玉米BADH基因的克隆与序列分析

Betaine was accumulated as a nontoxic and protective osmolyte in water-dificit and salt-stressed plants. Betaine aldehyde dehydrogenase for glycine betaine synthesis is an important enzyme. The BADH gene of Maize was cloned by RT-PCR and RACE. The gene is 1 762 bp in length, including an open reading frame of 1 515 bp.Its nucleotide sequence shares 95% with the the partial cDNA of BADHI from Sorghum bicolor. Its deduced amino acid sequence contains the conserved domain sequence “VTLELGGKSP“ of ALDH, and a tripeptide SKL at its C-terminal, a signal targetting to the microbodies. The phylogenetic tree of 20 BADHs was constructed,which corresponds to the classical botanical division of plant, the BADH of maize is closest to the one of Oryza Sativa.     

YADE, a New Method for PCR Walking of Genomic DNA and cDNA in Cotton

The isolation of flanking sequences is a crucialstep in the study of gene cloning and expression.On the basis of Y-shaped adaptor(Prashar andWeissman,1996),we designed a new methodwhich could be used to amplify the flankingregion from both genomic DNA and cDNA.Thismethod was designated as Y-shaped adaptordependent extesion(YADE).The schematic ofthis method is showed in fig.1.     

Methods for Plant Differential Expressed Gene Cloning and Progress

The research of plant genome has been transferred from the structural genomics focusing on determining the complete sequences of the genome to the functional genomics focusing on elucidating the biological function of genes. Now cloning differential expressed genes is of prime interest in molecular biology. In the past ten years, some methods used for studying changes of gene expression in plants have been developed. The authors review the developments of techniques and studies for the plants related gene cloning in RNA level and expound their principles, technical routes, improved methods, advantages and defects. Moreover, their applications and prospects in plants related studies are discussed.     

Functional Analysis of DHS by Virus-induced Gene Silencing in Nicotiana benthamiana

With the progress in plant genomics research, the study direction is turned into the discovery and functional analysis of important genes. But an effective method is needed to investigate the biological function of eDNA sequences in large-scale （Wang et al., 2005）. With the newly developed technique-virus-induced gene silencing （VIGS）, plants infected with virus vector carrying host-derived sequence inserts will show loss-of-function or reduced-expression mutants in the host gene. The symptoms will tell the functional information of the gene （Liu et al., 2004; Burton et al., 2000）. Normally the process of constructing a virus vector and monitoring symptoms on infected plants can be completed within a few weeks, such that VIGS provides a simple, rapid and high throughput means of analyzing the function of sequenced genes.     

Genetic Analysis of Rice Mutants Identifies a New Locus at the Pi-ta Region Controlling Pathogen Recognition

Understanding the molecular mechanisms of host and parasite interactions should facilitate the development of novel strategies to control plant diseases. Host interactions with biotrophic and hemi-biotrophic pathogens are known to follow a gene-for-gene specificity. The plant expresses a resistance （R） gene that is effective in preventing disease in response to pathogen races expressing the corresponding avirulence gene. We are studying the interaction mechanisms of the R gene, Pi-ta, in rice with the corresponding avirulence gene, A VR-Pita, found in the hemi-biotrophic pathogen, Magnaporthe oryzae （formerly Magnaporthe grisea）. Pi-ta is a putative cytoplasmic receptor with a centrally localized nucleotide-binding site and leucine rich domain at the carboxyl terminus （Bryan et al., 2000; Jia et al., 2000）. AVR-Pita is predicted to be a metalloprotease （Jia et al., 2006b）. The putative processed protein, AVR-Pita176, has been shown to interact with the Pi-ta protein （Bryan et al., 2000; Jia et al., 2000）.     

Progress of Research on Expressing Antibodies in Plants

One of burgeoning fields of biology is the use of transgenic plants for the expression of antibodies. It refers to introducing into plants and expressing in them the genes encoding antibodies or antibody fragments. The plant-derived antibodies can serve the fuction of antigen recognition and binding. A number of transformation techniques have been used to introduce antibody genes into plant cells. An important advantage of expressing antibodies in plants is that it can produce inexpensively therapeutic antibodies in large scale. Immunotherapy can also be aimed at plants themselves, that is, these recombinant antibody molecules can be effective in insect or disease resistance. Furthermore, intracellular expression of antibody molecules can be used to modulate the metabolism of the expressing cells. Several groups have expressed antibody molecules in plants, either to modify or improve plant performance and characteristics or with a view to harnessing plants as bioreactors for large-scale production of antibodies. The research and development of expressing antibodies in plants are reviewed.     

AFLP Analysis of Mitochondrial DNA from a Cytoplasmic Male Sterility Line and its Maintainer in Pepper （Capsicum annuum L.）

Cytoplasmic male sterility （CMS） is a maternally inherited trait characterized by the inability of a plant to produce functional pollen, which is widespread among higher plants. CMS system is a valuable tool for plant brc, ders to utilize in hybrid seed production and for molecular biologists to study the interaction between nuclear and mitochondrial genomes. Investigations into the molecular mechanism of CMS in several species have revealed that the incompatible nuclear-mitochondrial interactions leading to new chimeric mitochondrial genes such as T-urfl3 for Texas male sterility cytoplasm in maize, pcfin petunia, orf522 in sunflower, orf107 in sorghum and orf244 gene in Brassica may be responsible for CMS （Dewey et al., 1986; 1987; Young and Hanson,1987; Nivison and Hanson, 1989; Kohler et al., 1991; Moneger et al., 1994; Tang et al., 1996; Handa et al., 1995; Singh and Brown, 1991）.     

Brown Planthopper Resistance Genes in Rice： from Germplasm to Breeding

The brown planthopper （BPH）, Nilaparvata lugens Stal （Homoptera： Delphacidae）, is one of the most destructive and widespread insect pests of rice （Oryza sativa） that can be found throughout the rice-growing areas in Asia, causing significant yield loss in susceptible cultivars every year. In addition to causing physiological damage to the rice plant, BPH also causes indirect damage by acting as a vector for rice ttmgro virus, grassy stunt virus and ragged stunt virus. Planting the resistant variety can efficiently restrain the breaking-out of BPH and its damage to rice. Mapping and cloning the BPH resistance genes will be propitious to the development of resistant rice variety and understanding of BPH-resistance mechanism in rice.     

小麦质膜质子ATP酶基因和启动子序列(登录号:AY829002)

Plant plasma membrane H^＋ -ATPase fuels ATP and pumps protons from the cytoplasm to the cell exterior resulting in acidification of plant cell wall and alkalinization of cytoplasm. This electrochemical gradient across the PM plays an extremely important role in plant growth and development. We isolated promoter and complete gene of plasma membrane H^＋ -ATPase from common wheat （Triticum aestivum L.cv. yumai 18）. The genomic sequence of the enzyme includes fourteen introns and fifteen exons. Existence of GGGCGGG sequence in promoter, located upstream-135, indicates that the enzyme is typical of “housekeeping” genes expressed in most tissues.     

芥菜型油菜4-二氢黄酮醇还原酶基因的克隆和表达分析

利用同源克隆方法, 在芥菜型油菜中克隆了DFR基因。在DNA和cDNA中扩增的DFR基因大小分别为 1 612 bp和1 214 bp。该基因含有5个内含子, 开放阅读框为1 158 bp, 预计编码385个氨基酸, 预测分子量为42 886.0 Da, 推测的等电点为5.54。DFR基因在芥菜型油菜紫叶芥和黑籽近等基因系的叶片、胚和种皮中都表达, 在四川黄籽中只在叶片和胚中表达。DFR基因在四川黄籽种皮中不表达, 导致种皮中花色素和原花色素不能合成, 从而种皮透明, 形成黄籽, 因此DFR基因是油菜种皮颜色形成途径中一个关键基因。本研究为利用该基因与种子、种皮特异启动子构建反义表达载体或RNAi载体, 阐明油菜种皮颜色形成的分子机理和创造黄籽油菜新种质奠定了基础。     

圆果黄麻纤维素合成酶基因CcCesA1的克隆、反义载体构建及转化拟南芥

以圆果黄麻(Corchorus capsularis L.) “179”茎部韧皮为材料,利用同源克隆和RACE技术,克隆黄麻纤维素合成酶基因CcCesA1 5¢端500 bp序列外的全部cDNA。其序列长度为2529 bp,编码627氨基酸残基,经Blast基因比对和蛋白质结构分析,确定是黄麻纤维素合成酶基因。半定量RT-PCR分析表明,CcCesA1在植株不同部位表达量具组织差异性,依次为茎部韧皮>根>叶>顶芽>麻骨。利用CcCesA1基因部分cDNA序列和3¢UTR区,构建黄麻CcCesA1基因反义载体,用测序验证的阳性质粒转化模式植物拟南芥。Southern结果表明,外源基因以单拷贝方式整合进入基因组,转基因拟南芥生长严重受阻,植株变得矮小且茎部易弯曲倒伏,角果数量变少,长度变短,纤维素含量有不同程度的降低,本研究结果表明,所克隆的黄麻CcCesA1基因除了参与植物其他生理代谢过程外,还参与纤维素的生物合成。     

黄黑籽甘蓝型油菜类黄酮途径基因SNP位点检测分析

类黄酮物质在植物花、叶、果实和种子颜色变化的过程中起着至关重要的作用,本研究以不同黄黑籽种皮材料为研究对象,采用基因同源克隆方法,获得17个类黄酮基因全长ORF序列,在核酸和蛋白水平上分别序列差异比较表明,这些基因在不同黄黑籽材料中共存在41个不同拷贝成员。在核苷酸水平上,检测到BnTT3、BnTT18、BnTTG1和BnTTG2的单核苷酸位点数目介于16~52之间,且BnTTG2在3个不同的位置上还存在多个碱基的连续性缺失现象(119~121 bp,183~189 bp和325~330 bp),但在蛋白水平上仅存在2~16个氨基酸位点差异,说明BnTT3、BnTT18、BnTTG1和BnTTG2在不同甘蓝型黄黑籽材料中存在单核苷酸位点差异,而单核苷酸位点突变不一定导致氨基酸位点的变异。在不同黄黑籽材料中仅BnTT3和BnTT18存在一致性的氨基酸突变位点(252和87),推测BnTT3和BnTT18可能在黄黑籽甘蓝型油菜种皮颜色差异形成过程中发挥至关重要的作用。通过这些位点的等位特异PCR可以区分材料间透明种皮基因,为特异基因芯片的开发及阐明甘蓝型油菜种皮色泽性状的基因及其作用位点奠定基础。     

甘蓝KAPP编码基因的克隆与序列分析

采用PCR、RT-PCR及其他分子生物学方法,以甘蓝基因组DNA、花蕾RNA和叶片RNA为模板,分别对甘蓝KAPP gDNA和KAPP cDNA进行扩增,分别获得3247bp的KAPP gDNA片段、1699bp的KAPP cDNA片段、1578bp的花蕾KAPP2 cDNA片段和1581bp的叶片KAPP2cDNA片段。对克隆的甘蓝KAPP gDNA和cDNA(结合报道的KAPPcDNA)进行比对表明,甘蓝KAPP基因包含11个内含子,均符合"GU-AG"剪接规则,并且克隆得到的KAPP cDNA序列与报道的KAPP cDNA序列有6处单个碱基的差异,但两者的氨基酸序列完全一致。然而花蕾KAPP2 cDNA、叶片KAPP2 cDNA片段与报道的KAPP cDNA序列相似性分别为85.2%和85.0%。这两个序列分别在590bp和593bp处较早出现一个无义突变引起的终止密码子。Blast分析表明,两基因编码的氨基酸序列与拟南芥KAPP氨基酸序列相似性最高,其次为甘蓝KAPP氨基酸序列。以已报道的8种植物KAPP的CDS及本实验所克隆的两个KAPP2序列构建分子进化树,获得序列与甘蓝KAPP序列聚为一支。结合比较作图及分子进化树,推测KAPP基因在甘蓝基因组上有两个拷贝,而笔者克隆到的KAPP2 cDNA序列是其中一个拷贝,是KAPP进化过程中突变失活的拟基因。     

花生转录因子WRI1基因特征的in silico分析

本研究利用电子克隆的方法获得花生转录因子WRI1的cDNA序列(AhWRI1),采用生物信息学方法,预测和分析AhW RI1的序列特点、编码蛋白AhW RI1的特性以及与其他植物氨基酸序列的相似性。结果表明:AhW RI1含一个长度为780bp的完整开放阅读框架,编码259个氨基酸。编码蛋白AhWRI1包含2个典型的AP2功能域,是亲水性蛋白,在蛋白质的三级结构上与拟南芥和油菜的WRI1相似。AhW RI1与拟南芥、油菜WRI1氨基酸保守序列同源性在81.87%～100%之间。AhW RI1无序化程度为71.8%,比拟南芥低3.8%,比油菜高2.5%。亚细胞定位显示AhW RI1在细胞核内,并预测该蛋白具有转录复制,调控及转录与结合的可能性分别为0.244、0.226和0.152。研究结果为花生WRI1基因的分子克隆,功能鉴定提供理论基础。     

聚合酶链式反应扩增甘蔗钙调蛋白基因及其序列分析

从甘蔗茎顶端分生组织提取点ＲＮＡ,反转录合成ｃＤＮＡ第一条链,以此为模板,参考大麦钙调蛋白基因序列设计并合成５端和３端引物,ＰＣＲ扩增甘蔗钙调蛋白基因,与克隆载体ｐＧＥＭ－３Ｚｆ（＋）重组,转化Ｅ．ｃｏｌｉＨＢ１０１得到得组克隆。ＤＮＡ序列分析结果表明：甘蔗钙调蛋白基因同４５０个核甘酸组成,编码１４８个氨基酸;在核甘酸序列上与迄今知道的几种植物的钙调蛋白基因有很高的同源性,同源率在８０％以上,编码     

莲多酚氧化酶基因的克隆及序列分析

多酚氧化酶是一类普遍存在于植物、真菌、昆虫的质体中，由核基因编码能与铜相结合的金属蛋白酶。它是许多果蔬等农产品酶促褐变的主因，也在植物的光合作用、抗病虫害、生长发育以及花色的形成中起一定作用。用RT—PCR方法，从中国莲（Nelumbo nucifera）幼叶RNA中成功扩增出ppo的部分序列，经克隆测序，得到长度在978-1057bp的序列共7个，编码326～352个氨基酸。与其他物种PPO进行比较，其核苷酸序列相似性为83％-85％，氨基酸序列相似性为84％99％。莲ppo的成功克隆为抑制莲藕的酶促褐变，提高莲的抗虫抗病性等的研究打下了良好的基础。     

棉花TCP家族转录因子基因GhTCP1的克隆与表达分析

通过比对拟南芥、金鱼草、水稻和玉米等植物中已知TCP家族转录因子的氨基酸序列,根据保守区设计兼并引物,以陆地棉(Gossypium hirsutum L.)品种新陆早13号的DNA为模板,获得棉花转录因子基因GhTCP1的中间保守区序列。根据该段序列设计特异性引物,采用5'和3'RACE技术获得了全长cDNA序列,编码397个氨基酸。基因组DNA序列分析表明,GhTCP1基因含有一个内含子。棉花GhTCP1蛋白与其它植物中的TCP家族转录因子有较高的相似性,证明该基因在进化过程中是相当保守的。半定量RT-PCR表达分析结果显示,该基因在棉花的侧芽中特异表达。     

矮牵牛DFR-A基因的克隆与表达分析

通过分离矮牵牛DFR-A基因,研究其在不同部位的表达规律和酶活性变化,为花色素苷合成的结构基因分离及其他研究奠定基础。基于已公布的矮牵牛DFR序列,直接从矮牵牛花瓣中分离了DFRA基因序列,利用实时荧光定量PCR技术研究其在不同器官中的表达特性,同时测定了不同器官中的DFR酶活性。（1）矮牵牛DFR-A基因cDNA序列为1473 bp,该基因最大开放阅读框为1143 bp,编码380个氨基酸。（2）DFR-A基因在矮牵牛各种器官中均有表达,但不同组织中表达量存在差异,在花药中的表达量最高,其次是初开和盛开花瓣中,各期花蕾中的表达量都较低,而在叶中表达量最低。（3）DFR酶在叶片及花药中几乎无活性。在初开花瓣中达到最高峰。（4）除花药以外的部位,DFR酶活性与DFR mRNA浓度存在线性相关性。矮牵牛DFR酶活性主要受转录调控,活性在初开花瓣中最高。     

西伯利亚蓼PsLEA基因的克隆及在NaHCO_3胁迫下的表达

根据西伯利亚蓼叶片cDNA文库中获得的胚胎发育晚期丰富蛋白(late embryogenesis abundant proteins,LEA)基因的部分序列,采用cDNA末端快速扩增(RACE)技术成功克隆了胚胎发育晚期丰富蛋白基因(LEA),命名为PsLEA(GenBank登录号:FJ478172)。该基因全长686bp,5'非翻译区为102bp,3'非翻译区为131bp,开放读码框为453bp,编码150个氨基酸。荧光定量PCR分析表明:正常情况下PsLEA基因在西伯利亚蓼的叶、茎和地下茎中皆有表达,其中茎中表达量最高。3%NaHCO3诱导胁迫下,叶、茎、地下茎中PsLEA表达均受到影响且表达模式存在差异,说明PsLEA基因与西伯利亚蓼的耐盐性相关。