稻属中一个高度保守和组成型表达酪氨酸丝氨酸-酸酸双特性蛋白激酶基因的克隆与分析

从水稻中克隆了一个在稻属植物中高度保守和组成型表达的丝氨酸􊄯苏氨酸蛋白激酶基因(OsSTK)。该基因包含两个外显子和一个114 bp 的小内含子序列, 预测编码一个419 个氨基酸的蛋白质。该基因推导的氨基酸序列与其它已知序列的一致性均低于52%。利用从不同种和类型的野生稻克隆的部分该基因序列构建的系统树与野生稻的分类和进化关系相一致。OSPK N-端拥有一段富含丝氨酸、碱性氨基酸和带电荷氨基酸的特异性导肽序列, 其中包含“GDGDGDGDG”短重复序列。由于该基因蛋白激酶结构域中的VIb , VIII 和XI 亚结构域中同时具有酪氨酸蛋白激酶和丝氨酸􊄯苏氨酸蛋白激酶的特性, 推测该基因可能同时具有催化酪氨酸和丝氨酸、苏氨酸磷酸化的双重功能。     

玉米中一种新的蛋白激酶电子克隆与序列分析

目的：电子克隆玉米中一种新的蛋白激酶基因。方法：以拟南芥中一个蛋白激酶的氨基酸序列为探针,对玉米的EST数据库进行同源性检索和筛选并克隆。结果：序列分析显示该cDNA长1121bp,有一个450bp的开放阅读框,编码149个氨基酸,且具有保守苏氨酸/丝氨酸蛋白激酶结构域和TEY基元,说明所克隆的cDNA序列为玉米的MAPK全长cDNA。结论：所克隆的cDNA序列为玉米的MAPK全长cDNA。     

蛋白激酶研究进展：Ⅰ.结构和分类

蛋白激酶含有２５０～３００个氨基酸残基构成的催化功能域,包括１２个功能亚域．这些氨基酸序列折叠成为一个核心催化结构．根据蛋白激酶功能域氨基酸序列比较分析可以找出各蛋白激酶在进化上的亲缘关系,这是蛋白激酶的分类基础．蛋白激酶可分为二大类,一类是丝氨酸／苏氨酸蛋白激酶,另一类是酪氨酸蛋白激酶．丝氨酸／苏氨酸蛋白激酶在进化上出现较早,可分为几个组,每个组又分为许多家族．酪氨酸蛋白激酶在进化上出现较晚,亲缘关系较紧密,同属于一个组,该组也分为许多家族．最近发现的许多双底物特异蛋白激酶,分散在丝氨酸／苏氨酸蛋白激酶的不同家族中．     

小粒野生稻STK类抗病基因同源序列的克隆与分析

根据丝氨酸/苏氨酸蛋白激酶类(serine-threonine kinase,STK)抗病基因结构中保守结构域,设计引物,以小粒野生稻基因组DNA为模板,扩增获得10条STK类抗病基因同源序列.同源性分析表明其都具有STK保守结构域,与已克隆的STK类抗病基因有不同程度的相似性,为进一步克隆小粒野生稻中的STK类抗病基因提供依据.     

大叶落地生根类糖原合成激酶KdSK基因的克隆与表达分析

类糖原合成酶激酶(SKs)属于丝氨酸/苏氨酸类蛋白激酶,在植物器官发育、激素信号传导过程中十分重要,并参与生物胁迫、非生物胁迫的应答过程。大叶落地生根中的胎生苗发育过程,同时具备胚胎发生和器官发生的特征,是研究无性生殖的理想模型。为了更好地理解大叶落地生根中胎生苗发育的分子机制,该研究利用RACE-PCR技术,从大叶落地生根中克隆了1个新的基因KdSK。该基因具有423个氨基酸残基,分子量为47.79 kD,等电点为8.37,其开放阅读框长为1 272 bp。其蛋白与黄瓜的同源性最高,属于植物类GSK3/shaggy蛋白激酶家族的第Ⅳ类,与苜蓿(MSK4)蛋白在进化关系上最近,且与拟南芥(AtSK4-1、AtKSK4-2)聚为一枝。保守域结构分析表明,KdSK蛋白具有明显的蛋白激酶的结构域,包括蛋白激酶的ATP结构域和丝氨酸/苏氨酸蛋白激酶活化结构域。实时荧光定量PCR分析表明,该蛋白基因在大叶落地生根的根中表达量最高,且受渗透胁迫(甘露醇)的诱导上调表达。该研究首次从大叶落地生根中克隆出KdSK基因,该研究结果为进一步研究该基因的功能打下了基础。     

水稻蛋白激酶基因Ptil的克隆与分析

植物Ptil基因编码丝氨酸／苏氨酸蛋白激酶,是重要的抗病相关基因。在水稻抗褐飞虱基因Qtil所在的染色体区段存在一个与番茄Ptil基因高度同源的序列片段。从抗虫水稻B5中分离了Ptil基因的全长cDNA克隆,测定了基因组序列。分析发现,水稻中Ptil基因长度有5644bp,含有7个内含子,编码368个氨基酸的激酶蛋白。其蛋白质的C末端在不同植物之间具有高度的保守性,而N末端的变异则相对较大。对不同水稻材料Ptij基因的序列进行了比较,发现药用野生稻与栽培稻之间存在较大的差异,而栽培稻各品种之间的差异较小。讨论了Ptil基因在抗虫防卫反应中可能的作用。     

内蒙古白绒山羊erk2基因的克隆及表达模式分析

旨在克隆内蒙古白绒山羊erk2基因cDNA并分析其基本表达模式。采用RT-PCR方法克隆白绒山羊erk2基因cDNA。通过在线软件Blast进行核酸序列分析,用SMART与Psite进行氨基酸序列分析。定量RT-PCR检测erk2基因在绒山羊组织中的表达特异性。免疫组化法检测绒山羊睾丸中erk2表达。克隆到的内蒙古白绒山羊erk2基因cDNA片段 (GenBank Accession No.JX569765) 长1 083 bp,包含了编码360个氨基酸残基的全长ORF,氨基酸序列与牛的ERK2 (Bos Taurus BC133588.1) 同源性为100％。SMART分析表明,ORF编码的蛋白包含了活化位点“TEY”及具有丝氨酸/苏氨酸激酶催化活性的S-TKc结构域。Psite分析表明,含2个N-糖基化位点、1个依赖于cAMP/cGMP的蛋白激酶磷酸化位点、3个蛋白激酶c磷酸化位点、5个酪蛋白激酶Ⅱ磷酸化位点、2个N-豆蔻酰化位点、2个异戊二烯基结合区 (CAAX box)、7个微体羧基端靶向信号、2个蛋白激酶ATP结合区标记及一个丝/苏氨酸蛋白激酶活性区域标记。PSORT (k-NN prediction) 程序预测其定位于细胞质中。定量RT-PCR分析显示erk2基因mRNA丰度在心脏、皮肤以及乳腺组织中mRNA丰度较高,脾、肾中的表达相对较低。在睾丸中检测到ERK2蛋白表达。     

一个小麦丝氨酸—苏氨酸蛋白激酶基因的克隆和分析

用mRNA差异显示技术在含有抗白粉病基因Pm2 1的小麦 (TriticumaestivumL .)_簇毛麦 (Haynaldiavillosa)6VS/ 6AL易位系 92R137中分离与抗白粉病相关的基因 ,获得一个命名为TaPK1的全长cDNA克隆。序列分析表明 ,它与大豆 (Glycinemax (L .)Merr.)蛋白激酶基因GmPK6高度同源。经推测 ,TaPK1编码 416个氨基酸的多肽 ,属丝氨酸_苏氨酸蛋白激酶家族 ,并具酪氨酸激酶特性。TaPK1是从小麦中分离的新基因。     

棘孢木霉丝裂原活化蛋白激酶基因task1的克隆及序列分析

为深入研究丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)的功能,从棘孢木霉(Tricho-derma asperellum)中克隆了丝裂原活化蛋白激酶(MAPK)基因task1,并对其序列进行分析。该基因编码355个氨基酸,全长1 757 bp,理论分子质量41.1 kD,理论等电点为6.64,与深绿木霉(T.atroviride)MAPK基因tmk1、里氏木霉(T.reesei)MAPK基因tmkA和绿色木霉(T.virens)MAPK基因tmkA在氨基酸和核苷酸水平上同源性都很高,蛋白结构预测为丝氨酸/苏氨酸蛋白激酶。     

水稻蛋白激酶基因Pti1的克隆与分析

植物Pti1基因编码丝氨酸/苏氨酸蛋白激酶,是重要的抗病相关基因。在水稻抗褐飞虱基因Qbp1所在的染色体区段存在一个与番茄Pti1基因高度同源的序列片段。从抗虫水稻B5中分离了Pti1基因的全长cDNA克隆,测定了基因组序列。分析发现,水稻中Pti1基因长度有5644bp,含有7个内含子,编码368个氨基酸的激酶蛋白。其蛋白质的C末端在不同植物之间具有高度的保守性,而N末端的变异则相对较大。对不同水稻材料Pti1基因的序列进行了比较,发现药用野生稻与栽培稻之间存在较大的差异,而栽培稻各品种之间的差异较小。讨论了Pti1基因在抗虫防卫反应中可能的作用。     

Cloning and Analyses of a Dual Specific Serine􊄯Thronine Protein Kinase Gene with High Conservative and Constitutive Expression in Oryza (OsSTK)

A cytoplasmic serine􊄯thronine protein kinase gene (OsSTK), had been cloned from Oryza genus. It was found high conservative and constitutive expression in Oryza. OsSTK gene had two exons, separated by 114 bp short intron. The open reading frame of OsSTK gene that predicted encoded a 419 amino acids protein . The amino acid sequence of OsSTK had low identities ( less than 53% ) with any other known protein kinase . The phylogenetic tree based on the partial DNA sequences of OsSTK from different species and types of wild rice and cultivated rice, was close to the taxation system ofrice . Interestingly , OsSTK had a serine, including basic amino acids and charged amino acids abundant polypeptide with a “GDGDGDGDG”sequence at N- terminal that had not been found in any other genes. OsSTK may play dual specificity that phosphorylates both serine􊄯thronine and tyrosone, because the amino acids module of VIb , VIII and XI catalytic domain have both the serine􊄯thronine and tyrosine kinase characters .     

Rice serine/threonine kinase 1 is required for the stimulation of OsNug2 GTPase activity

Several GTPases are required for ribosome biogenesis and assembly. We recently identified rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), a YlqF/YawG family GTPase, as having a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating OsNug2 function, yeast two-hybrid screens were performed using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a candidate interacting protein. OsSTK1 appeared to interact with OsNug2 both in vitro and in vivo. OsSTK1 was found to have no effect on the GTP-binding activity of OsNug2; however, the presence of recombinant OsSTK1 in OsNug2 assay reaction mixtures increased OsNug2 GTPase activity. A kinase assay showed that OsSTK1 had weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Using yeast complementation testing, we identified a GAL::OsNug2(S209N) mutation-harboring yeast strain that exhibited a growth-defective phenotype on galactose medium at 39 °C, which was divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of OsNug2(S209N), which was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings indicate that OsSTK1 functions as a positive regulator of OsNug2 by enhancing OsNug2 GTPase activity. In addition, phosphorylation of OsNug2 serine 209 is essential for its complete function in biological functional pathway.     

对水稻4号染色体一个编码S位点相关的受体样蛋白激酶基因簇的分析

通过对水稻 (OryzasativaL .) 4号染色体一段 32 3kb的序列测定和分析 ,在其中 10 8kb的区域内发现了一个由 14个编码S位点相关的受体样蛋白激酶 (SRK)基因组成的基因簇。RT_PCR实验证明了这 14个基因中有 9个基因表达 ,并且这 9个基因有不同的表达模式 :其中 2个基因主要在生殖器官中表达 ,而另外 7个基因在水稻的营养和生殖器官中均有表达。对这些基因的预测的氨基酸序列进行分析表明他们的细胞外受体部分均和甘蓝的SLG蛋白高度同源 ,而细胞内的激酶区都包含有丝氨酸 /苏氨酸激酶中特异的氨基酸。     

A novel transmembrane serine/threonine protein kinase gene from a rifamycin SV-producing amycolatopsis mediterranei U32.

Genomic DNA sequencing in the vicinity of methylmalonyl-CoA mutase gene (mutAB) from a rifamycin SV-producing Amycolatopsis mediterranei U32 allowed us to clone, sequence, and identify a gene encoding a novel serine/threonine protein kinase (amk). The sequence contains a complete ORF of 1821 base pairs encoding a predicted protein of 606 amino acids in length. The N-terminal domain of the protein shows significant homology to the catalytic domain of other protein kinases from both prokaryotic and eukaryotic sources. It also contains all the structural features that are highly conserved in active protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP-binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein kinase. This protein kinase gene was expressed in Escherichia coli and was shown to have the ability of autophosphorylation. The autophosphorylated site was found to be the threonine at position 164 by labeled phosphoamino acid analysis and site-directed mutagenesis. The C-terminal half of protein kinase was found to contain strong transmembrane structures by PhoA fusion protein analysis, suggesting that Amk protein kinase is a transmembrane protein. A Southern hybridization experiment showed that this type of protein kinase is distributed ubiquitously and might play significant physiological roles in the various species of streptomycetes. However, overexpression of amk gene in Streptomyces cinnamonensis showed no effect on methylmalonyl-CoA mutase activity, monensin production and the hyphae morphology. Although its biological role is still unknown, Amk protein kinase is the first transmembrane serine/threonine protein kinase described for genus Amycolatopsis.     

Two novel protein kinase genes,OsMSRPK1 andOsMSURPK2, are regulated by diverse environmental stresses in rice

Two novel rice (Oryza sativa L.) protein kinase (PK) genes have been isolated.OsMSRPK1 andOsMSURPK2, which most likely exist as single-copy genes in the rice genome, encode 693 and S03 amino acids polypeptide, respectively, and have the serine/threonine kinase domain of cyclin dependent protein kinase (OsMSRPK1), or the serine/threonine kinase domain and NAF domain (OsMSURPK2). Steady-state mRNA analyses of these PKs, with constitutive expression in the leaves of two-week-old seedlings, revealed thatOsMSRPK1 is up-regulated upon exposure to environmental stresses, whereasOsMSVRPK2 is down-regulated by these same stresses. Furthermore, the two PKs are developmentally regulated in both young and mature rice plants, including in the panicles. These results strongly suggest that the genes have roles in both plant development and in their defense/stress-signaling pathways.     

水稻中水杨酸和茉莉酸特异诱导蛋白激酶基因OsSJMK1的分离和鉴定

裂原激活蛋白激酶（MAPK）级联系统负责把接受自胞外或胞内的信号进一步传递和放大而最终作用于特异的转录因子,从而启动或调控基因的表达。MAPK信号级联系统在细胞分裂、分化、生物胁迫和非生物胁迫等多种信号传递途径中起着十分重要的作用。该文以一个茉莉酸（JA）诱导的表达序列为基础,在水稻中分离到了一个裂原激活蛋白激酶基因OsSJMK1的全长cDNA。序列比较分析表明该基因编码498个氨基酸,蛋白质等电点为8．43,包括完整的MAPK家族的蛋白激酶结构域。OsSJMK1与所有物种的MAPK一样包括蛋白激酶的全部11个次级结构域,在Ⅶ和Ⅷ次级结构域间一个双磷酸化位点;该位点苏氨酸（T）和酪氨酸（Y）残基之间为天冬氨酸（D）,而不是其他MAPK中常见的谷氨酸（E）、脯氨酸（P）或甘氨酸（G）。除了典型的MAPK激酶功能域外,在羧基端还有一段长约150个氨基酸残基的可能参与蛋白互作的结构域。以上这些结构特征表明OsSJMK1属于植物中第v类MAPK家族成员。蛋白激酶结构域序列比较表明OsSJMK1与报道的稻瘟病菌和机械伤害诱导的BWMK1的序列相似性高达81％,而且基因内含子和外显子的组成也非常相似,属于同一亚类,但在蛋白质序列的C端差异却很大。与BWMK1不同,OsSJMK1的表达不受伤害诱导,而受稻瘟病轻微诱导,但在JA和SA（水杨酸）处理早期表达量却迅速升高。在JA处理后1h,OsSJMK1转录水平升高到最大,而12h后回落到处理前的本底水平;在SA处理后30min转录水平就开始上升,2h达到最高值,而随后开始下降到处理前的本底水平。SA类似物BTH也能诱导OsSJMK1的表达。其他一些激素处理（如ABA）和非生物胁迫（如干旱、盐胁迫）都不对基因的表达产生任何影响,而且在植物大部分组织中的表达量都非常低。这些结果说明OsSJMK1可能特异性的参与JA和SA介导的防卫反应。     

NPK15, a tobacco protein-serine/threonine kinase with a single hydrophobic region near the amino-terminus

A cDNA clone (cNPK15) was isolated from tobacco cells in suspension culture, which encodes a predicted protein kinase of 422 amino acids. The predicted NPK15 protein consists of a hydrophobic region near the amino-terminus, a linker domain and the catalytic domain of a protein-serine/threonine kinase in the carboxyl-half. NPK15 was not found to be closely related to any reported protein, but its putative catalytic domain shares some structural similarity with those of receptor-like protein kinases of plants, such as ZmPK1 from Zea mays and TMK1 from Arabidopsis, even though no receptor-like domain is found in NPK15. Recombinant NPK15 expressed in Escherichia coli as a fusion protein was found capable of autophosphorylation and of phosphorylation of the histone H1 protein on both serine and threonine residues. Upon overexpression of cNPK15 under control of the promoter of cauliflower mosaic virus 35S RNA in tobacco cells, into which it had been introduced by Agrobacterium-mediated transformation, the NPK15 gene acted as a suicide gene and blocked proliferation of the host cells. By contrast, such a suicide effect was not observed with the gene for a kinase-negative mutant protein in which the nucleotide sequence for the ATP-binding site had been mutated or with a mutant derivative encoding a protein in which the hydrophobic region had been deleted. Thus, the protein kinase activity of NPK15 and the hydrophobic region of the protein are responsible for the suicide effect. The NPK15 protein kinase seems to be associated with specific cellular functions. Southern blot analysis with cNPK15 as the probe detected several fragments in restriction digests of genomic DNAs from both tobacco and other members of the Solanaceae. This result suggests that NPK15-related genes constitute a small gene family in the genomes of Solanaceae.     

云南元江普通野生稻中Pi-ta和Pib同源基因的克隆和分析

用高保真PCR技术从云南元江普通野生稻中克隆了抗稻瘟病Pi-ta同源基因的编码区及Pib基因的部分同源序列。Pi-ta同源基因的编码区序列与报道的栽培稻有99.7%的同源性。根据前人的结果,从元江普通野生稻的Pi-ta基因推导的氨基酸序列中918位点为丝氨酸,属于Pi-ta~-等位基因,不能对含有AVRPita基因的稻瘟病菌产生抗性。与Pi-ta基因相比,元江普通野生稻中的Pib同源基因第一外显子与栽培稻的相应序列间存在较大差异,其中有一段87 bp的DNA序列缺失,而且不能按正常的Pib基因序列的阅读框进行翻译。因此认为,元江普通野生稻不具有基于Pi-ta和Pib基因的抗稻瘟病遗传基础。     

Cloning and expression of a lipase gene from rice (Oryza sativa cv. Dongjin)

Lipases are useful enzymes that are primarily responsible for the hydrolysis of acylglycerides during lipid processing. We have cloned a lipase gene from a rice seed coat cDNA library (Oryza sativa cv. Dongjin). The cDNA was 1,445 bp in length and encoded 361 amino acid residues (GenBank accession No. AY580163). The deduced amino acid sequence had 82 and 56% identity to Oryza sativa (cv. Chuchung) and Arabidopsis thaliana lipase genes, respectively, and there was a GxSxG consensus motif near the catalytic triad at the active serine site. The deduced sequence had little homology to mammalian and microbial lipases. When the Oryza sativa lipase gene was expressed in Escherichia coli with the pET expression system, activity was found mainly in the pellet fraction. The purified product had lipolytic activity towards tributyrin and was about 40 kDa in size.