烟草乙烯受体类似物NTHK2全长基因的克隆及其激酶结构域生化特性

应用5′-ARCE方法克隆到烟草NTHK2的全长cDNA。其全长cDNA共有3216bp,其中5′非编码区为509bp,3′非编码区为427bp,编码区为2280bp,编码产物为760个氨基酸。NTHK2氨基酸序列与植物中的许多杂合型的两组分乙烯受体基因有较高的同源性,具有推测的组氨酸激酶结构域和接受域。但是,在激酶结构域中没有保守的组氨酸,而是被一个天冬氨酸残基所替代。为了研究其生化特性,在酵母中以融合蛋白的形式表达了激酶结构域,体外激酶分析表明,当有Mg^2 存在的情况下NTHK2能够自我磷酸化。进一步的研究应阐明NTHK2在植物体内是否能够作为乙烯受体。参与乙烯的信号传导过程。     

猪苓菌丝形成菌核的MAPK基因克隆及表达分析

【目的】克隆药用真菌猪苓MAPK基因并进行生物信息学分析及表达模式研究。【方法】利用5′-RACE-PCR技术从猪苓菌丝中克隆得到MAPK基因全长,利用生物信息学软件推测蛋白的理化性质、结构域;DNA Star对氨基酸进行多序列比对;用MEGA 5.0做进化关系分析;借助实时定量PCR检测基因表达模式。【结果】猪苓MAPK基因的全长cDNA为1 293 bp,其中编码区占1 161 bp,共编码386个氨基酸,推测分子量为43.872 kD,理论等电点为6.68。猪苓的MAPK有MAPK中ERK1/2类型的保守区。系统进化树结果显示猪苓MAPK蛋白属于担子菌类群。实时荧光定量PCR分析结果表明猪苓菌核形成初期,菌核中的MAPK表达量显著高于菌丝组织,随着菌核的快速生长而减少。【结论】猪苓MAPK基因PuMAPK的分子特征为进一步研究其在猪苓菌丝形成菌核过程中的作用奠定基础。     

水稻乙烯受体类似物基因的克隆及其表达特性

乙烯在植物的生长发育以及对逆境的反应中起着重要作用. 乙烯受体基因在拟南芥、 烟草和番茄等双子叶植物中已有一些研究, 但到目前为止还没有见到关于单子叶植物乙烯受体研究的报道. 我们从水稻中克隆了一个乙烯受体基因OSPK2, 发现它所编码的蛋白与双子叶植物的乙烯受体有所不同. OSPK2的N端较长, 其后是3个跨膜区、一个GAF结构域、一个推测的激酶结构域和接受器结构域. 虽然大多数的结构域都是保守的, 但预期的磷酸化位点His和磷酸基团接受位点Asp在OSPK2中分别被Gln和Asn取代. 这一事实说明, OSPK2可能不是以组氨酸激酶的磷酸转移方式进行作用, 而是以其他的机制, 如具有丝/苏激酶的活性. 应用RT-PCR方法对不同条件下OSPK2基因的表达进行了研究, 结果表明, OSPK2受伤害和PEG处理诱导, 但盐及ABA处理对其没有显著影响. OSPK2基因的差异表达可能反映了它在介导不同非生物逆境反应中的作用, 这与我们以前关于烟草乙烯受体的研究结果是一致的.     

蜻蜓凤梨花发育相关B类MADS-box基因克隆及表达分析

为探索MADS-box基因在凤梨花发育过程中的调控机制,通过设计简并引物,利用RACE技术,从蜻蜓凤梨花蕾中分离得到2个花发育相关B类MADS-box基因,分别命名为AfAP3和AfPI;AfAP3cDNA全长957bp,编码区编码226个氨基酸;AfPI cDNA全长808bp,编码区编码198个氨基酸,二者均具有典型的植物MADS-box蛋白结构.RT-PCR分析结果表明,AfAP3和AfPI基因主要在花器官中表达,在根系中也有微量表达;乙烯诱导后7d,AfPI基因在茎尖处开始有表达,表明此时蜻蜓凤梨花芽分化可能已经完成,AfAP3基因表达晚于AfPI.     

高山离子芥MAP激酶基因CbMAPK3的克隆

从高山离子芥中克隆得到了一种新的MAPK基因的cDNACbMAPK3,全长1419bp,其中包括1110bp的开放阅读框、91bp的5＇非翻译区（5＇UTR）和218bp的3＇非翻译区（3＇UTR）。CbMAPK3基因编码369个氨基酸的蛋白质,分子量为42.5kDa,等电点为5.79,含有MAP激酶所具有的11个保守序列区以及MAP激酶的磷酸化位点TEY基序。CbMAPK3蛋白与响应环境胁迫有关的植物MAPK亚类有很高的同源性。半定量分析表明,该基因表达受低温胁迫诱导。     

西伯利亚蓼钙调蛋白基因的克隆及其在盐胁迫下的表达

已从西伯利亚蓼叶中cDNA文库中获得的钙调蛋白EST序列,采用cDNA末端快速扩增（RACE）技术克隆了具有完整编码区的钙调蛋白基因的cDNA序列（GenBank登录号GQ988382）,命名为PsCaM。该基因全长615bp,编码区为450bp,编码149个氨基酸,5＇非翻译区为63bp,3＇非翻译区为102bp。同源性分析表明,该蛋白与其他植物钙调蛋白高度保守,氨基酸同源性高达98%。用实时荧光定量PCR研究3%NaHCO3胁迫下西伯利亚蓼基因表达的结果显示,自然条件下,该基因在叶中表达量最高,地下茎次之,茎中最低;盐胁迫下CaM在西伯利亚蓼的地下茎、茎和叶中均有表达,表达模式不同。     

一种新的甘露糖结合凝集素--朱顶兰凝集素基因的克隆及序列分析

运用同源克隆的方法设计简并引物,通过3′和5′RACE技术,从石蒜科植物朱顶兰(Amaryllis vittata Ait)总RNA中克隆了编码此凝集素(AVA)的全长cDNA序列.该基因全长686 bp,起始密码子位于第41～43 bp,终止密码子位于515～517 bp处,开放阅读框长474 bp,编码158个氨基酸,包含信号肽序列、成熟蛋白序列和C-末端剪切序列的前体蛋白.成熟蛋白由109个氨基酸残基组成,分子量为11.9kD.成熟蛋白在氨基酸水平上与雪花莲凝集素、水仙凝集素、石蒜凝集素和君子兰凝集素分别有73.4%、85.3%、80.7%和83.5%的同源性;朱顶兰凝集素的分子模式显示其与雪花莲凝集素有极其相似的三维结构;在Blocks数据库中检索AVA蛋白氨基酸序列的结构域,发现有3个凝集素功能结构域,并具有3个典型的甘露糖专一结合位点盒(QDNY).     

编码人分化抗原5D4的全长cDNA克隆及 其编码蛋白质的二级结构分析*1)

用3D5细胞染色强阳性,而Daudi与Jurkat细胞染色弱阳性的单克隆抗体5D4,从一个人扁桃体细胞λgt11 cDNA文库(ATCC No.37546)筛选到一个长1 846 bp的cDNA克隆.RT-PCR显示3D5细胞、Daudi细胞和Jurkat细胞5D4 mRNA均阳性,但3D5细胞明显强于后两者.5D4 cDNA中88～1 209 bp为一个开放阅读框架,编码374个氨基酸的蛋白质.Northern blot分析结果和起始密码子前5′端非编码区有2个连续的终止密码子表明,这是一个全长cDNA.从其开放阅读框架推断的蛋白质的氨基酸序列中295～334 aa为一强疏水区,预测313～334 aa为一分值极高的跨膜螺旋区,其方向最可能是由膜内到膜外.     

栀子ERF基因的克隆及其在果实成熟过程中的表达

乙烯反应元件因子(ethylene-responsive element-binding factor,ERF)是一类植物DNA结合蛋白,是乙烯信号传导过程中的一类转录因子,与植物的生长发育和生理过程有关。从栀子(Gardenia jasminoidesEllis)果实的cDNA文库中筛选获得栀子乙烯反应元件因子GjERF的cDNA。该基因全长962 bp,5’端非翻译区长82 bp,3’端非翻译区长100 bp,预测ORF为780 bp,编码259个氨基酸,分子量为28.6 kD。序列分析表明GjERF含有59个氨基酸构成的AP2/ERF结构域及N端的MC(MCGGAII)模体,它属于ERF家族的第Ⅶ亚类。RT-PCR分析表明GjERF基因在栀子成熟叶片中的表达高于在果实中的表达,并且在果实中的表达与果实的成熟过程无关。     

编码人分化抗原5D4的全长cDNA克隆及其编码蛋白质的二级结构分析

用 3D5细胞染色强阳性 ,而Daudi与Jurkat细胞染色弱阳性的单克隆抗体5D4,从一个人扁桃体细胞λgt1 1cDNA文库 (ATCCNo .375 46 )筛选到一个长 1 846bp的cDNA克隆 .RT_PCR显示 3D5细胞、Daudi细胞和Jurkat细胞 5D4mRNA均阳性 ,但 3D5细胞明显强于后两者 . 5D4cDNA中 88～ 1 2 0 9bp为一个开放阅读框架 ,编码374个氨基酸的蛋白质 .Northernblot分析结果和起始密码子前 5′端非编码区有 2个连续的终止密码子表明 ,这是一个全长cDNA .从其开放阅读框架推断的蛋白质的氨基酸序列中 2 95～ 334aa为一强疏水区 ,预测 31 3～ 334aa为一分值极高的跨膜螺旋区 ,其方向最可能是由膜内到膜外 .     

Spatial expression and characterization of a putative ethylene receptor protein NTHK1 in tobacco

A putative ethylene receptor gene NTHK1 encodes a protein with a putative signal peptide, three transmembrane segments, a putative histidine kinase domain and a putative receiver domain. The receiver domain was expressed in an Escherichia coli expression system, purified and used to generate polyclonal antibodies for immunohistochemistry analysis. The spatial expression of the NTHK1 protein was then investigated. We found that NTHK1 was abundant during flower and ovule development. It was also expressed in glandular hairs, stem, and in leaves that had been wounded. The NTHK1 gene was further introduced into the tobacco plant and we found that, in different transgenic lines, the NTHK1 gene was transcribed to various degrees. Upon ACC treatment, the etiolated transgenic seedlings showed reduced ethylene sensitivity when compared with the control, indicating that NTHK1 is a functional ethylene receptor in plants.     

Serine/threonine kinase activity in the putative histidine kinase-like ethylene receptor NTHK1 from tobacco

A histidine kinase-based signaling system has been proposed to function in ethylene signal transduction pathway of plants and one ethylene receptor has been found to possess His kinase activity. Here we demonstrate that a His kinase-like ethylene receptor homologue NTHK1 from tobacco has serine/threonine (Ser/Thr) kinase activity, but no His kinase activity. Evidence obtained by analyzing acid/base stability, phosphoamino acid and substrate specificity of the phosphorylated kinase domain, supports this conclusion. In addition, mutation of the presumptive phosphorylation site His (H378) to Gln did not affect the kinase activity whereas deletion of the ATP-binding domain eliminated it, indicating that the conserved His (H378) is not required for the kinase activity and this activity is intrinsic to the NTHK1-KD. Moreover, confocal analysis of NTHK1 expression in insect cells and plant cells suggested the plasma membrane localization of the NTHK1 protein. Thus, NTHK1 may represent a distinct Ser/Thr kinase-type ethylene receptor and function in an alternative mechanism for ethylene signal transduction.     

Roles of ethylene receptor NTHK1 domains in plant growth, stress response and protein phosphorylation

Ethylene receptors sense ethylene and regulate downstream signaling events. Tobacco ethylene receptor NTHK1, possessing Ser/Thr kinase activity, has been found to function in plant growth and salt-stress responses. NTHK1 contains transmembrane domains, a GAF domain, a kinase domain and a receiver domain. We examined roles of these domains in regulation of plant leaf growth, salt-stress responses and salt-responsive gene expressions using an overexpression approach. We found that the transgenic Arabidopsis plants harboring the transmembrane domain plus kinase domain exhibited large rosettes, had reduction in ethylene sensitivity, and showed enhanced salt sensitivity. The transgenic plants harboring the transmembrane domain plus GAF domain also showed larger rosettes. Truncations of NTHK1 affected salt-induced gene expressions. Transmembrane domain plus kinase domain promoted RD21A and VSP2 expression but decreased salt-induction of AtNAC2. The kinase domain itself promoted AtERF4 gene expression. The GAF domain itself enhanced Cor6.6 induction. Moreover, the NTHK1 functional kinase domain phosphorylated the HIS and ATP subdomains, and five putative phosphorylation sites were identified in these two subdomains. In addition, the salt-responsive element of the NTHK1 gene was in the transmembrane-coding region but not in the promoter region. These results indicate that NTHK1 domains or combination of them have specific functions in plant leaf growth, salt-stress response, gene expression and protein phosphorylation.     

烟草乙烯受体研究进展(综述)

对近几年有关烟草乙烯受体基因研究的最新进展作简要介绍,并就今后该领域的研究方向进行探讨。已知烟草乙烯受体家族至少包括NtETR1、NtERS1、NTHK1和NTHK2等4种基因,其中NTHK1和NTHK2同源且有相似结构,两者的激酶活性与细菌双组分调节系统非常相似,激酶活性需要一些二价阳离子的参与。烟草乙烯受体在细胞内的作用位点还缺少研究。     

A two-component gene (NTHK1) encoding a putative ethylene-receptor homolog is both developmentally and stress regulated in tobacco

The full-length of a two-component gene NTHK1 (Nicotiana tabacum histidine kinase-l) was isolated from tobacco (N. tabacum var. Xanthi) using a previously obtained NTHK1 cDNA fragment as a probe. Sequence analysis revealed that NTHK1 shared high homology with LeETR4 from tomato and encoded an ethylene- receptor homolog. The predicted NTHK1 protein had a putative signal peptide, three transmembrane domains, a histidine kinase domain and a receiver domain. The putative autophosphorylation site at His378 and the phosphate receiver site at Asp689 were also identified. By using the in situ hybridization technique, NTHK1 mRNA was detected during flower organ development. It is also highly expressed in the processes of pollen formation and embryo development. The expression of NTHK1 in response to wounding and other stresses was investigated using competitive RT-PCR. The results demonstrated that NTHK1 was inducible upon wounding (cutting). Floating of the cut leaf pieces in 0.5× MS, with shaking, led to a relatively rapid and strong expression. This phenomenon was confirmed by the in situ hybridization results. In addition to the up-regulation by wounding, NTHK1 expression was also induced following NaCl and PEG treatment, indicating a possible role for NTHK1 in multiple stress responses. Received: 28 June 2000 / Accepted: 1 August 2000     

黄粉虫热休克蛋白70基因的克隆、序列分析与表达（英文）

为给黄粉虫Tenebrio molitor抗逆机理研究提供理论依据, 本研究采用PCR和RACE法从黄粉虫幼虫中克隆出一个热休克蛋白70基因Tmhsp70, 并运用半定量RT-PCR法检测其在黄粉虫不同发育阶段的mRNA表达水平。结果表明： 克隆出的Tmhsp70 序列全长2 282 bp, 具有一个富含A的115 bp 5′ 非翻译区和一个1 935 bp的开放阅读框及一个富含A、 T的232 bp 3′-非翻译区。5′-非翻译区含有7个热休克元件nGAAn, 3′-非翻译区末端有长22 bp的Poly(A)尾。Tmhsp70编码的黄粉虫热休克蛋白（TmHSP70）具有3个典型的HSP70特征基序（IDLGTTYS, IFDLGGGTFDVSIL和IVLVGGSTRIPKIQQ）和1个胞质HSP70末端特征基序（EEVD）, 无信号肽和跨膜区域, 包含2个主要的结构域, 即： N-端42 kDa的高度保守ATPase功能域和C-端18 kDa的保守多肽结合功能域。ATPase功能域的三级结构由2个大球形亚功能域组成, 具有1个核苷酸结合中心; 多肽结合功能域形成1个双层4股β-折叠片样的三明治结构和2个α-螺旋, 内含1个多肽结合通道。此外, 黄粉虫Tmhsp70 mRNA的表达具有热激诱导和发育调控的特征。半定量RT-PCR分析表明, 42℃热激1 h的黄粉虫各发育阶段Tmhsp70 mRNA的表达量上升了1.4~26.9倍。25℃下1日龄黄粉虫蛹中的Tmhsp70 mRNA 表达量要高于其余各发育阶段的累积表达量; 42℃热激1 h 后90日龄幼虫中的Tmhsp70 mRNA 表达量最丰富, 既高于30日龄和60日龄幼虫中的累积表达量, 也高于15日龄和30日龄成虫中的累积表达量。这些结果为进一步研究黄粉虫热休克蛋白的结构、 功能和表达调控及其与抗逆性的关系奠定了基础。     

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。