毛白杨PtTFL1.1和PtTFL1.2基因的克隆和表达模式分析

以毛白杨为材料,利用同源基因克隆法设计引物,分别以毛白杨基因组DNA和RNA为模板,分离克隆了毛白杨中TFL1(TERMINAL FLOWER 1)基因两个成员,分别命名为PtTFL1.1和PtTFL1.2。序列分析发现,PtTFL1.1序列全长976 bp,编码区长度为525 bp,可编码 174个氨基酸;PtTFL1.2序列全长1 090 bp,编码区长度为522 bp,可编码173个氨基酸。二者都包含4个外显子和3个内含子,具有TFL1的典型保守的PEBP结构域,所推测的氨基酸序列具有TFL1特异关键的His88(H)和Asp141(D)氨基酸残基。同源蛋白比对结果显示,PtTFL1.1和PtTFL1.2与拟南芥、葡萄、柑橘、苹果等物种中的TFL1蛋白同源性都在80%以上。系统进化分析表明,PtTFL1.1和PtTFL1.2都属于FT/TFL1家族中的TFL1亚家族。荧光定量PCR实验表明,PtTFL1.1和PtTFL1.2的表达模式存在差异,PtTFL1.1在茎中的表达量要高于根和叶,在不同生长时期的花芽中,随着季节光照时间的递减,表达量呈现明显下降趋势,推测毛白杨中PtTFL1.1能通过响应日照长短,参与调控开花的光周期途径,PtTFL1.1可能是花的诱导初期主要的开花调控子;而PtTFL1.2在根茎叶以及各个时期的花芽中表达量都极低,且未检测到差异性变化。这些研究有助于探索TFL1在毛白杨花发育以及开花调控过程的重要作用,也将为后期深入研究毛白杨成花调控的分子机制奠定一定基础。     

大豆开花基因GmCO和GmFT的克隆及表达

为了研究大豆光周期反应是否受开花基因CO(CONSTANS)和FT(FLOWERING LOCUS T)调控,采用同源序列法从大豆中分离了CO和FT的同源物GmCO和GmFT.GmCO和GmFT分别编码151和109个氨基酸,与水稻和拟南芥中相关蛋白的氨基酸序列同源性达到70%以上.通过RT-PCR分析GmCO和GmFT在短日照(short daylength,SD)、自然光照(natural light,NL)和长日照(long daylength,LD)处理大豆不同发育阶段叶片中的表达发现,GmCO在LD处理大豆早期发育的叶片中高丰度表达,GmFT在SD和NL处理大豆开花时期的叶片中高丰度表达.上述结果表明,GmCO和GmFT的表达与大豆开花时间及光照长度密切相关,且GmCO抑制GmFT的表达.     

毛白杨蔗糖合酶基因PtSS2克隆与表达分析

为了解毛白杨蔗糖合酶(sucrose synthase,SS)基因功能和表达模式,以毛白杨茎段来源的cDNA为模板,根据毛果杨PtrSS2CDS序列信息设计特异引物,采用RT-PCR技术分离克隆了PtSS2基因。测序分析表明,PtSS2基因序列全长为2 412bp,编码803个氨基酸,蛋白大小为92.14kD,理论等电点6.00,属于酸性蛋白;氨基酸序列同源性分析表明,PtSS2与毛果杨PtrSS2和PtrSS1一致性分别高达100%和98.63%;结构域分析表明,PtSS2含有2个高度保守的功能域,即蔗糖合酶(5~552)和糖基化转移酶(554~744)功能域。qRT-PCR检测表明,PtSS2在毛白杨根、茎、叶、营养芽、雄雌花芽等各个组织中均有表达,但在营养芽和花芽中表达量较高,根部相对较低,总体呈组成型表达模式;在干旱胁迫下,PtSS2表达水平提升。研究结果推测,蔗糖合酶基因PtSS2在毛白杨各组织器官发育过程及干旱胁迫响应过程中具有重要功能。     

文心兰COL基因的分离及其表达分析

CONSTANS(CO)及CONSTANS-like(COL)基因在光周期调控植物开花中起到重要的作用。该研究以文心兰(Oncidium)品种‘金辉’为材料,分离了CO同源基因OnCOL2及另外2个COL基因(OnCOL8和OnCOL9),它们分别编码326、411和291个氨基酸;生物信息分析预测它们均定位于细胞核;OnCOL2、OnCOL8有2个锌指B-box结构域和1个CCT结构域,而OnCOL9缺少B-box结构域。多序列比对及进化树分析结果表明,所有COL蛋白可划分为3组,OnCOL2与OnCOL8、OnCOL9分到了2个不同组中。OnCOL2与建兰(Cymbidium ensifolium)CeCOL(90.77%)高度相似;OnCOL8与OnCOL9在进化关系上更为接近,分别与拟南芥(Arabidopsis thaliana)AtCOL9和AtCOL10关系最近,它们在B-box和CCT结构域都极为保守。表达分析结果表明OnCOL2、OnCOL8与OnCOL9分别在花、根和假鳞茎中表达量最高,在叶片中的表达呈周期性变化趋势,且在长、短日照条件下表达的峰值及时间均存在差异,在花芽分化时期的叶片中表达量均显著上调。研究表明,OnCOL2、OnCOL8与OnCOL9基因均受到生物钟和日长调节,在光周期途径中,可能通过上调它们的表达以促进文心兰花芽的形成。该研究结果为进一步研究基因功能及光周期调控文心兰开花机制奠定了基础。     

高羊茅FaFT基因表达、蛋白互作及生物学功能分析

FLOWERING LOCUS T(FT)对植物开花有重要的调控作用.为探索高羊茅FaFT基因的生物学功能,从前期高羊茅转录组中克隆得到FaFT基因.qRT-PCR分析表明FaFT基因在长日照、短日照、连续光照、连续黑暗、昼夜颠倒和长日照下不同发育阶段都保持一定的昼夜节律并受光周期的调控.亚细胞定位显示FaFT基因编码...     

毛白杨(Populus tomentosa)中2个谷甾醇糖基转移酶基因的克隆与表达分析

谷甾醇糖基转移酶(sitosterol glycosyltransferase,SGT)是能与膜结合催化植物谷甾醇糖基化的一种糖基转移酶。谷甾醇–葡萄糖苷(sitosterol-glucoside,SG)能在纤维素合酶(cellulose synthase,Ces A)的作用下,作为纤维素合成的引物,以UDP-葡萄糖(UDP-Glucose)为底物合成葡聚糖链。为了研究毛白杨(Populus tomentosa)PtSGT基因的功能,根据毛果杨(Populus trichocarpa)的同源基因Ptr SGT1和Ptr SGT3设计了PCR引物,以毛白杨组培苗叶片c DNA为模板克隆了毛白杨PtSGT1和PtSGT3 CDS序列,长度分别为1 851 bp、1 911 bp,分别编码616个氨基酸、636个氨基酸。结构分析表明,毛果杨(P.trichocarpa)同源基因Ptr SGT1和Ptr SGT3均含有14个外显子和13个内含子,结构与长度差异明显,分别定位于14号和2号染色体上。氨基酸序列同源性分析和进化分析表明,毛白杨(P.tomentosa)的PtSGT1和PtSGT3均与毛果杨和胡杨(Populus euphratica)相应蛋白具有较高的同源性。Real-time PCR分析显示,PtSGT基因家族的所有基因在根、茎、叶中均有表达,总体上呈组成型表达模式;PtSGT3在各组织中表达量显著高于其他成员,在茎中表达量最高,推测PtSGT3基因在谷甾醇–葡萄糖苷合成中有着重要作用。该实验研究结果为进一步解析毛白杨纤维素合成中这2个基因的功能奠定了基础。     

砂梨CONSTANS基因的克隆及原核表达分析

为了探索梨树植物的开花调控机制,本研究以砂梨(Pyrus pyrifolia Nakai)叶片为材料,采用同源序列克隆法,进行了开花调控相关基因CONSTANS的克隆,命名为PyCO,GenBank登录号为KF246572。序列分析表明,该基因包含一个1023 bp的开放阅读框,编码340个氨基酸,推测蛋白质分子量为37.81 kD,等电点为5.95。PyCO蛋白具有典型的植物CO家族的结构特征,含有2个高度保守的B-box及CCT结构域。进化树分析表明,该氨基酸序列与苹果的同源性接近93%,与碧桃、可可树、草莓等其他高等植物的CO类蛋白同源性也在70%以上。原核表达获得了具有较高表达水平的融合蛋白,分子量约为58.5 kD,为进一步探索梨树开花调控机理奠定了基础。     

甘菊ClCOL16基因的克隆与功能初步研究

CO基因通过光周期途径整合昼夜规律、光信号以及分生组织相关基因来调节开花时间,在植物成花过程中起着至关重要的作用。该研究利用RT-PCR法从甘菊(Chrysanthemum lavandulifolium)中克隆得到一个CO家族基因,命名为ClCOL16(GenBank登录号：AOA52174);采用农杆菌介导的花序浸染法转化拟南芥,抗性筛选并经过PCR鉴定得到T3代转基因植株;随机抽取6个转基因拟南芥株系与野生型(WT)共同于长日照环境(16 h/8 h光暗交替)培养,观察其表型,并于定植后37 d检测WT和转基因拟南芥植株叶片的ClCOL16表达,以明确甘菊ClCOL16基因在植物光周期调控开花中的作用,为进一步解析菊花花期调控机理奠定基础。结果显示：(1)甘菊ClCOL16基因含有一个1 278 bp的编码框,编码425个氨基酸;ClCOL16编码蛋白含有一个B-box和一个CCT保守结构域,为典型的CO家族第Ⅲ组成员。(2)NCBI同源比对分析显示,ClCOL16蛋白与除虫菊COL16蛋白(GenBank登录号：GEZ38716.1)的一致性最高,达到94.38%。(3)荧光定...     

粉菠萝FVE同源基因的克隆及表达分析

FV E是植物自主开花途径中的关键基因,主要通过抑制FLC的表达来调控花发育过程。本研究利用粉菠萝(Aechmea fasciata)茎尖组织转录组序列数据,通过RACE技术克隆获得了粉菠萝中FVE同源基因AfF-V E的cDNA全长序列。该序列全长为1672 bp,开放阅读框为1404 bp,编码由468个氨基酸残基组成的蛋白,该蛋白序列中含有6个保守的WD重复区域。氨基酸序列比对结果表明,A fFV E基因编码的氨基酸序列与其它物种中FV E的同源序列具有较高的相似性。qRT-PCR (实时荧光定量PCR)分析结果显示,粉菠萝中A fFV E基因对外源乙烯的刺激产生了应答。在采用乙烯利灌心处理不同时间的过程中,发现A fFV E基因的转录水平均在处理后1 d时达到最大,推测AfFVE可能参与乙烯信号反应,该基因在粉菠萝自主开花途径中的调节作用可能受乙烯影响。     

玉米生物钟基因ZmPRR1-2的克隆及表达分析

为探究玉米生物钟基因ZmPRR1-2的功能及表达特性,解析玉米光周期途径调控开花的机理,该研究以玉米骨干自交系‘黄早4’为材料,克隆ZmPRR1-2基因的cDNA序列并进行生物信息学分析;利用qRT-PCR技术对该基因进行组织特异性表达分析和48 h的昼夜节律表达分析。结果表明：(1)成功克隆获得ZmPRR1-2基因的编码区全长1 554 bp,编码517个氨基酸,编码的蛋白属于PRR基因家族,含有1个REC结构域和1个CCT结构域,多序列比对和系统进化分析显示ZmPRR1-2基因在禾本科植物中高度保守;ZmPRR1-2蛋白属亲水性蛋白,不包含跨膜结构域和信号肽。(2)ZmPRR1-2基因在玉米叶片中的表达量最高,显著高于其他7个组织,表明该基因主要在叶片中发挥功能,而在果穗和花丝中表达量相对较低,且显著低于雄穗中的表达量。(3)昼夜节律表达分析显示,在短日照条件下,ZmPRR1-2基因的表达量于光照3 h后开始逐渐上升,在光照结束后3 h时达到表达高峰;在长日照条件下,于光照6 h后ZmPRR1-2基因的表达量才开始逐渐上升,且在光照结束时达到表达高峰。研究认为,ZmPRR1-2基因...     

Molecular cloning and characterization of the cDNA coding for the biotin-containing subunit of the chloroplastic acetyl-coenzyme A carboxylase.

We report the molecular cloning and sequence of the cDNA coding for the biotin-containing subunit of the chloroplastic acetylcoenzyme A (CoA) carboxylase (ACCase) of Arabidopsis thaliana (CAC1). The 3' end of the CAC1 sequence, coding for a peptide of 94 amino acids, which includes a putative biotinylation motif, was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. The resulting GST-CAC1 fusion protein was biotinylated in vivo, indicating that CAC1 codes for a biotin-containing protein. Antibodies generated to the GST-CAC1 protein reacted solely with the 38-kD biotin-containing polypeptide of Arabidopsis. Furthermore, these antibodies inhibited ACCase activity in extracts from Arabidopsis leaves. The deduced amino acid sequence of CAC1 has an apparent N-terminal chloroplast-targeting transit peptide. The CAC1 protein is coded by a single Arabidopsis gene, and its mRNA accumulates to the highest levels in organs that are undergoing rapid growth. The amino acid sequence of the CAC1 protein is most similar to the biotin carboxyl-carrier protein component of eubacterial ACCases. These characterizations identify CAC1 as the biotin-containing subunit of the plastidic, heteromeric ACCase of Arabidopsis. The results support the ancient origin of the two structurally distinct ACCases of plants.     

Arabidopsis profilins are functionally similar to yeast profilins: identification of a vascular bundle-specific profilin and a pollen-specific profilin

Four members of the Arabidopsis profilin ( pfn ) multigene family have been cloned, sequenced and analyzed. By RNA gel blot analysis it has been shown that these four genes fall into two groups: one group ( pfn 1 and pfn 2) is expressed in all organs of the plant and the other group ( pfn 3 and pfn 4) in floral tissues only. Based on amino acid sequence alignment Arabidopsis profilins can be divided into the same two groups: PFN1 and PFN2 are 89% identical and PFN3 and PFN4 are 91% identical. Between these two groups they are 71–75% identical. The Arabidopsis profilins bind poly- l -proline and can complement both the Saccharomyces cerevisiae profilin deletion mutant and the Schizosaccharomyces pombe cdc3-124/profilin mutation, showing that the plant profilins are functionally similar to yeast profilins despite the low amino acid sequence homology. Analysis of pfn promoter-GUS fusion genes in transgenic Arabidopsis shows that pfn 2 is specifically expressed in the vascular bundles of roots, hypocotyls, cotyledons, leaves, sepals, petals, stamen filaments and stalks of developing seeds, whereas expression of pfn 4 is restricted to mature and germinating pollen grains.     

Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2

We report the cloning of both the cDNA and the corresponding genomic sequence of a new PP2C from Arabidopsis thaliana, named AtP2C-HA (for homology to ABI1/ABI2). The AtP2C-HA cDNA contains an open reading frame of 1536 bp and encodes a putative protein of 511 amino acids with a predicted molecular mass of 55.7 kDa. The AtP2C-HA protein is composed of two domains, a C-terminal PP2C catalytic domain and a N-terminal extension of ca. 180 amino acid residues. The deduced amino acid sequence is 55% and 54% identical to ABI1 and ABI2, respectively. Comparison of the genomic structure of the ABI1, ABI2 and AtP2C-HA genes suggests that they belong to a multigene family. The expression of the AtP2C-HA gene is up-regulated by abscisic acid (ABA) treatment.     

Acceleration of flowering by overexpression of MFT (MOTHER OF FT AND TFL1)

MFT (MOTHER OF FT AND TFL1) is a member of a gene family that includes two important regulators, FT (FLOWERING LOCUS T) and TFL1 (TERMINAL FLOWER 1), in determination of flowering time in Arabidopsis. Although the functions of FT and TFL1 are assigned in the family, the roles of other members are largely unknown. Especially the sequence of MFT is homologous to both FT and TFL1, which act as a floral promoter and an inhibitor, respectively, making it difficult to predict the role of MFT. We performed genetic analyses of MFT to understand its role in floral development. Constitutive expression of MFT led to slightly early flowering under long days. However, a T-DNA insertion allele of MFT did not show obvious phenotype. Further genetic analyses with the loss-of-function alleles of FT, TFL1, and ATC (Arabidopsis Thaliana CENTRORADIALIS homologue) showed that a decrease of MFT activity did not enhance the phenotypes of the single mutants. Taken together, we suggest that MFT functions as a floral inducer and that it may act redundantly in determination of flowering time in Arabidopsis.     

Biosynthesis of Lipoic Acid in Arabidopsis: Cloning and Characterization of the cDNA for Lipoic Acid Synthase

Lipoic acid is a coenzyme that is essential for the activity of enzyme complexes such as those of pyruvate dehydrogenase and glycine decarboxylase. We report here the isolation and characterization of LIP1 cDNA for lipoic acid synthase of Arabidopsis. The Arabidopsis LIP1 cDNA was isolated using an expressed sequence tag homologous to the lipoic acid synthase of Escherichia coli. This cDNA was shown to code for Arabidopsis lipoic acid synthase by its ability to complement a lipA mutant of E. coli defective in lipoic acid synthase. DNA-sequence analysis of the LIP1 cDNA revealed an open reading frame predicting a protein of 374 amino acids. Comparisons of the deduced amino acid sequence with those of E. coli and yeast lipoic acid synthase homologs showed a high degree of sequence similarity and the presence of a leader sequence presumably required for import into the mitochondria. Southern-hybridization analysis suggested that LIP1 is a single-copy gene in Arabidopsis. Western analysis with an antibody against lipoic acid synthase demonstrated that this enzyme is located in the mitochondrial compartment in Arabidopsis cells as a 43-kD polypeptide.     

ANT1, an aromatic and neutral amino acid transporter in Arabidopsis

A new amino acid transporter was identified from the Arabidopsis expressed sequence tag cDNAs by expressing the cDNA in a yeast amino acid transport mutant. Transport analysis of the expressed protein in yeast showed that it transports aromatic and neutral amino acids, as well as arginine. This transporter (ANT1, aromatic and neutral transporter) also transports indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. The cDNA is 1.6 kb in length with an open reading frame that codes for a protein with 432 amino acids and a calculated molecular mass of 50 kD. Hydropathy analysis showed ANT1 is an integral membrane protein with 11 putative membrane-spanning domains. Southern analysis and a BLAST search of the Arabidopsis genome database suggests that ANT1 is part of a small gene family containing at least five members. Phylogenetic comparisons with other known amino acid transporters in plants suggests that ANT1 represents a new class of amino acid transporter. RNA gel-blot analysis showed that this transporter is expressed in all organs with highest abundance in flowers and cauline leaves.     

OsHT, a Rice Gene Encoding for a Plasma-Membrane Localized Histidine Transporter

Using a degenerative probe designed according to the most conservative region of a known Lys- and His-specific amino acid transporter (LHT 1) from Arabidopsis, we isolated a full-length cDNA named OsHT (histidine transporter of Oryza sativa L.) by screening the rice cDNA library. The cDNA is 1.3 kb in length and the open reading frame encodes for a 441 amino acid protein with a calculated molecular mass of 49 kDa. Multiple sequence alignments showed that OsHT shares a high degree of sequence conservation at the deduced amino acid level with the Arabidopsis LHT1 and six putative lysine and histidine transporters. Computational analysis indicated that OsHT is an integral membrane protein with 11 putative transmembrane helices. This was confirmed by the transient expression assay because the OsHT-GFP fusion protein was, indeed, localized mainly in the plasma membrane of onion epidermal cells. Functional complementation experiments demonstrated that OsHT was able to work as a histidine transporter in Saccharomyces cerevisiae, suggesting that OsHT is a gene that encodes for a histidine transporter from rice.This is the first time that an LHT-type amino acid transporter gene has been cloned from higher plants other than Arabidopsis.     

甘蓝型油菜Toc33基因编码区的分离及蛋白序列的比较

以甘蓝型油菜新鲜嫩叶为实验材料提取其总DNA,以其为模板,根据拟南芥Toc33基因编码区序列设计引物,PCR扩增甘蓝型油菜叶绿体外膜蛋白转运机器的构件蛋白基因Toc33,得到两条扩增带,测序结果显示克隆到的两个片段分别长1370bp、1490bp,将这两个片段分别命名为Bn Tpc33-1,Bn Toc33-2,序列比较发现它们之间的同源性为78％,其中外显子的同源性为96％,而内含子的同源性仅为60％。为研究Toc33与同一基因家族的Toc34基因功能间的关系,对拟南芥、油菜、诸葛菜等植物的Toc33、Toc34蛋白序列进行比较分析并构建了分子系统进化树。