序。然后在体外对aac(3)-I基因进行易错PCR改造,筛选到一株阳性克隆测序后发现7处碱基变异,其中C272T、T373C引发了氨基酸的变异,分别对应:Ser91Phe、Tyr125His。以琼脂稀释法检测不同克隆的最低抑菌浓度(minimum inhibitory concentration,MIC)。实验结果表明变异株MIC较突变前下降64倍(庆大霉素)、4倍(阿米卡星)、8倍(依替米星)。为进一步了解和阐明耐药酶AAC(3)-I与底物的作用机制打下基础。     

番茄红素β-环化酶基因的玉米转化及 后代遗传分析

利用农杆菌介导法将番茄红素β-环化酶基因(Lycb)转入由玉米自交系天塔五号植株,分析基因在T0转化及后代的遗传情况,结果表明,在27株T0转基因植株中,PCR初步检测后8株呈阳性;将T1代转基因植株以株系为单位用200mg/L草铵膦抗性筛选后,收获抗性植株种子。T2代转基因植株进一步进行PCR、RT-PCR和田间草铵膦涂抹检测,结果表明,PCR、RT-PCR为阳性的6个株系植株均具有草铵膦抗性。选取6株阳性植株提取叶片总类胡萝卜素,经HPLC分析其β-胡萝卜素含量显著高于野生型,表明目的基因Lycb成功的转入玉米,并得到了稳定遗传。     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

Chemical composition, in situ ruminal degradability and post-ruminal disappearance of dry matter and crude protein from the halophytic plants Kochia scoparia, Atriplex dimorphostegia, Suaeda arcuata and Gamanthus gamacarpus

Samples of Kochia (K. scoparia), Atriplex (A. dimorphostegia), Suaeda (S. arcuata) and Gamanthus (G. gamacarpus) were collected and analyzed for chemical composition including crude protein (CP), ether extract (EE), ash, neutral detergent fiber (NDFom), acid detergent fiber (ADFom), non-protein N (NPN), Ca, P, Na, K, Cl, Mg, Fe, Cu and Se. In addition, in situ ruminal degradability and post-ruminal disappearance of dry matter (DM) and CP of the samples using a mobile bag technique were determined. Results indicate that the chemical composition of Kochia and Atriplex was notably different from those of Suaeda and Gamanthus. All of these halophytic plants had high concentrations of Na, K, Cl, Cu and Se, and low levels of Ca, P and Mg. The rapidly degradable fractions of DM and CP (g/g) of Kochia (0.31 and 0.35, respectively) and Atriplex (0.39 and 0.50, respectively) were lower than for Suaeda (0.53 and 0.55, respectively) and Gamanthus (0.56 and 0.66, respectively). Ruminal DM and CP disappearance of Kochia (444 and 517 g/kg, respectively) and Atriplex (472 and 529 g/kg, respectively) were lower (P<0.05) than those of Suaeda (553 and 577 g/kg, respectively) and Gamanthus (663 and 677 g/kg, respectively) (P<0.05) using the mobile bag technique. Suaeda had the lowest (P<0.05) NDFom and ADFom disappearance (214 and 232 g/kg, respectively) in the rumen. Kochia scoparia and Atriplex dimorphostegia have more beneficial chemical nutritive components and digestible values versus Suaeda arcuata and Gamanthus gamacarpus.     

Flavonoids of Astilbe and Rodgersia compared to Aruncus

The flavonoid profiles of Astilbe (four taxa studied) and Rodgersia (two taxa studied) are based on simple flavonol glycosides. Astilbe has 3-O-mono-, 3-O-di-, and 3-O-triglycosides of kaempferol, quercetin, and myricetin, while Rodgersia has only mono- and diglycosides of kaempferol and quercetin. Astilbe×arendsii was also shown to accumulate dihydrochalcone glycosides. The flavonoid profile of Rodgersia is the simplest recorded so far in the herbaceous Saxifragaceae. The flavonoids of two species of Aruncus were shown to be based upon kaempferol and quercetin 3-O-mono- and 3-O-diglycosides. One of the species also exhibited an eriodictyol glycoside. The triglycoside differences were not considered important, but the differences in myricetin occurrences were taken as evidence against derivation of Saxifragaceae from an Aruncus-like ancestor. Should such an event be proposed, however, serious consideration would have to be given to the current pattern of myricetin occurrence in the two families.     

Multiple invasions of Gypsy and Micropia retroelements in genus Zaprionus and melanogaster subgroup of the genus Drosophila

Background  

The Zaprionus genus shares evolutionary features with the melanogaster subgroup, such as space and time of origin. Although little information about the transposable element content in the Zaprionus genus had been accumulated, some of their elements appear to be more closely related with those of the melanogaster subgroup, indicating that these two groups of species were involved in horizontal transfer events during their evolution. Among these elements, the Gypsy and the Micropia retroelements were chosen for screening in seven species of the two Zaprionus subgenera, Anaprionus and Zaprionus.     

百合LbCAT和LbGPX基因的克隆与表达分析

以切花百合(Lilium brownii var. viridulum)‘卡瓦纳’cDNA为模板,克隆了过氧化氢酶(LbCAT)和谷胱甘肽过氧化物酶(LbGPX)基因。序列分析表明,这2个基因分别包含1 479 bp和519 bp的开放阅读框(ORF),编码492个和172个氨基酸。进化分析结果表明,LbCAT蛋白与岷江百合CAT蛋白的氨基酸序列相似性最高(99.19%),且亲缘关系最近;LbGPX蛋白与油棕GPX蛋白的氨基酸序列相似性最高(78.61%),亲缘关系最近。qRT PCR结果显示,LbCAT和LbGPX在百合根、鳞茎、叶和花中都有表达。LbCAT在叶中表达量最高,LbGPX在花中表达量最高。这2个基因在百合花蕾的生长发育过程中均有表达,且表达量逐渐增加;在PEG处理后2个基因的转录水平升高,但独角金内酯(SLs)处理却显著降低了这2个基因的转录水平;该结果为百合抗逆性机理研究以及抗逆育种奠定了基础。     

Quinua and Relatives (Chenopodium sect.Chenopodium subsect.Celluloid)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

Cloning and characterization of femA and femB from Staphylococcus epidermidis

A DNA fragment was identified and cloned from Staphylococcus epidermidis (Se) using femA from S. aureus (Sa) as a heterologous hybridization probe. DNA sequence analysis of a portion of this clone revealed two complete ORFs highly related to femA and femB of Sa. The genomic arrangement of the Se femA/B complex was nearly identical to that observed in Sa. Intra- and interspecies relatedness of these genes and conservation of genomic organization were consistent with gene duplication of one of these genes in an ancestral organism. Recombinant FEMA, produced in Escherichia coli (Ec), was purified to near homogeneity. Identity of the purified protein was verified by N-terminal amino acid (aa) sequence analysis.