甘蔗全长丙酮酸磷酸二激酶（PPDK）基因的克隆

和C3植物相比,C4植物具有明显的生长优势及水分和营养利用率,生物产量也较高。甘蔗是典型的C4作物之一。以甘蔗叶片提取的基因组DNA为模板,以GenBank公布的甘蔗PPDK基因cDNA序列设计引物,进行LA-PCR(Long Acute PCR)扩增。将PCR产物克隆到pMD18-T载体中,转化大肠杆菌JM109,测序,得到了13.5Kb的甘蔗全长PPDK基因序列。为方便后续实验,在引物中引入可利用的XhoI和NotI酶切位点,将全长PPDK基因分两段克隆到pMD18-T Simple载体中,转化大肠杆菌JM109,完成了甘蔗全长丙酮酸磷酸二激酶（PPDK）基因的完整克隆,为将其导入C3作物中奠定了研究基础,实验室保藏菌种。     

流感病毒基因的密码子偏好性及聚类分析

流行性感冒病毒是一种造成人类及动物患流行性感冒的RNA病毒,它造成急性上呼吸道感染,并由空气迅速传播,在世界各地常有周期性的大流行。根据该病毒的基因组CDS序列,探讨了基因组序列密码子的使用模式和特性,并进行了病毒间的聚类分析。结果表明:流感病毒的G+C含量均低于A+U含量,偏向使用以A、U结尾的密码子的程度比使用以G、C结尾的较高,CUG、UCA、AGU、AGC、AGA、AGG、GUG、CCA、ACA、GGA、GCA、AUU、UGA、CAU、CAA、AAU、AAA、GAA等18个密码子为流感病毒共有的偏好性密码子,且以A结尾的居多,尤其偏爱AGA、GGA。聚类结果表明首先亚洲流感病毒H2N2和香港流感病毒H2N2聚为一类,亚洲流感病毒H1N1和俄罗斯流感病毒H1N1聚为一类,1997年和2003年~2004年发生的人禽流感聚为一类,说明它们的密码子使用的偏好性相似;而2009年爆发的甲型H1N1流感和任何一个流感的距离都比较远,说明甲型H1N1流感病毒是一种新型的病毒,不同于以往任何一种流感病毒。     

金针菇高表达基因的密码子偏好性及特征分析

为探究金针菇的密码子偏好性,挖掘高表达基因的特征信息,以金针菇基因组及转录组数据为材料,分析金针菇的密码子偏好性及其影响因素,并对发育阶段高表达基因进行功能注释和顺式元件分析。分析表明,金针菇高表达基因表现出较强的密码子偏好性,且其偏好密码子多以胞嘧啶(C)结尾,此外在高表达基因中存在6种氨基酸的最优密码子较为保守。在进化过程中,金针菇高表达基因密码子偏好性受到自然选择压力的影响较大。功能注释分类表明,高表达基因多为核糖体通路相关的基因,与蛋白质翻译和生物合成相关。顺式元件分析表明,高表达基因启动子区域大多存在MeJA响应元件、ABA响应元件、光响应元件及MYB转录因子结合元件。研究结果可为提高金针菇异源表达效率和挖掘强启动子提供理论基础和思路。     

籽粒苋中PPDK基因的克隆及表达特性分析

为寻找能提高植物光合效率的基因资源,以高光效植物籽粒苋(Amaranthus hypochondriacus L.)为试材,利用同源克隆和RACE技术克隆了丙酮酸磷酸二激酶(Pyruvate orthophosphate dikinase, PPDK)基因,基因cDNA全长为3 224 bp,其中5′非翻译区为71 bp,阅读框为2 868 bp,3′非翻译区为285 bp,推导的蛋白质为956个氨基酸,分子量约106 kDa。序列分析表明,克隆的基因含有PPDK基因的功能结构域。表达模式分析显示克隆的PPDK基因在绿色组织中特异表达,为PPDK基因的长转录本,初步确定已克隆得到为籽粒苋中的PPDK基因,将其命名为AhPPDK。     

子囊菌延长因子1 alpha基因密码子偏好性分析(英文)

文中对子囊菌代表类群的延伸因子1 alpha基因密码子的使用模式进行了研究。结果表明:该基因的密码子使用偏好性不仅与核酸碱基组成密切相关,也受到其他选择性压力的影响。统计分析揭示了子囊菌各类群该基因的密码子组成和编码特点,在同义密码子的选择模式上,酵母纲(Saccharomycetes)的成员具有较独特的偏好性。基于密码子用法分歧度的聚类分析方法较合理地反映了大部分类群的分类学地位,但在各个纲的内部,密码子偏好性的变化程度存在差异。     

紫花苜蓿叶绿体基因组密码子偏好性分析

为分析紫花苜蓿叶绿体基因组密码子偏好性的使用模式,该文以紫花苜蓿叶绿体基因组中筛选到的49条蛋白质编码序列为研究对象,利用CodonW、CUSP、CHIPS、SPSS等软件对其密码子的使用模式和偏好性进行研究。结果表明:(1)紫花苜蓿叶绿体基因的第3位密码子的平均GC含量为26.44%,有效密码子数(ENC)在40.6～51.41之间,多数密码子的偏好性较弱。(2)相对同义密码子使用度(RSCU)分析发现,RSCU>1 的密码子数目有30个,以A、U结尾的有29个,说明了紫花苜蓿叶绿体基因组A或U出现的频率较高。(3)中性分析发现,GC3与 GC12的相关性不显著,表明密码子偏性主要受自然选择的影响; ENC-plot 分析发现一部分基因落在曲线的下方及周围,表明突变也影响了部分密码子偏性的形成。此外,有17个密码子被鉴定为紫花苜蓿叶绿体基因组的最优密码子。紫花苜蓿叶绿体基因组的密码子偏好性可能受自然选择和突变的共同作用。该研究将为紫花苜蓿叶绿体基因工程的开展和目标性状的遗传改良奠定基础。     

滇楸叶绿体基因组密码子偏好性分析

为分析滇楸(Catalpa fargesii)叶绿体基因组密码子的使用模式,本研究以滇楸叶绿体基因组密码子为研究对象,筛选出了38条蛋白编码序列,并利用CodonW和CUSP在线软件对其进行了中性绘图分析、ENC-plot和PR2-plot分析。结果表明：滇楸叶绿体基因组密码子平均GC含量为39.03%,不同位置上的GC含量依次是GC₁(47.51%)>GC₂ (40.80%)>GC₃(28.78%),说明叶绿体基因组密码子末位碱基偏好以A和U结尾;其有效密码子数(effective number of codons, ENC)的范围为34.93～55.78,平均值为46.61,有25个ENC值大于45,表明其密码子的偏好性较弱;同义密码子相对使用度(relative synonymous codon usage, RSCU)分析表明,RSCU>1的密码子中有30个以A或U作为结尾,说明其密码子偏好以A和U结尾;中性绘图分析显示,GC₁₂和GC₃的相关性不显著...     

普通野生稻线粒体蛋白质编码基因密码子使用偏好性的分析

以普通野生稻(Oryza rufipogon Griff.)线粒体基因组为对象,分析其蛋白质编码基因的密码子使用特征及与亚洲栽培稻(O. sativa L.)的差异,探讨其密码子偏性形成的影响因素和进化过程。结果显示:普通野生稻线粒体基因组编码序列第1、第2和第3位碱基的GC含量依次为49.18%、42.67%和40.86%;有效密码子数(Nc)分布于45.32～61.00之间,其密码子偏性较弱; Nc值仅与GC_3呈显著相关,密码子第3位的碱基组成对密码子偏性影响较大;第1向量轴上显示9.91%的差异,其与GC_3s、Nc、密码子偏好指数(CBI)和最优密码子使用频率(Fop)的相关性均达到显著水平;而GC_3和GC₁₂的相关性未达到显著水平。因此,普通野生稻线粒体基因组密码子的使用偏性主要受自然选择压力影响而形成。本研究确定了21个普通野生稻线粒体基因组的最优密码子,大多以A或T结尾,与叶绿体密码子具有趋同进化,但是与核基因组具有不同的偏好性。同义密码子相对使用度(RSCU)、PR2偏倚分析和中性绘图分析显示,普通野生稻线粒体基因功能和其密码子使用密切相关,且线粒体密码子使用在普通野生稻、粳稻(O. sativa L. subsp. japonica Kato)和籼稻(O. sativa L. subsp.indica Kato)内具有同质性。     

普通野生稻线粒体蛋白质编码基因密码子使用偏好性的分析

以普通野生稻（Oryza rufipogon Griff.）线粒体基因组为对象,分析其蛋白质编码基因的密码子使用特征及与亚洲栽培稻（O.sativa L.）的差异,探讨其密码子偏性形成的影响因素和进化过程。结果显示：普通野生稻线粒体基因组编码序列第1、第2和第3位碱基的GC含量依次为49.18%、42.67%和40.86%;有效密码子数（Nc）分布于45.32~61.00之间,其密码子偏性较弱;Nc值仅与GC₃呈显著相关,密码子第3位的碱基组成对密码子偏性影响较大;第1向量轴上显示9.91%的差异,其与GC_3s、Nc、密码子偏好指数（CBI）和最优密码子使用频率（Fop）的相关性均达到显著水平;而GC₃和GC₁₂的相关性未达到显著水平。因此,普通野生稻线粒体基因组密码子的使用偏性主要受自然选择压力影响而形成。本研究确定了21个普通野生稻线粒体基因组的最优密码子,大多以A或T结尾,与叶绿体密码子具有趋同进化,但是与核基因组具有不同的偏好性。同义密码子相对使用度（RSCU）、PR2偏倚分析和中性绘图分析显示,普通野生稻线粒体基因功能和其密码子使用密切相关,且线粒体密码子使用在普通野生稻、粳稻（O.sativa L.subsp.japonica Kato）和籼稻（O.sativa L.subsp.indica Kato）内具有同质性。     

红松查尔酮合成酶基因CHS密码子偏好性分析

查尔酮合成酶(Chalcone synthase,CHS)广泛存在于植物体内,是花色素形成过程中一种重要的酶,可以进一步催化生成黄酮类化合物。本研究采用Codon W和EMBOSS在线软件对红松查尔酮合成酶基因CHS的密码子使用偏好性进行分析,并与北美乔松等其他24种植物的CHS基因以及模式植物基因组进行比较,对认识红松CHS基因的密码子使用偏好性,为选择适宜的表达系统奠定了一定的基础。研究结果表明:红松CHS基因编码区的有效密码子数(ENC)和GC含量分别为48.92和0.548,C+G含量高于A+T含量,密码子偏好以A/T结尾;多数植物CHS基因的G+C含量高于A+T含量,且密码子更偏好C/G结尾;聚类分析表明,红松与马尾松和赤松的密码子使用偏好性的相似性较高;密码子使用频率研究发现,红松CHS遗传转化与异源表达较优的受体可能是大肠杆菌和拟南芥。     

植物丙酮酸磷酸双激酶研究进展

丙酮酸磷酸双激酶(pyruvate orthophosphate dikinase,PPDK)作为C4光合途径中一个非常重要的限速酶,其功能已经清楚,但在C3植物中以及逆境条件下的作用尚不明确。在阐述PPDK基本生物学特征的基础上,重点介绍了PPDK在C4植物和C3植物中的功能、活性调控、基因工程以及PPDK对逆境胁迫应答的研究进展,以期为植物抗逆基因挖掘及抗逆种质创制提供参考。     

植物丙酮酸磷酸双激酶（PPDK）研究进展

丙酮酸磷酸双激酶（PPDK）是C4植物和景天科酸代谢（CAM）植物光合作用的关键酶,催化形成固定CO2的初始分子受体磷酸烯醇式丙酮酸（PEP）。本文重点比较了植物的ppDK基因结构及分子进化关系,综述了PPDK在C4植物和C3植物中的功能、PPDK的调控机理、PPDK在胁迫条件下的功能以及转PPDK基因等在近年来的研究进展。     

Immunological Studies on Pyruvate Orthophosphate Dikinase in C3 Plants

Pyruvate orthophosphate dikinase (PPDK) was detected in someC₃ plants, wheat, barley, rice and tobacco, by protein blottingusing an antibody against maize PPDK, although the amounts weremuch lesser than those of C₄ plants. The PPDK activity in immaturegrains of rice was specifically immunoprecipitated by the anti-(maize)PPDK antibody. The molecular weight of the subunit of PPDK inall tested C₃ plants was similar (ca. 95 kD) to that of maizePPDK, and the fragment patterns of the C₃ PPDKs in peptide mappingwere also similar to that of maize PPDK. These results suggestthat C₃ PPDKs have a primary structure similar to that of maizePPDK. In order to obtain information about the expression of PPDKin C₃ plants, changes in the enzyme activity and in the amountof PPDK protein were investigated during the greening of riceseedlings. PPDK, which was found in the etiolated seedlings,decreased temporarily in an early stage of greening and thenincreased. The mechanism of this variation is discussed. ¹ To whom correspondence should be addressed. (Received December 9, 1986; Accepted March 12, 1987)     

The Gene for Pyruvate,Orthophosphate Dikinase in C4 Plants: Structure, Regulation and Evolution

Pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1 [EC] ) is a keyenzyme in photosynthesis in plants that exploit the C4 photosyntheticpathway for the fixation of CO₂. This review focuses on thestructure, regulation and evolution of the C4-type ppdk genein the maize genome. The C4-ppdk gene in maize consists of 19exons spanning about 12 kbp. The gene is transcribed from twodifferent initiation sites under the control of two promotersto produce two mRNAs of different sizes. The larger one containsthe exon 1 sequence that encodes the chloroplast transit peptideand its product acts as C4-PPDK in chloroplasts, while the smallerone does not contain the sequence and its product may functionas a C3-enzyme in the cytosol. This unusual dual promoter systemis not unique to the maize C4-type ppdk gene since the sameorganization is also observed in the rice (C3 plant) ppdk geneand in Flaveria. Thus, the two-promoter system is common toplant ppdk genes from C3 and C4, monocot and dicot plants. Adiscussion is also presented of the generation of a system forregulation of the expression of the C4-type ppdk gene. A chimericgene consisting of a reporter gene under the control of thepromoter of maize CA-ppdk is exclusively expressed in photosynthetictissues and not in roots or stems of transgenic rice. The expressionof the introduced gene is also regulated by light: it is lowin etiolated leaves and is enhanced by illumination. These resultsindicate that the regulatory system that controls ppdk expressionin maize is not unique to C4 plants. ¹Recipient of the JSPP Young Investigator Award, 1995.     

Structural Basis of Reversible Phosphorylation by Maize Pyruvate Orthophosphate Dikinase Regulatory Protein

Pyruvate orthophosphate dikinase (PPDK) is one of the most important enzymes in C₄ photosynthesis. PPDK regulatory protein (PDRP) regulates the inorganic phosphate-dependent activation and ADP-dependent inactivation of PPDK by reversible phosphorylation. PDRP shares no significant sequence similarity with other protein kinases or phosphatases. To investigate the molecular mechanism by which PDRP carries out its dual and competing activities, we determined the crystal structure of PDRP from maize (Zea mays). PDRP forms a compact homo-dimer in which each protomer contains two separate N-terminal (NTD) and C-terminal (CTD) domains. The CTD includes several key elements for performing both phosphorylation and dephosphorylation activities: the phosphate binding loop (P-loop) for binding the ADP and inorganic phosphate substrates, residues Lys-274 and Lys-299 for neutralizing the negative charge, and residue Asp-277 for protonating and deprotonating the target Thr residue of PPDK to promote nucleophilic attack. Surprisingly, the NTD shares the same protein fold as the CTD and also includes a putative P-loop with AMP bound but lacking enzymatic activities. Structural analysis indicated that this loop may participate in the interaction with and regulation of PPDK. The NTD has conserved intramolecular and intermolecular disulfide bonds for PDRP dimerization. Moreover, PDRP is the first structure of the domain of unknown function 299 enzyme family reported. This study provides a structural basis for understanding the catalytic mechanism of PDRP and offers a foundation for the development of selective activators or inhibitors that may regulate photosynthesis.In C₄ plants, pyruvate orthophosphate dikinase (PPDK), which catalyzes the reversible phosphorylation of pyruvate to phosphoenolpyruvate, is the most important rate-limiting C₄ cycle enzyme (Edwards et al., 1985). Phosphorylation and dephosphorylation of residue Thr-527 of PPDK lead to its inactivation and activation, respectively. This process is accomplished by a single, bifunctional protein, namely PPDK regulatory protein (PDRP; Burnell and Hatch, 1985), and the process is light intensity dependent (Hatch and Slack, 1969; Chen et al., 2014b). PDRP is an unusual enzyme in three respects. First, PDRP is a bifunctional enzyme that catalyzes both phosphorylation and dephosphorylation, and these activities are usually catalyzed by separate kinases and phosphatases. Second, PDRP uses ADP as the phosphoryl donor for kinase activity, while most kinases utilize ATP. Third, unlike most phosphatases that dephosphorylate substrates to yield inorganic phosphate (Pi), PDRP employs a Pi-dependent, pyrophosphate (PPi)-forming dephosphorylation mechanism (Ashton et al., 1984).Owing to the importance in regulating C₄ photosynthetic cycle, the preliminary studies on maize (Zea mays) PDRP carried out in the 1980s were widely considered to represent a significant breakthrough in the field of C₄ photosynthesis research. Burnell and Hatch (1983) identified PDRP as a regulator of PPDK. They used in vitro activity assays to detect the reversible phosphorylation of PPDK by PDRP (Burnell and Hatch, 1985). By selectively substituting Ser or Tyr for the targeted Thr residue in the active site of PPDK, Chastain et al. (2000) discovered that Ser but not Tyr was functionally similar to the Thr residue (Chastain et al., 2000). This led to the conclusion that PDRP was a member of the Ser/Thr family of protein kinases (Scheeff and Bourne, 2005).Unfortunately, extensive studies on PDRP have been hindered by the low protein abundance in vivo and difficult protein purification in vitro, and our understanding of the mechanism of the dual enzymatic activities was not significantly advanced. More recently, PDRP was cloned from both maize and Arabidopsis (Arabidopsis thaliana), and bioinformatics analysis only predicted a phosphate binding loop (P-loop) domain and a conserved domain of unknown function 299 (DUF299) domain (Burnell and Chastain, 2006; Chastain et al., 2008). Moreover, multiple sequence alignment and molecular phylogenetic analysis has indicated that PDRP from many species appears to lack a canonical protein kinase subdomain and defined protein phosphatase motifs, and the PDRPs of plants may belong to the DUF 299 family (Chastain et al., 2008). Surprisingly, a DUF299 of Escherichia coli regulates the reversible phosphorylation of the target Thr residue in the active site of PEP synthetase (PEPS, homolog of PPDK), and the process is also ADP and Pi dependent just as is maize PDRP. This DUF299 gene was subsequently established as PSRP (EC 2.7.11.33 and 2.7.4.28), the PEPS regulatory protein (Burnell, 2010). However, the detailed catalytic mechanism of PSRP, as with PDRP, has remained obscure.It is also unclear whether PDRP uses two separate or one shared active site to perform phosphorylation and dephosphorylation activities. Differential effects on the enzymatic activities in thermolysin studies indicated two separate sites (Burnell and Hatch, 1986). However, the respective inhibition by phosphorylated and nonphosphorylated PPDK suggested that PDRP may contain separate active sites in relatively close proximity (Burnell and Hatch, 1985).In this study, we determined the crystal structure of PDRP and identified clear electron density corresponding to a bound AMP molecule. Combined structural analysis and enzymatic experiments suggest PDRP uses a single active site to perform both phosphorylation and dephosphorylation activities. Structural alignment and activity assays of site-directed mutagenesis provided comprehensive insight into the evolutionary relationships with other bifunctional protein kinase-phosphatases and the catalytic mechanism that may prove useful for the development of selective activators and inhibitors.     

Factor(s) Protecting Pyruvate Orthophosphate Dikinase of Panicum maximum against Cold-Inactivation

An apparent seasonal change in the cold-lability of pyruvateorthophosphate dikinase was observed in Panicum maximum thatsuggests a climatic change in the amount of the factor(s) whichprotects the enzyme against cold-inactivation. This factor(s)was partially characterized as a large molecule(s) with a heat-labilemoiety essential for its function. (Received May 23, 1984; Accepted July 25, 1984)     

丙酮酸磷酸双激酶及其基因结构

丙酮酸磷酸双激酶（PPDK）在植物C4光合途径中催化CO2原初受体磷酸烯醇式丙酮酸（PEP）的形成。本文介绍PPDK的类型、分布、生理功能、活性调节、基因结构和C4植物的PPDK基因转化C3植物及其与转基因植株光合作用之间关系的研究进展。     

籽粒苋苹果酸酶基因克隆及分析

NAD/NADP-苹果酸酶(NAD-ME/NADP-ME)是C4植物光合途径的关键酶。采用RT-PCR技术对籽粒苋NAD-ME基因进行克隆,获得了籽粒苋NAD-ME基因的cDNA序列。结果表明,该序列开放可读框长度为1 872 bp,编码623个氨基酸;多序列比对和进化树分析表明,该基因核苷酸序列与其他植物已报道的NAD-ME/NADP-ME基因的核苷酸序列一致性高达75.1%~80.6%,其氨基酸序列与其他植物的NAD-ME/NADP-ME蛋白一致性为73.2%~80.3%。对推断氨基酸序列的蛋白保守区、疏水性/亲水性、潜在跨膜片段、信号肽、蛋白固有无序化和蛋白二级结构分析表明,该蛋白具有苹果酸酶的保守区、兼具亲水性和疏水性,并且含有无序结构域,可能是一种跨膜的非分泌性蛋白。     

Correlation of the Activities of Phosphoenolpyruvate Carboxylase and Pyruvate,Orthophosphate Dikinase with Biomass in Maize Seedlings

The relations of three carbon-assimilating enzymes in maizeto biomass productivity were studied. There was no significantcorrelation between biomass and the amount of fraction I protein(RuBP carboxylase/oxygenase protein). In contrast, both theactivities of phosphoenolpyruvate carboxylase and pyruvate,P₁dikinase were highly correlated to the biomass. (Received February 7, 1983; Accepted March 26, 1983)     

苋菜凝集素基因的克隆及在转基因烟草中抗蚜性研究

通过ＰＣＲ从苋属植物千穗谷（Ａmaranthus hypochondriacus)的总ＤＮＡ中扩增出苋菜凝集素（ＡＨＡ）的核基因片段。序列分析结果表明该基因为２４５３bp,含有－１５３８bp的内含子和两个分别为２１２bp和７０３bp的外显子。采取反向ＰＣＲ的方法获得仅含该基因的编码区克隆。以此为基础与二元表达载体pBin438构建含内含子与不含内含子ＡＨＡ基因的植物表达载体pBAHAg和pBAHAc并通过土壤农杆菌介导转了化烟草,转化再生植株的ＰＣＲ和Southern blot分析表明,ＡＨＡ基因已整合到烟草的染色体中,有单贝和多拷贝的整合。用与ＡＨＡ蛋白高度同源的ＡＣＡ蛋白的抗血清进行了免疫斑点（Immunodot blot)检测,结果初步表明转基因烟草有ＡＨＡ蛋白的表达,虫试结果表明转pBAHAg和pBAHAc烟草对蚜虫的平均抑制率分别达５７．２％和４８．８％,有的高达９０％以上,含内含子和不含内含子的ＡＨＡ基因在转基因植株中的抗蚜性不同。