Functional Analysis of the N-Terminal Prepeptides of Watermelon Mitochondrial and Glyoxysomal Malate Dehydrogenases*

Mitochondrial and glyoxysomal malate dehydrogenase (mMDH; gMDH; L-malate: NAD⁺ oxidoreductase; EC 1.1.1.37) of watermelon (Citrullus vulgaris) cotyledons are synthesized with N-terminal cleavable presequences which are shown to specify sorting of the two proteins. The two presequences differ in length (27 or 37 amino acids) and primary structure. Precursor proteins of the two isoenzymes with site-directed mutations in their presequences and hybrid precursor proteins with reciprocally exchanged presequences were analyzed for proper import using two approaches, namely in vitro using isolated watermelon organelles or in vivo after synthesis in the heterologous host, Hansenula polymorpha. The mitochondrial presequence is essential and sufficient to target the mature glyoxysomal isoenzyme into mitochondria (Gietl et al., 1994). As to the function of the mitochondrial presequence a substitution of ^?3R (considered important for one step precursor cleavage in yeast and mammals) with ^?3L permitted import into mitochondria but cleavage of the transit peptide and conversion into active mature enzyme was impeded. Substitution of ^?13R^?12S (in a sequence reminiscent of the octapeptide motif serving as a substrate for the mammalian and yeast intermediate peptidase) into ^?13L¹²F permitted mitochondrial import and processing like the wild type transit peptide. Purified rat mitochondrial processing protease, which can effect single step cleavage of mitochondrial protein precursors, cleaves in vitro translated watermelon mMDH precursor into its mature form. The glyoxysomal presequence is essential and sufficient to target the mature mitochondrial isoenzyme into peroxisomes of Hansenula polymorpha, but these peroxisomes lack a processing enzyme to cleave the presequence (Gietl et al., 1994). We here show that isolated watermelon organelles also import the hybrid proteins in vitro and process the glyoxysomal presequence. Site directed mutations within the conserved RI-X₅-HL-motif impede efficiency of import and cleavage by watermelon organelles.     

甘蔗细胞质型苹果酸脱氢酶基因的克隆及其表达分析

对甘蔗(Saccharum officinarum L.)叶片全长cDNA文库进行测序,获得了1个细胞质型苹果酸脱氢酶(cMDH)基因的全长cDNA序列,命名为Sc-cMDH。生物信息学分析表明,该基因全长1314 bp,开放阅读框为999 bp,编码332个氨基酸。Sc-cMDH与其他植物cMDH的氨基酸序列同源性高达86.5%～97.0%。Sc-cMDH包含典型的NAD+结合基元T11GAAGQI17和催化基元I184WGNH188,还有相当保守的6个半胱氨酸残基,因此推断该基因为细胞质型NAD-MDH。定量PCR分析结果表明,该基因在甘蔗叶片和根中的表达量高于茎。     

玉米苹果酸脱氢酶基因的分离与结构分析

以一个玉米（ＺｅａｍａｙｓＬ．）杂种一代超亲表达的ｃＤＮＡ片段为探针,从玉米幼苗期ｃＤＮＡ文库中筛选到一个全长１２８７ｂｐ的ｃＤＮＡ克隆。序列分析表明,该ｃＤＮＡ编码细胞质苹果酸脱氢酶,推导的氨基酸序列与龙须海棠（Ｍｅｓｅｍｂｒｙａｎｔｈｅｍｕｍ　ｃｒｙｓｔａｌｌｉｕｍ　Ｌ．）及拟南芥（Ａｒａｂｉｄｏｐｓｉｓ　ｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）同一编码基因的氨基酸序列同源性分别为９０％和８４％。这是禾谷类作物中首次克隆的编码细胞质苹果酸脱氢酶的完整基因。     

cDNA Cloning of Thylakoid-Bound Ascorbate Peroxidase in Pumpkin and Its Characterization

A cDNA for thylakoid-bound ascorbate peroxidase of pumpkin wascloned and characterized. Thylakoid-bound ascorbate peroxidasehad a high similarity to cytosolic ascorbate peroxidases, andthe precursor contained a transit peptide to chloroplasts atits ammo-terminus and a putative membrane-spanning region atits carboxy-terminus. (Received February 23, 1996; Accepted March 25, 1996)     

Isolation and Characterization of a cDNA Encoding Maize Cyto solic Malate Dehydrogenase

A cDNA fragment derived from a gene over-expressing in hybrid maize ( Zea mays L. ) was isolated with RT-PCR and used as probe to screen cDNA library of hybrid maize seedlings. A positive cDNA clone ZH02 corresponding to the full-length mRNA sequence was obtained, which was shown to have an open reading frame encoding 332 a.a. DNA and proteinase database search revealed that the deduced amino acid sequence of ZH02 has high similarity with the cMDH of Mesembryaathemum crystallium L. and Arabidopsis thaliana (L.) Heynh. up to 90% and 84%, respectively. This is the first report of the full-length gene coding for the cereal cMDH.     

一个深黄被孢霉苹果酸脱氢酶基因的克隆与表达

根据转录组测序结果设计特异性引物,以深黄被孢霉(Mortierella isabellina)M6-22的c DNA为模板,PCR扩增苹果酸脱氢酶基因MIMDH2,测序结果显示该序列长1 017 bp,编码338个氨基酸。序列分析表明该序列与已报道的烟曲霉(Aspergillus fumigates)线粒体苹果酸脱氢酶的相似性最高,达71.26%,且含有苹果酸脱氢酶的保守辅酶结合位点、底物结合位点和催化活性位点。将MIMDH2片段连接到表达载体pET32a(+)中,构建重组表达质粒pET32a-MIMDH2,并转化至大肠杆菌BL21中进行诱导表达,SDS-PAGE电泳检测结果显示在50 k D左右处有一蛋白质条带,酶活分析结果显示经镍柱亲和层析纯化的重组蛋白酶活高达271.33 U/mg。以上结果说明所克隆的MIMDH2为一个新的潜在的苹果酸脱氢酶基因,所编码的蛋白质具有苹果酸脱氢酶的活性。     

一类依赖于NAD的玉米胞质型苹果酸脱氢酶基因的克隆及其序列分析

苹果酸脱氢酶普遍存在于各种生物中,它负责催化草酰乙酸和苹果酸之间的相互转换.根据其辅酶的特异性和在细胞内的分布及其生理功能的不同,苹果酸脱氢酶在高等植物中可以区分出不同的类型,依赖于NAD的细胞质型苹果酸脱氢酶是其中研究较少的一类.根据已发表的其他高等植物的依赖于NAD的胞质型苹果酸脱氢酶基因的保守序列,运用SMART RACE RT-PCR技术,从玉米叶片中分离了cyMDH 的1 264 bp全长cDNA序列,通过生物信息学分析发现,该序列含有一个999 bp的完整的开放阅读框,其共编码332个氨基酸(GenBank登陆号 EU625276).序列联配与树状分析结果表明,该玉米cyMDH 序列与多个物种的cyMDH 基因具有高度的同源性.组织特异性表达分析显示MDH基因在玉米叶片中表达量最高,在茎、根中亦有低水平表达.本研究将为更深入的研究玉米cyMDH 基因的分子调控机理奠定基础.     

水稻乙醛脱氢酶基因的克隆及其在不育系中的表达

从水稻中分离了乙醛脱氢酶基因 (Osaldh)的全长cDNA ,序列分析显示OsaldhcDNA含有一个完整的编码 5 49个氨基酸的开放阅读框 ,其编码蛋白OSALDH的N端为预期的线粒体前导肽 ,中部具有醛类脱氢酶基因家族的酶催化活性中心 ,OSALDH与玉米、烟草、人类的线粒体乙醛脱氢酶同源性分别高达 87%、77%、5 9%。Northern分析显示 ,幼根中Osaldh的表达水平高于幼苗、幼穗 ,不育品种幼穗中Osaldh的转录水平普遍高于对应的可育恢复系。植物线粒体乙醛脱氢酶的功能及其与雄性不育的关系值得进一步研究     

黄瓜胞质6-磷酸葡萄糖酸脱氢酶基因克隆及序列分析

根据6-磷酸葡萄糖酸脱氢酶(6-phosphogluconate dehydrogenase,6PGDH)基因的保守氨基酸序列设计简并引物,应用RT-PCR技术从黄瓜栽培种品种'北京截头'(Cucumis sativus 'Beijingjietou')叶片中获得了640 bp的特异片段,以该序列在EST数据库进行同源检索筛选,发现甜瓜EST序列AM715537.2与之高度一致,据此设计引物经RT-PCR扩增、分子克隆和序列拼接,获得了黄瓜6-磷酸葡萄糖酸脱氢酶基因全长序列,命名为Cs6PGDH(GenBank登录号FJ610345).序列分析表明,该基因全长1 829 bp,其中开放读码框(ORF)长1 488 bp编码495个氨基酸组成的多肽,编码区内无内含子存在,5'、3'端非翻译区长度各为70 bp和271 bp.Blast同源性分析显示该基因编码的氨基酸序列与拟南芥、大豆、水稻、玉米、菠菜等物种6PGDH 基因有74%以上的一致性.由于与其他物种胞质6PGDH相类似氨基酸N端都缺少长度约为40aa的转运肽,推断Cs6PGDH为黄瓜胞质6-磷酸葡萄糖酸脱氢酶基因.     

Purification and Characterization of Glyoxysomal Enzymes from Germinating Pumpkin Cotyledons

Four glyoxysomal enzymes, malate synthase, malate dehydrogenase,3-hydroxyacyl-CoA dehydrogenase and citrate synthase, were purifiedfrom glyoxysomes of germinating pumpkin cotyledons. Molecularweights of their subunits were as follows: malate synthase,60,000; malate dehydrogenase, 33,000; 3-hydroxyacyl-CoA dehydrogenase,72,000 and citrate synthase, 45,000. Malate synthase and 3-hydroxyacyl-CoAdehydrogenase activities were exclusively localized in glyoxysomes,whereas malate dehydrogenase and citrate synthase activitieswere found in both glyoxysomes and mitochondria. Monospecificantibodies against malate dehydrogenase and citrate synthaseinhibited their activities present in glyoxysomes but in mitochondria.Immunocytochemical analysis using the protein A-gold techniquecombined with Lowicryl K4M embedding showed that the antigenicsites for these enzymes were found exclusively in glyoxysomes.These data indicates that malate dehydrogenase and citrate synthasepresent in glyoxysomes are immunologically different from thosein mitochondria, respectively. ¹ This is paper No. 9 in the series "Analytical Studies on MicrobodyTransition". ³ Present address: Meiji Institute of Health Science, Naruta,Odawara, Kanagawa 250, Japan. ⁵ Present address: Department of Biology, Faculty of Science,Kobe University, Rokkoudai, Nada, Kobe 657, Japan. (Received December 23, 1987; Accepted January 27, 1988)     

小球藻NADP-谷氨酸脱氢酶的cDNA克隆及转基因烟草分析

用RT-PCR方法从小球藻(Chlorella sorokiniana)中克隆了铵诱导表达的以辅酶Ⅱ为辅基的谷氨酸脱氢酶(NADP-GDH)基因的cDNA片段,DNA测序分析表明与已报道的该基因c DNA序列同源性为94%.将NADP-GDH基因先插入到SPDK621质粒的2CaMV35S启动子和Ω增强序列之后,然后将2CaMV35S-Ω-GDH-NOS表达单元构建到RokⅡ质粒的HindⅢ与Eco RⅠ之间,从而获得高效植物表达载体.将RokⅡ-GDH质粒转移到根癌土壤杆菌(Agro bacterium tumefaciens (Smith et Townsend) Conn) EHA105中,对烟草(Nico tiana tabacum L.)进行转化并得到阳性转化后代.对转基因烟草分析表明,在低氮培养基或在低氮蛭石中其生长速度和叶片数明显高于对照;铵毒性实验表明,无论在低铵或高铵条件下,接种在MS固化培养基上的转基因绿叶圆片存活时间长,叶绿素含量高.这些结果说明外源NADP-GDH增强了植物对氮素的吸收和利用.另外,转化后代还表现了对除草剂膦化麦黄酮(PPT)具有较强的抗性;培养在含有不同浓度PPT的MS固化培养基上的转基因绿叶圆片,其愈伤化程度明显高于对照;在MS培养基中用0.5 μg/mL 的PPT可以代替卡那霉素对转化后代进行筛选,这暗示 NADP-GDH基因可以作为一种新的选择标记用于植物基因工程的研究.     

小球藻NADP—谷氨酸脱氢酶的cDNA克隆及转基因烟草分析

用RT_PCR方法从小球藻 (Chlorellasorokiniana)中克隆了铵诱导表达的以辅酶Ⅱ为辅基的谷氨酸脱氢酶(NADP_GDH)基因的cDNA片段 ,DNA测序分析表明与已报道的该基因cDNA序列同源性为 94%。将NADP_GDH基因先插入到SPDK6 2 1质粒的 2CaMV35S启动子和Ω增强序列之后 ,然后将 2CaMV35S_Ω_GDH_NOS表达单元构建到RokⅡ质粒的HindⅢ与EcoRⅠ之间 ,从而获得高效植物表达载体。将RokⅡ_GDH质粒转移到根癌土壤杆菌(Agrobacteriumtumefaciens (SmithetTownsend)Conn)EHA10 5中 ,对烟草 (NicotianatabacumL .)进行转化并得到阳性转化后代。对转基因烟草分析表明 ,在低氮培养基或在低氮蛭石中其生长速度和叶片数明显高于对照 ;铵毒性实验表明 ,无论在低铵或高铵条件下 ,接种在MS固化培养基上的转基因绿叶圆片存活时间长 ,叶绿素含量高。这些结果说明外源NADP_GDH增强了植物对氮素的吸收和利用。另外 ,转化后代还表现了对除草剂膦化麦黄酮 (PPT)具有较强的抗性 ;培养在含有不同浓度PPT的MS固化培养基上的转基因绿叶圆片 ,其愈伤化程度明显高于对照 ;在MS培养基中用 0 .5 μg/mL的PPT可以代替卡那霉素对转化后代进行筛选 ,这暗示NADP_GDH基因可以作为一种新的选择标记用于植物基因工程的研究。     

一种人肝辅酶Ⅱ依赖性视黄醇脱氢酶剪接体cDNA的克隆及特征分析

在用RT-PCR法局部扩增人与小鼠肝脏635bp的NRDR DNA时,在人肝中发现了另一短序列PCR产物,克隆后测序显示其整个序列与NRDR cDNA编码区的前后序列完全一致。采用3’-Race和5’-Race方法,从人肝组织细胞中扩增得到两个全长cDNA,除1261bp的NRDR cDNA外,另一个为全长1003bp、编码区长为525bp的NRDRiso(GenBank登录号：AY071856)。数据库分析表明,NRDRiso编码区是由人NRDR8个外显子中的第1、2、3、7、8外显子选择性剪接而成。缺失的NRDR第4、5、6外显子共258bp,编码86个氨基酸。因此,与人NRDR的260个氨基酸残基相比,NRDRiso由174个氨基酸残基组成,分子量为18．6kDa,并且NRDRiso的组织表达与NRDR明显不同。     

Comparative Genome-Wide Analysis of the Malate Dehydrogenase Gene Families in Cotton

Malate dehydrogenases (MDHs) play crucial roles in the physiological processes of plant growth and development. In this study, 13 and 25 MDH genes were identified from Gossypium raimondii and Gossypium hirsutum, respectively. Using these and 13 previously reported Gossypium arboretum MDH genes, a comparative molecular analysis between identified MDH genes from G. raimondii, G. hirsutum, and G. arboretum was performed. Based on multiple sequence alignments, cotton MDHs were divided into five subgroups: mitochondrial MDH, peroxisomal MDH, plastidial MDH, chloroplastic MDH and cytoplasmic MDH. Almost all of the MDHs within the same subgroup shared similar gene structure, amino acid sequence, and conserved motifs in their functional domains. An analysis of chromosomal localization suggested that segmental duplication played a major role in the expansion of cotton MDH gene families. Additionally, a selective pressure analysis indicated that purifying selection acted as a vital force in the evolution of MDH gene families in cotton. Meanwhile, an expression analysis showed the distinct expression profiles of GhMDHs in different vegetative tissues and at different fiber developmental stages, suggesting the functional diversification of these genes in cotton growth and fiber development. Finally, a promoter analysis indicated redundant but typical cis-regulatory elements for the potential functions and stress activity of many MDH genes. This study provides fundamental information for a better understanding of cotton MDH gene families and aids in functional analyses of the MDH genes in cotton fiber development.     

粉尘螨线粒体样苹果酸脱氢酶基因cDNA克隆和分子特征分析

摘要 目的：获取粉尘螨线粒体样苹果酸脱氢酶蛋白（mitochondrial-like malate dehydrogenase，mMDH）的编码基因并了解其分子特征。方法：以粉尘螨Total RNA为模板，RT-PCR扩增获得mMDH编码基因后、构建原核表达质粒，转化E.coil BL21(DE3)感受态细胞中，IPTG诱导表达，SDS-PAGE和Western Blot鉴定目的蛋白表达情况。采用NCBI、EXPASY在线生物信息学软件分析该基因编码蛋白质的生物学特征。结果：获得粉尘螨线粒体样苹果酸脱氢酶蛋白的编码基因，全长1032bp。构建的原核表达质粒pET28a(+)-mMDH，经转化和诱导表达后，SDS-PAGE和Western Blot可见目的蛋白条带。该基因编码的蛋白质由343个氨基酸组成，相对分子质量（Mr）36014.54Da，亚细胞定位主要在细胞质和细胞核，含有34个磷酸化位点（19个丝氨酸，12个苏氨酸和3个酪氨酸）。糖基化预测结果显示其含有2个N-糖基化位点（123位存在糖基化位点NASI和151位存在糖基化位点NSTV）和1个0-糖基化位点。二级结构主要为?琢-螺旋和无规则卷曲。三维建模可观察到该蛋白为二聚体结构，CD-Search保守区域分析后显示其属于NADB-Rossmann家族，具有MDH-glyoxysomal-mitochondrial结构域。PyMol可视化后可在三维结构中观察到保守区域位点。将该基因推导出的氨基酸序列进行Blast获得同源基因，粉尘螨与屋尘螨、梅氏嗜霉螨进化关系较近，独成一簇。结论：获得粉尘螨线粒体样苹果酸脱氢酶蛋白的编码基因全长及其原核表达质粒，并对其生物学特征进行分析，为进一步探讨该基因的生理功能、开发尘螨控制措施奠定基础。     

Polymorphism of Microbody Malate Dehydrogenase in Opuntia basilaris

Electrophoretic survey of malate dehydrogenase (EC 1.1.1.37) in Opuntia basilaris showed intraspecific polymorphism. Further experiments with microbody malate dehydrogenase-specific antiserum suggest that the polymorphism occurs in microbody malate dehydrogenase independent of the soluble and mitochondrial forms. The pattern of polymorphism is one expected from a two-allele Mendelian system.