一种硫酸盐还原菌的亚硫酸盐还原酶基因(dsrA)的克隆和序列分析

硫酸盐还原菌是一类具有重要生态学意义的微生物，从一种具有还原六价金属铬能力的复合硫酸盐还原菌SRBⅢ中克降了它的亚硫酸盐还原酶A亚基的完整基因，该序列带有亚硫酸盐还原酶-亚硝酸盐还原酶中保守的CX5CXNCX3C的序列，与巳报道的同类基因相比，该基因与脱硫脱硫弧菌（Essex6株）的异化型硫酸盐还原菌最为接近，核苷酸序列的同一性为89%，氨基酸序列的同一性约为94%。     

褐飞虱NADH泛醌氧化还原酶51kDa亚基cDNA片段的克隆及表达分析

NADH泛醌氧化还原酶是动物体内呼吸链电子传递系统的第一个酶，克隆水稻害虫褐飞虱的NADH泛醌氧化还原酶基因，及研究其在褐飞虱与水稻互作中的表达变化，将为科学防治褐飞虱提供新的线索。利用反转录多聚酶链式反应（RT-PCR）技术克隆了褐飞虱NADH泛醌氧化还原酶51kDa亚基基因的cDNA片段，并进行了序列测定；使用NoRhem杂交技术检测了该基因对两种不同抗性水稻的分子反应。分子杂交结果表明，在取食抗性水稻品种B5后，褐飞虱的NADH泛醌氧化还原酶51kDa亚基基因表达水平明显升高，而取食感虫水稻TN1后，该基因的表达水平没有明显变化。     

日本蛇根草OjDFR5基因的克隆与真核表达载体的构建

二氢黄酮醇4-还原酶具有明显的底物特异性，是类黄酮代谢途径中的关键酶，对不同花青素的合成与积累起到决定作用，直接影响植物的颜色性状。日本蛇根草是一种具有较好药用价值的研究材料。通过分子生物学方法克隆获得日本蛇根草中的二氢黄酮醇4-还原酶的编码基因，OjDFR5,并对该基因的序列进行分析，同时将其与真核表达载体pBI121进行连接，获得真核重组表达质粒pBI121-OjDFR5,并将重组质粒转入农杆菌GV3101感受态细胞中。研究结果一方面为该基因的功能解析奠定基础，同时也为探究日本蛇根草类黄酮代谢机制研究提供基因资源。     

小麦液泡质子焦磷酸酶基因EST的克隆与分析

通过RT-PCR的方法在矮苏3小麦的总cDNA中克隆到一个与大麦液泡质子焦磷酸酶基因(VP)高度同源的EST。以该序列为基础,利用生物信息学的方法构建了一个编码小麦VP蛋白的全长EST重叠群,长2764bp,其包含一个长2355bp的完整开放读码框(ORF),编码785个氨基酸多肽。通过Southern杂交,将该VP基因定位在小麦染色体的第7同源群上。     

棉纤维醛酮还原酶基因促进拟南芥主根伸长

为了研究棉纤维醛酮还原酶基因在参与细胞伸长发育中所起的作用,本研究利用RT-PCR技术从处于快速伸长发育时期的棉花纤维组织中克隆得到棉花醛酮还原酶(aldo/keto reductase,AKR)基因c DNA,该基因全长读码框为1134 bp,编码377个氨基酸。氨基酸序列生物信息学分析表明Gh AKR蛋白具有较高的保守性,包含氧化还原酶、醛酮还原酶结合位点和跨膜信号序列等功能序列和位点;进化树分析显示Gh AKR与可可(Tc AKR)的亲缘关系较近。q RT-PCR和酶活性分析表明Gh AKR基因与纤维细胞的快速伸长发育密切相关,尤其在开花后5-15 d纤维快速伸长发育时期具有较高的表达。构建35S::Gh AKR过量表达载体并转化野生型拟南芥(哥伦比亚生态型),转基因拟南芥的主根伸长发育获得了显著的促进,与野生型相比,转基因拟南芥主根伸长增加了约1.4倍。拟南芥根中甲醇和乙醇含量分析显示,转基因拟南芥根中具有较高的甲醇和乙醇积累。这些结果表明棉纤维Gh AKR基因与纤维细胞伸长发育联系紧密,可能通过增加细胞内的醇含量进一步促进细胞的伸长。本研究可为棉花Gh GAKR基因参与纤维伸长发育分子机制解析,以及利用基因工程培育优质棉花品种提供一定参考。     

高温胁迫下菜心SSH cDNA文库的建立

为发现与菜心耐热性相关的差异表达基因,该研究以耐热性强的"四九19号菜心"和耐热性弱的"3T6"作实验材料,采用抑制差减杂交(SSH)技术,构建了一个高温胁迫下在"四九19号菜心"中特异表达的SSH c DNA基因文库.该文库共有154个contigs,经测序和拼接获得99条unique ESTs.这些EST序列长度在122~819 bp之间,平均344 bp.BLAST分析发现99条ESTs代表81个基因,其中68个基因有功能注释、9个无功能注释和4个未匹配的新基因.这些基因可能与菜心的耐热性相关.     

水分胁迫下马铃薯萌芽出苗期 根系转录组差异表达分析

以马铃薯‘克新1号’为材料,通过Illumina HiSeqTM 4000高通量测序技术,对不同水分胁迫后的马铃薯萌芽出苗期根系cDNA文库进行了RNA-Seq分析,拟探明马铃薯感应水分胁迫的分子机制,挖掘出与抗旱密切相关的功能基因.将比对到基因组上的基因利用RPKM衡量各样本间的基因表达丰度,以P0.05筛选出差异表达基因,通过GO (基因本体,gene ontology)数据库、KEGG代谢途径数据库,对不同水分胁迫样本差异表达基因的功能和参与的调控路径进行分析;选择6个差异表达基因,利用实时荧光定量PCR验证RNASeq结果的可靠性.结果表明:(1)DLWR(重度水分胁迫)样本中,比对到基因组的表达基因30 114 133个,差异表达基因5 241个;LWR(中度水分胁迫)样本中,比对到基因组的表达基因32 858 890个,差异表达基因1 488个.2个样本中同时存在的差异基因369个,其中上调表达基因78个,下调表达基因291个;显著差异表达基因主要与蛋白激酶、水解酶、氧化还原酶、水通道蛋白、转运蛋白、转录因子等相关.(2)在GO富集分析中,2个水分胁迫处理样本在生物学功能中富集的类别并不完全相同,但显著富集的主要是与氧化还原、转运、膜结合、转录调节子等相关的基因.(3)KEGG代谢分析显示,差异表达基因显著富集在谷胱甘肽代谢、植物激素信号转导等途径,与植物抗逆能力密切相关.(4)利用qRT-PCR验证的6个基因表达模式与RNA-Seq分析结果一致,证实了RNA-Seq结果的可靠性.通过研究得到了不同水分胁迫下马铃薯根系转录组信息,获得了差异基因及其功能信息,阐明了差异基因所参与的代谢通路及与植物水分胁迫的关系.     

鲤NADH泛醌氧化还原酶亚基3基因的克隆及低温适应相关性分析

根据鲤低温下特异表达的基因（片段）序列设计引物,采用2轮菌落PCR方法对鲤脑全长cDNA文库中的克隆进行筛选,确定了基因序列152在文库中的位置,经生物信息学比对分析确定其为NADH泛醌氧化还原酶亚基3基因.这一基因在低温下的特异表达,对于低温下鱼类机体的供能,起着重要作用.结果还表明,NADH泛醌氧化还原酶亚基3基因在鲤氧化呼吸链电子传递系统中与低温适应性状在一定程度上存在相关性.     

辣椒单脱氢抗坏血酸还原酶基因的克隆与序列分析

以番茄单脱氢抗坏血酸还原酶基因序列为信息探针，在GenBank dbEST数据库中找到高度同源的辣椒EST序列，通过人工序列拼接及RT-PCR得到了辣椒该基因的cDNA序列，命名为CaMDHAR，CaMDHAR与番茄单脱氢抗坏血酸还原酶cDNA序列一致率为92％，包含一个长为1302bp的完整的开放读码框．推导的氨基酸序列与番茄、黄瓜、花椰菜、豌豆的胞质单脱氢抗坏血酸还原酶基因的一致率分别为92％、77％、76％、76％。     

淞江鲈肝脏组织cDNA文库的构建与部分EST测序

以成年雌性淞江鲈肝脏组织为材料,用TRIzol Reagent试剂提取总RNA.以总RNA为模板,先用逆转录酶合成cDNA第一链,再通过LD-PCR合成cDNA第二链,所得双链DNA分子用SfiⅠ酶切处理后,连接到同样经SfiⅠ酶切处理的改良pUC19载体上,获得重组质粒.将重组质粒导入大肠杆菌DH5α感受态细胞中,建成质粒cDNA文库.经鉴定,该文库库容为1.5×106,重组率为94.5%,平均插入片段长度为1.1kb,插入片段大小分布为0.5~2.0kb.从文库中随机挑选2 200个克隆进行5’端单向测序,剔除不合格序列后,得2 002条高质量表达序列标签(Expressed Sequence Tag,EST)序列,提交dbEST数据库.经筛选,从这些EST序列中共发现471个基因,分别对其进行了KOG功能分类预测(216个蛋白被注释上21种KOG分类)、KEGG注释(186个基因注释上175个KO,139个基因注释上195个pathway)和GO功能分类注释(312个蛋白被注释上GO分类).     

水稻S5-ORF3蛋白质的进化分析

生殖隔离在形成新物种和物种同一性的保持中有重要作用,因而在水稻优化育种中有重要的意义。S5位点是一个已克隆的控制水稻生殖隔离的基因,它产生的S5-ORF3,ORF4和ORF5蛋白质共同调控着indica-japonica杂交后代的育性,特别是S5-ORF3蛋白质在打破水稻亚种indica-japonica之间的生殖隔离与促进物种间的基因交流中发挥重要作用。针对S5-ORF3的进化分析对于研究它的功能和起源很重要,但目前尚无报道。本文基于序列和进化分析,提出S5-ORF3为一种定位在内质网中的新的HSP70家族蛋白,但缺乏HSP70的C端的多肽结合结构域,推测S5-ORF3可能通过影响alpha-淀粉酶的合成来作用于内质网压力;找到了ORF3的19条同源蛋白,并用极大似然算法构建了可靠的进化树,发现水稻的S5-ORF3与节节麦的luminal-binding蛋白进化关系最为密切;作了置信度100%的S5-ORF3蛋白质的三维结构预测,预测了居中的配体绑定位点;发现了水稻的S5-ORF3与其他HSP70蛋白相比独特的基序,为进一步研究S5-ORF3的作用机理和演化历史提供了线索和数据支持。     

Genetic and physical finemapping of the Sc locus conferring indica-japonica hybrid sterility in rice （Oryza sativa L.）

Hybrid sterility is a major hindrance to utilizing the heterosis in indica-japonica hybrids. To isolate a gene Sc conferring the hybrid sterility, the locus was mapped using molecular markers and an F2 population derived from a cross between near isogenic lines. A primary linkage analysis showed that Sc was linked closely with 4 markers on chromosome 3, on which the genetic distance between a marker RG227 and Sc was 0.07 cM. Chromosome walking with a rice TAC genomic library was carried out using RG227 as a starting probe, and a contig of ca. 320 kb covering the Sc locus was constructed. Two TAC clones, M45EI4 and M90J01 that might cover the Sc locus, were partially sequenced. By searching the rice sequence databases with sequences of the TACs and RG227 a japonica rice BAC sequence, OSJNBb0078P24 was identified. By comparing the TAC and BAC sequences, six new PCR-based markers were developed. With these markers the Sc locus was further mapped to a region of 46 kb. The results suggest that the BAC OSJNBb0078P24 and TAC M45EI4 contain the Sc gene. Six ORFs were predicted in the focused 46-kb region.     

Genetic analysis and gene fine mapping for a rice novel mutant （rl9〈t）） with rolling leaf character

Much attention has been paid to leaf shape of rice in the process of ideotype breeding[1]. Several authors have reported that the rolling of leaf in some degree helps keep it erect, consequently optimizing canopy light transmission condition, which is good for dry matter accumulation and for high yield[2―6]. Rice as a polymorphic crop has many types of vari- ety with different morphologies. In terms of leaf shape, different cultivars with rolling leaf have been identifiedin rice germplasm. Le…     

Morphology and mapping analysis of rice (Oryza sativa L.) clustered spikelets( Cl) mutant

The rice clustered spikelets (Cl) mutant exhibits a phenotype that most of branch apical have 2-3 spikelets clustered together,SEM (scanning electron microscope )observation suggested that the Cl gene controlled branch apical development,and influenced the terminal spikelets elongation,The spikelet number was reduced in mutant,indicating that Cl may also have an effect on spikelet number,To map Cl locus,two F2 mapping populations derived from the crosses between the Cl and ZhongHua11,and Cl and ZheFu802 were constructed ,respectively,The Cl locus was roughly mapped between two CAPS markers CK0214 and SS0324,A further fine mapping analysis showed that the Cl locus was mapped between makers R0674E and Cl12560,with genetic distances of 0.2 and 2.1 cM,respectively ,Then we found a PAC conting spanning Cl locus,the region was delimited to 196 kb.This results was useful for cloning of the Cl gene,Allelism test demonstrated that Cl was allelic to Cl2 another rice clustered spikelets mutant.     

Fine mapping of a semidwarf gene sd-g in indica rice（Oryza sativa L.）

The semidwarf gene sd-g which has been usedin indiea rice breeding in southern China is a new one, non-allelic to sd-1. To map sd-g, an F2 population derived fromthe cross between Xinguiaishuangai and 02428 was con-structed. The sd-g was roughly mapped between two mi-crosatellite markers RM440 and RM163, with genetic dis-tances of 0.5 and 2.5 cM, respectively. Then nine new poly-morphic microsatellite markers were developed in this region.The sd-g was further mapped between two microsatellitemarkers SSR5-1 and SSR5-51, with genetic distances of 0.1and 0.3 cM, respectively, while cosegregated with SSR418. ABAC contig was found to span the sd-g locus, the region be-ing delimited to 85 kb. This result was very useful for cloningof the sd-g gene.     

A family of candidate taste receptors in human and mouse

The gustatory system of mammals can sense four basic taste qualities, bitter, sweet, salty and sour, as well as umami, the taste of glutamate. Previous studies suggested that the detection of bitter and sweet tastants by taste receptor cells in the mouth is likely to involve G-protein-coupled receptors. Although two putative G-protein-coupled bitter/sweet taste receptors have been identified, the chemical diversity of bitter and sweet compounds leads one to expect that there is a larger number of different receptors. Here we report the identification of a family of candidate taste receptors (the TRBs) that are members of the G-protein-coupled receptor superfamily and that are specifically expressed by taste receptor cells. A cluster of genes encoding human TRBs is located adjacent to a Prp gene locus, which in mouse is tightly linked to the SOA genetic locus that is involved in detecting the bitter compound sucrose octaacetate. Another TRB gene is found on a human contig assigned to chromosome 5p15, the location of a genetic locus (PROP) that controls the detection of the bitter compound 6-n-propyl-2-thiouracil in humans.     

Construction of the primary physical map of rice chromosome 12

A primary physical map of rice chromosome 12 was constructed using marker-based chromosome landing and chromosome walking. A BAC library from IR64 was screened using 84 RFLP markers, 4 STS markers and 6 microsatellite markers on chromosome 12 by colony hybridization and polymerase chain reaction (PCR) amplification. A total of 59 contigs consisting of 419 BAC clones including 5 single-clones were physically aligned on rice chromosome 12 with the largest BAC contig covering 855 kb. The whole physical map had a size of ∼16 Mb and covered about 52% of rice chromosome 12. This physical map will be certainly helpful for map-based gene cloning of agronomically and biological important genes and understanding the genome structure of the chromosome. Foundation item: Supported by Rockefeller Foundation Biography: FU Bin-Ying (1965-), male, Ph. D. candidate, Reseach direction: plant molecular genetics.     

肢带型肌营养不良一家系致病基因排除性定位

为了定位一个常染色体显性遗传肢带型肌营养不良家系的致病基因（ADLGMD）,采用13个荧光微卫星标记对收集到的一个包括4代33人的ADLGMD家系进行连锁分析,所选择的标记覆盖了3个已知ADL—GMD致病基因位点和4个已报道的致病基因定位区段．通过Linkage 5．1软件包计算连锁概率,各位点连锁分析所得的LOD值均小于-3,显示该家系致病基因与这7个位点均不连锁．该家系的肌营养不良症致病基因不在已知的位点内,很可能是一个新致病基因．