杜梨叶绿体基因组分析

利用高通量测序平台对杜梨（Pyrus betulaefolia）进行测序，并对其叶绿体全基因组序列的结构特征进行分析。结果表明，杜梨叶绿体基因组与大多数高等植物一样，具有典型的环状双链四分体结构。其叶绿体基因组大小为160 058 bp，共检测到61 个SSR 位点（58 个单核苷酸重复和3 个二核苷酸重复）和56 个RNA 编辑位点，这些RNA 编辑位点均引起了氨基酸的变化。与砂梨（Pyrus pyrifolia）、川梨（Pyrus pashia）和桃叶梨（Pyrus spinosa）的叶绿体基因组比较，其基因种类和数量都比较保守，表明了梨属植物进化的缓慢性。在4 个梨属植物中共检测到351 个SNP 和217 个InDel。     

大花君子兰叶绿体基因组及其特征

采用Illumina MiSeq测序平台对大花君子兰（Clivia miniata）叶片总DNA进行测序，通过组装获得了其叶绿体基因组（cpDNA）全长序列（158 114 bp）。对其cpDNA注释得到135个基因，包含87个蛋白编码基因、40个tRNA基因和8个rRNA基因。采用生物信息学方法对获得的cpDNA进行简单序列重复（SSR）分析和密码子偏好性分析。结果显示：①大花君子兰cpDNA中共有61个SSR位点，其中单核苷酸、二核苷酸、三核苷酸、四核苷酸、五核苷酸和六核苷酸重复数分别为38、9、2、8、3和1个，多数SSR分布在基因间隔区；②大花君子兰cpDNA密码子偏爱以A或U（T）结尾，亮氨酸使用频率最高，半胱氨酸使用频率最低。基于24种植物的cpDNA全长和23种植物的叶绿体ycf2基因序列进行系统发育分析，结果显示大花君子兰与石蒜科植物在同一分支，显示最近的亲缘关系，支持大花君子兰属于石蒜科。基于叶绿体ycf2的系统发育分析结果与基于cpDNA全长的系统发育分析研究结果大部分相同，支持ycf2基因可以代替cpDNA全长用于植物系统发育分析。     

芜菁二氢黄酮醇4–还原酶基因的克隆与功能鉴定

二氢黄酮醇4–还原酶（dihydroflavonol 4-reductase,DFR）是花青素生物合成晚期阶段的关键酶,属于NAD/NADP依赖型还原酶家族,催化从二氢黄酮醇转变成无色花色素苷的反应。利用UV-A处理‘津田’芜菁（‘Tsuda’turnip）和‘赤丸’芜菁（‘Yurugi Akamaru’turnip）块根24 h后提取总RNA,通过RT-PCR方法分别克隆了‘津田’芜菁BrDFR1和‘赤丸’芜菁BrDFR2基因。BrDFR1和BrDFR2的开放读码框分别为1 158 bp和999 bp,分别编码385和332个氨基酸。氨基酸序列分析显示,BrDFR1和BrDFR2与大白菜DFR具有高度同源性,从第8到第302位氨基酸的肽段具有FR_SDR_e结构域。BrDFR1和BrDFR2基因组全长序列均含有5个位置与序列完全相同的内含子。Southern杂交结果显示,‘津田’芜菁BrDFR1和‘赤丸’芜菁BrDFR2基因均为单一拷贝。UV-A可以诱导BrDFR1基因表达,对BrDFR2基因表达的诱导效果不明显。BrDFR1和BrDFR2基因在大肠杆菌细胞中可以表达并纯化出分子量约为42.8 kD的BrDFR1蛋白和37.5 kD的BrDFR2蛋白。过量表达BrDFR1和BrDFR2基因的烟草花色加深。芜菁DFR基因的RNAi载体遗传转化烟草后,转基因植株的花色变浅。这些工作将为阐明依光型和非依光型花青素生物合成机理奠定研究基础。     

贵州威宁红花油茶的叶绿体基因组特征分析

对威宁红花油茶（Camellia weiningensis）的叶片高通量测序数据进行叶绿体全基因组的组装和注释分析。其叶绿体全基因组长为156 490 bp,包含典型的四分体结构,序列已登录GenBank（MK820035）。叶绿体全基因组比较分析表明,与其他山茶属物种一样,其结构与基因排序均保守,但在基因组的反向重复区边界处表现出明显区别。简单重复序列（SSR）分析表明,全基因组仅包含51个单核苷酸重复的SSR,且仅为A/T重复类型。系统发育分析表明,威宁红花油茶与腾冲红花油茶亲缘关系最近,但其所处分支包含的4个山茶属物种的分组与传统分类不一致。     

草菇丝/苏氨酸蛋白激酶同源基因的克隆及序列分析

根据担子菌丝/苏氨酸蛋白激酶同源基因编码的氨基酸序列设计两对简并引物,通过巢式简并PCR方法获得草菇(Volvariella volvacea)丝/苏氨酸蛋白激酶同源基因(vv-stpk1)的保守片段,然后通过基因组步行的方法获得了vv-stpk1全长序列。vv-stpk1全长为2 003 bp,含有8个内含子,长度分别为72、57、53、54、45、50、55和48 bp,可编码522个氨基酸残基的多肽。推定的氨基酸序列与玉米黑粉菌(Ustilagomaydis)、木糖发酵酵母(Pichia stipitis)、新型隐球菌(Cryptococcus neoformans)中与细胞形态发生相关的丝/苏氨酸蛋白激酶Orb6的相似性分别为81%、77%和76%,对VV-STPK1蛋白的系统发生学分析的结果表明,VV-STPK1与Orb6同源蛋白聚在同一进化枝上,这些数据都支持VV-STPK1蛋白为与细胞形态发生相关同源蛋白的推定。     

Spacing Experiments on Vegetables: V. The Effect of Spacing and Manuring on the Composition of the Foliage of Shallots and Globe Beet,Considered in Relation to the Growth of the Plants

Summary

A leaf curl disease was observed on croton (Codiaeum variegatum L.), a popular ornamental plant in botanical, home, and office gardens in and around Bengaluru, South India. Diseased plants showed typical symptoms of vein thickening, severe inward curling and a reduction in leaf size, and stunting. The pathogen responsible was transmitted to healthy croton plants by grafting of infected scions, and through the whitefly vector, Bemisia tabaci, suggesting that the disease was caused by a begomovirus. The association of a begomovirus with the disease was further confirmed by the amplification of viral DNA fragments of ca. 520 bp and 575 bp derived from the coat protein (CP) gene of DNA-A using degenerate primers and total DNA extracted from infected, but not from healthy croton plants. The 575 bp fragment corresponding to the core region of the CP gene was cloned and sequenced. Phylogenetic analysis of the core CP sequence grouped the croton-infecting begomovirus, which we tentatively called croton leaf curl virus (CrLCuV), with Ageratum yellow vein virus (AJ810825), with which it shared the highest nucleotide identity (95%). The core CP sequence was similar (90 – 95%) to many other begomoviruses from the Indian sub-continent that infect tomato, tobacco, cotton, and papaya. Thus, its precise taxonomic denomination will require sequencing of the complete ssDNA viral genome.     

糙皮侧耳凝集素基因Plectin的克隆和表达分析

基于cDNA末端快速扩增(RACE)技术克隆了两个糙皮侧耳凝集素基因Plectin1和Plectin2。结构分析显示Plectin1和Plectin2基因全长分别为1 371和1 359 bp,二者均含有5个外显子和4个内含子;Plectin1开放阅读框长1 134 bp,编码377个氨基酸,Plectin2开放阅读框长1 122 bp,编码373个氨基酸。Southern杂交试验证实Plectin1和Plectin2在糙皮侧耳基因组中均只有1个拷贝。利用qRT-PCR分析了Plectin1和Plectin2在糙皮侧耳不同生长阶段的表达量,结果显示Plectin1和Plectin2分别在成熟子实体阶段和幼嫩子实体阶段表达量最高,这表明Plectin1和Plectin2基因可能在糙皮侧耳子实体生长发育中起到一定的调控作用。     

茶树CsMAPK3的全长克隆及其逆境表达分析

以‘福鼎大白’茶树为材料,克隆了CsMAPK3的全长cDNA序列(GenBank登录号:MF034662)、基因组序列及其启动子序列。CsMAPK3的cDNA序列全长1700 bp,含有1119 bp的ORF序列,编码373个氨基酸;CsMAPK3蛋白预测为亲水性蛋白,含有多个磷酸化位点;多序列比对和进化树分析表明CsMAPK3 C–末端含有保守的CD结构域,属于TEY类型的A亚家族MAPK;亚细胞定位预测CsMAPK3主要定位于细胞质和细胞核中。CsMAPK3基因组全长4930 bp,包含5个内含子和6个外显子,第1个内含子和第2个内含子较大,分别为1608和1318 bp,外显子长度在130~350 bp之间。克隆获得起始密码子上游1125 bp的启动子区序列,该启动子上含有干旱、低温、高温以及ABA等相关的顺式作用元件。荧光定量表达分析显示,ABA、低温和盐胁迫均能显著上调茶树叶片中CsMAPK3的表达。蛋白互作预测表明CsMAPK3可能与MYBR1互作来响应ABA依赖途径的非生物胁迫过程。综上表明,CsMAPK3可能与茶树抗逆响应密切相关。     

High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns

Background  

Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression.     

连翘叶绿体基因组特征分析

为探究连翘叶绿体基因组特征及该属物种的系统进化发育关系,利用高通量测序技术对连翘长花柱与短花柱植株叶绿体基因组进行测序和功能注释,利用PCR对高通量测序结果进行验证.结果 显示:连翘长花柱与短花柱植株叶绿体基因组全长均为156386 bp,为典型的四段式结构.连翘叶绿体基因组注释到114个基因,包括80个蛋白编码基因,...     

An intact mitochondrial cox1 gene and a pseudogene with different genomic configurations are present in apple cultivars ‘Golden Delicious’ and ‘Delicious’: Evolutionary aspects

We have characterized the mitochondrial cox1 gene copies in two apple cultivars ‘Golden Delicious’ and ‘Delicious’. Both the cultivars contained an intact copy and a truncated copy of cox1. The intact ‘Golden Delicious’ and ‘Delicious’ cox1 genes, designated G-cox1 and D-cox1, respectively, were both found to be actually transcribed to give an RNA of approximately 1.7 kb. The two intact cox1 and two truncated copies (G-φcox1 and D-φcox1) shared a common 1115-bp segment flanked by four combinations of two different 5′- and 3′-sequences. PCR assay demonstrated that the configurations bearing G-cox1 and G-φcox1 existed in substoichiometric amounts within the mitochondrial genome of ‘Delicious’ whereas substoichiometric molecules carrying D-φcox1 were present in the ‘Golden Delicious’ mitochondrial genome. Although ancestor/descendant relationships cannot be inferred between the G-cox1 and D-cox1 arrangements, the results led us to hypothesize that (1) the 1115-bp segment containing part of the progenitor cox1 was duplicated, thereby generating a pseudo-cox1 copy, and (2) this was followed by homologous recombination across a portion of the 1115-bp repeats which gave rise to the descendant cox1 and pseudo-cox1 arrangements.     

Identification of quantitative trait loci for bolting and flowering times in Chinese kale (Brassica oleracea var. alboglabra) based on SSR and SRAP markers

SUMMARY

Expressed sequence tag-simple sequence repeat (EST-SSR), simple sequence repeat (SSR), and sequence-related amplified polymorphism (SRAP) markers were used to construct the first intra-specific genetic linkage map for B. oleracea var. alboglabra. In addition, QTL locations were determined for bolting and flowering traits. A total of 189 polymorphic marker loci were detected in the two parents, with 45 marker loci not located on any linkage group (LG). The remaining 144 loci defined ten LGs and included 69 EST-SSR loci, three SSR loci, and 72 SRAP loci. The length of the map was 1,173.8 cM, the average distance between loci was 8.15 cM, and the proportion of segregation distortion loci was 25.7%. Three QTLs controlling bolting time and two QTLs controlling flowering time were detected in the F2:3 generation, which explained 37.07% and 18.44% of the phenotypic variation, respectively. Two QTLs controlling bolting time and two QTLs controlling flowering time were detected in the F2 generation, which explained 27.90% and 25.59% of the phenotypic variation, respectively. In both generations, QTLs ftB.1 and ftA.1 were located on LG5, and the distance between the two QTLs was only 1.0 cM, suggesting that they could be at an identical locus. These results provide a useful reference for future QTL studies on bolting and flowering time in B. oleracea var. alboglabra, and can be used to identify markers linked to these QTLs for marker-assisted selection in commercial breeding programmes.     

Recent progress in the molecular biology of tea (Camellia sinensis) based on the expressed sequence tag strategy: a review

Summary

The expressed sequence tag (EST) technique provides a quick, efficient, and inexpensive route for gene cloning, gene expression profiling and regulation analysis, genome mapping, and functional annotation of genome sequences. Although an important cash crop, research on the molecular biology of tea (Camellia sinensis) started later and progressed more slowly than similar advances in major cereal crops and woody species. Most recently, progress has been made on the molecular biology of tea based on the strategies of EST sequencing and annotation. Advances have included the elucidation of gene expression profiling, the establishment and use of cDNA microarrays, data mining for EST-SSR (simple sequence repeat; microsatellite) and STR (short tandem repeat) loci, and cloning and expression analysis of genes involved in secondary metabolism, and stress defense.All these advances have contributed to a better understanding of the molecular mechanisms underlying growth, development, metabolism, and responses to the environment in tea plants, as well as to the promotion of molecular biology research in tea. New high-throughput sequencing technology will further accelerate genome sequencing of this species in the future.     

Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> and oat

Background  

Gene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species.     

芋花叶病毒的RT-PCR检测及外壳蛋白基因序列分析

 采用RT-PCR技术对采自中国湖北、浙江和山东的91份芋[Colocasia esculenta（L.）Schott]样品的芋花叶病毒（Dasheen mosaic virus,DsMV）进行了检测,检出率为26.4%。对其中14个DsMV分离物的317 bp扩增产物（为外壳蛋白基因的一部分）序列分析的结果显示,各分离物内的核苷酸变异相对较低,而分离物间存在较大的分子变异,相似性为68.3% ~ 97.8%。对来自湖北和浙江的2个DsMV分离物DsMV-SCS和DsMV-JH的外壳蛋白基因（coat protein gene,cp）进行了测序,全长分别为951 bp和987 bp,二者cp核苷酸和氨基酸序列相似性分别为79.0%和82.3%,与已报道DsMV的cp核苷酸和氨基酸序列相似性分别为73.0% ~ 92.1%和74.8% ~ 98.2%,在构建的系统发育树上聚在两个不同的簇,各DsMV分离物的系统进化关系与其寄主和地理来源无显著相关性。     

CmRT1, a Ty3-gypsy-like retrotransposon in Castanea mollissima,is associated with a short-catkin mutation

Summary

Retrotransposons are major components of the genomes of most eukaryotic organisms and have resulted in the introduction of desirable traits in many crops, including fruit trees. Here, we describe a Ty3-gypsy-like retrotransposon associated with a short-catkin mutant in Chinese chestnut (Castanea mollissima), resulting in catkins that are < 20% the length of normal staminate catkins. A partial sequence of the retrotransposon, named CmRT1, detected by amplified fragment length polymorphism (AFLP) analysis, and its complete sequence were determined from the genome of Chinese chestnut (Castanea mollissima) using improved Tail-PCR. CmRT1 was 10,067 bp in length and shared high homology in its predicted amino acid sequence and motifs with other Ty3/gypsy-like retrotransposons. The 5’ long terminal repeat (LTR) of CmRT1 contained a TATA box and several cis-elements that were predicted to be important for processes involving abscisic acid, gibberellic acid, and auxins and in stress-mediated responses. Further characterisation of the transposition event that led to the short-catkin phenotype was performed using two pairs of primers that aligned with the flanking region of the LTRs. The expected PCR bands were observed only in genomic DNA from plants that showed the mutation. Finally, cloning and real-time qPCR analysis of an NADP-dependent alkenal double-bond reductase (CmADBR) target gene that was adjacent to CmRT1, revealed that CmADBR expression was significantly down-regulated in the short-catkin mutant. Taken together, these results suggest that the CmRT1 retrotransposon is responsible for the short-catkin phenotype.     

香菇乳清酸核苷-5'-单磷酸脱羧酶编码基因le-pyrG的克隆

通过巢式简并PCR和基因组步行技术从香菇(Lentinula edodes)单核体中克隆到乳清酸核苷-5′-单磷酸脱羧酶(orotidine-5'-monophosphate decarboxylase,OMPDC)的编码基因le-pyrG,长度为946 bp.经生物信息学分析,香菇le-pyrG基因含有2个长度分别为67 bp和51 bp的内含子,该基因可以编码1个含有275个氨基酸残基的蛋白质序列,该序列经比对分析显示与裂褶菌(Schizophyllum commne)和灰盖鬼伞(Coprinus cinereus) OMPDC序列的相同性分别为73%和70%,相似性分别为84% 和83%.这是首次报道从栽培食用真菌中克隆出乳清酸核苷-5′-单磷酸脱羧酶编码基因的全序列.     

柑橘及其近缘属植物LFY同源基因的比较

LFY基因处于成花调控网络的关键位置,不仅调控开花时间和花转变,而且在花序和花的发育中也起重要作用。为了进一步探讨柑橘及其近缘属植物开花的分子机理,利用PCR技术分别从兴津温州蜜柑(Citrus unshiuMarcovitch)、无核椪柑(Citrus reticulata Blanco)、沙田柚[Citrus grandis(L.)Osbeck]、融安金柑(Fortunella crassifoliaSwing)和无核黄皮[Clausena lansium(Lour.)Skeels]叶片中分离克隆了LFY全长同源基因。结果表明兴津温州蜜柑、无核椪柑、沙田柚、融安金柑和无核黄皮中的LFY全长同源基因的核苷酸长度分别为2090、2086、2092、2081、2089bp,分别编码398、398、398、398和397个氨基酸,这些同源基因均由3个外显子和2个内含子组成。同源性分析发现,这些LFY全长同源基因的核苷酸序列和氨基酸序列同源性高,分别为92%～99%和95%～100%。亲缘关系分析结果与当前的植物学分类结果一致。