美洲黑杨雄性花芽cDNA克隆测序及表达序列标签(ESTs)特性分析

采用SMART技术构建美洲黑杨雄性花芽cDNA文库.随机挑选4 200个克隆进行序列测定,获得原始序列3 092个ESTs,去除插入片段短于150 bp的序列和污染序列后,获得有效ESTs序列3 087个,平均长度515 bp.对聚类后的416个Clusters分别进行单独拼接,得到相应的Contigs和Singletons分别为451和1 104个,并通过与NCBI等核酸数据库进行比对、查询和注释,获得已知功能和未知功能的基因1 015个,无序列相似性的新基因540个.这些数据为进一步研究高等植物花发育提供了一个极有价值的资源.     

Construction and Characterization of cDNA Library from Water-Stressed Plantlets Regenerated in vitro of Populus hopeiensis

In order to isolate and clone water-stress-responsive genes, total RNA was extracted from water-stressed plantlets regenerated in vitro of Populus hopeiensis using a QIAGEN RNeasy Plant Mini Kit. CDNA, synthesized by LD-PCR with the SMART cDNA Library Construction Kit, was n vitro packaged into a phage λTriplEx2 vector. The resulting primary library and amplified library have a titer of 1.68×10i6 and 1.69×109 pfu·mL–1 respectively. The combination ratio reached 98.8% and the average size of inserts was about 800 bp. In addition, the percentage of inserted fragments (> 400 bp) was approximately 90%. The results indicate that a cDNA library has been successfully constructed.     

毛竹简易基因组文库的快速构建

以毛竹（Phyllostachys edulis）当年所发新叶为材料,用改良的CTAB法提取基因组DNA,通过超声波震断DNA,琼脂糖凝胶电泳回收大小为700bp-2000bp的片段,经T4DNA聚合酶末端补平,与经过牛小肠碱性磷酸酶处理的pUCl9载体连接,通过电转化转染受体菌XL-1 Blue,构建了毛竹基因组文库。文库容量为2．8×10^7,通过PCR验证,插入片段大小为750 bp-2000bp,插入效率为91％,符合基因组文库构建的要求,为毛竹特异功能基因的克隆和分子标记的研究奠定了基础。     

矿山型东南景天cDNA表达文库构建与耐镉基因筛选

以筛选出的镉超积累矿山型东南景天为材料,经镉胁迫后提取总RNA,纯化mRNA,利用改良SMART技术合成了全长cDNA,回收500 bp以上cDNA大片段克隆到改造过的大肠杆菌/酵母穿梭载体pYES2.0G中,建成东南景天镉全长cDNA文库,对随机挑取的阳性克隆进行PCR鉴定,插入片断大小在1 000 bp左右,说明所构建的文库达到了用于目的基因分离筛选和表达的建库要求,为将来筛选克隆与东南景天抗重金属相关基因奠定了基础。     

枣抑制消减cDNA文库构建与分析

枣树具有独特的开花结果特性,为获得枣花发育基因,以金丝4号枣不同发育时期的花蕾和同时期的叶片cDNA互为试验方和驱动方,利用抑制消减杂交技术分别构建了枣花和枣叶抑制消减杂交cDNA文库(SSH文库)。枣花和枣叶SSH文库重组率分别是92.05%和90.26%,插入片段长度均介于200~2 000 bp之间,主要集中于1 000 bp左右。分别从枣花和枣叶SSH文库中随机挑选1 000个阳性克隆进行DNA测序、组装拼接,结果显示枣花SSH文库中获得96条unigene,其中contigs 43个,singletons 53个,96条unigene预测得到73个ORF,经过同源比对分析获得有注释的序列20条,按功能分为7类;枣叶SSH文库中获得86条unigene,其中contigs 45个,singletons 41个,86条unigene预测得到70个ORF,经过同源比对分析获得有23条注释的序列,按功能分为6类。     

小胡杨对滨海苏打盐渍土改良效果研究

滨海苏打盐渍土修筑台田后种植小胡杨可以成活,18年后小胡杨林分基本郁闭。以未种植树木的台田作为对照(CK),对小胡杨林地内植被演替规律、土壤水分物理性质进行研究。结果表明,小胡杨成林后,林地内植被向良性方向发展,群落生态型由中度耐盐群落演替成轻度耐盐群落,群落高度也由0.2～0.6 m增加到0.5～0.8m,盖度则达到80%～90%,土壤也由苏打盐渍土转变成轻盐碱土;小胡杨可显著降低土壤容重,改善土壤孔隙状况,增加土壤持水性能,使土壤水分物理性质得到显著改良,其中,对0～10 cm土层的改良效果要好于10～30 cm土层。     

休眠期珙桐种子cDNA文库构建及EST分析

以休眠期的珙桐种子为材料,利用SMARTTM cDNA Library Construction Kit构建珙桐种子的cDNA文库.结果表明:该文库的滴度为1.5×106 pfu·mL-1,重组率为98%,cDNA文库插入片段大小主要集中在0.5～1.0 kb.随机测序获得148个EST序列,拼接成127个uniEST,经网上BlastN及BlastX分析,其中75个序列是已知功能的基因标签,相似性较高,而32个序列在GenBank中相似性较低,13个序列在GenBank中没有匹配.这些数据有助于对珙桐种子的相关功能蛋白及分子机制的研究.     

辽河三角洲盐碱地树种优化配置模式研究

在辽河三角洲盐碱地台田上,布置小胡杨纯林、白蜡纯林、小胡杨×刺槐混交林、白蜡×刺槐混交林4种树种配置模式,对不同配置模式下的树木生长和土壤水、盐、温度进行了研究。结果表明,小胡杨、白蜡、刺槐在辽河三角洲盐碱地上造林均能成活,成活率达92%～98%。混交配置模式对树木生长具有显著的促进作用,优于纯林,其中小胡杨×刺槐混交模式好于白蜡×刺槐模式。与CK相比,4种模式均能改善土壤水分状况,降低土壤盐分,调控土壤温度,防止盐分表聚,4种模式间比较顺序是小胡杨×刺槐混交林>白蜡×刺槐混交林>白蜡纯林>小胡杨纯林。可见,小胡杨×刺槐混交模式可作为该地区防护林营建的首选优化配置模式。     

Isolation and sequence analysis of a Dof protein gene （PtDof1） in Populus

Both cDNA and DNA clones of PtDof1(GenBank Accession No. FJ402844 and FJ402845) were isolated from plants grown in tissue culture of Populus tomentosa. The DNA sequence is 1597 bp including two exons and one intron. The cDNA is 969 bp in length with a 765 bp open reading frame which is capable of encoding 255 amino acids. The deduced amino acids sequence of the PtDof1 protein shares 65%,56% and 55% identity with Vitis vinifera(CAO48618) ,Nicotiana tabacum(CAA08755) and Glycine max(ABI16022) Dof protein by b...     

油茶EST文库中不饱和脂肪酸合成关键酶基因的序列分析

采用生物信息学方法,从随机测序而构建的油茶优良无性系湘林1号种子EST文库中初步鉴定了与茶油脂肪酸形成相关的大部分关键酶基因,并对其中几个直接控制不饱和脂肪酸的形成与转化的基因进行了相应的序列分析．     

Toward a Pinus pinaster bacterial artificial chromosome library

Conifers are of great economic and ecological importance, but little is known concerning their genomic organization. This study is an attempt to obtain high-quality high-molecular-weight DNA from Pinus pinaster cotyledons and the construction of a pine BAC library. The preparation incorporates modifications like low centrifugation speeds, increase of EDTA concentration for plug maintenance, use of DNase inhibitors to reduce DNA degradation, use of polyvinylpyrrolidone and ascorbate to avoid secondary metabolites, and a brief electrophoresis of the plugs prior to their use. A total of 72 192 clones with an average insert size of 107 kb, which represents an equivalent of 11X pine haploid genomes, were obtained. The proportions of clones lacking inserts or containing chloroplast DNA are both approximately 1.6%. The library was screened with cDNA probes for seven genes, and two clones containing Fd-GOGAT sequences were found, one of them seemingly functional. Ongoing projects aimed at constructing a pine bacterial artificial chromosome library may benefit from the methods described here.     

胡杨盐诱导基因与盐抑制基因的差减杂交显示研究

基因筛选是基因克隆与遗传转化的基础工作 ,其目的是估测在环境刺激或诱导条件下基因表达水平上的变化。即估测直接或间接表达基因的数目 ,并进一步作为分子标记进行基因分离与克隆。胡杨在盐诱导条件下蛋白质电泳结果显示 ,与对照 (0 % )相比 ,既有增加的条带 ,也有减少的条带 ,客观上证明了盐胁迫关闭了一些基因 (抑制基因 )的转录 ,也开启了一些基因 (诱导基因 )的转录。本研究旨在将这些盐诱导及盐抑制基因筛选出来 ,以便进一步进行抗盐基因的筛选与克隆。1　材料与方法胡杨 2ａ生盆栽实生苗 ,分别用 0 % ,0 5 % ,1 %和 2 %的ＮａＣｌ…     

Molecular cloning of two classes of Em-like proteins from the seeds of the leguminous tree Robinia pseudoacacia

To check for the presence of Em-like proteins in seeds of the leguminous tree Robinia pseudoacacia L. (black locust), a cDNA library constructed from mRNA isolated from developing seeds was screened using radiolabeled cDNA encoding the Em protein from Vigna radiata as a probe. Sequence analysis of the identified cDNA clones revealed two classes of Em proteins in Robinia. The nucleotide sequence data for each class have been submitted to Genbank/EMBL Data library and are available under the accession numbers U40820 and U40821. Northern blot analysis demonstrated that the Robinia Em proteins are translated from mRNAs of approximately 800 nucleotides, which are abundant in the seed but are not expressed in the bark. Southern blot analysis indicated that the Em proteins in Robinia are encoded by at least two genes. The Robinia Em proteins have a high degree of sequence homology with previously characterized Em-like proteins from both monocots and dicots.     

毛竹快速拔节过程节间组织cDNA文库的构建与分析

以同一棵毛竹笋上快速伸长生长的节间组织和基本停止伸长生长的节间组织为材料,分别提取总RNA,纯化成mR-NA后合成cDNA双链;以Uni-ZAP XR vector为载体,构建了2个cDNA文库。快速伸长生长的、停止伸长生长的节间组织cDNA文库的滴度分别为9.7×109、8.4×109pfu.mL-1,含插入片段的频率均达到99%以上,插入片段的大小均在500～3000 bp。cDNA文库的成功构建为探究毛竹快速生长的分子机理奠定了基础。     

油茶查尔酮合酶和异构酶基因的cDNA克隆

查尔酮合酶和查尔酮异构酶是类黄酮代谢与色素苷代谢的关键酶.以油茶近成熟种子cDNA文库和EST文库为材料,通过分子克隆方法鉴定了1条查尔酮合酶基因全长cDNA和1条查尔酮异构酶全长cDNA.结果表明:油茶查尔酮合酶基因的cDNA含1479 bp,编码412个氨基酸,为目前最长的查尔酮合酶,与其它物种的查尔酮合酶具有极高的相似性,在进化上高度同源;油茶查尔酮异构酶基因的cDNA含899 bp,编码206个氨基酸,与茶的查尔酮异构酶基因高度同源,但与其它物种的相似性较低.