两个大豆类受体蛋白激酶基因的克隆及其结构和功能的初步分析

利用高等植物类受体蛋白激酶基因的保守域设计简并引物的通过RT-PCR方法,从大豆叶片中克隆到两个新的,可能的类受体蛋白激酶基因的部分cDNA片段。对其基因结构的分析表明：在RLPK2的激酶保守域Vib与Ⅸ之间有一个407bp长的内含子。利用RT-PCR方法对它们的表达特性进行初步研究。发现这两个基因可能参与了对大豆叶片衰老和/或细胞分裂素延缓衰老过程的调节机制。     

植物体细胞胚胎发生受体类蛋白激酶的生物学功能

体细胞胚胎发生受体类蛋白激酶(Somatic embryogenesis receptor-like kinases,SERKs)属于膜富亮氨酸重复序列受体类蛋白激酶(Leucine-rich repeat sequence receptor-like kinase,LRR-RLK)家族的第二亚类。SERK具有典型的胞外信号受体结构域、跨膜结构域和胞内激酶活性结构域,研究发现SERKs在植物生命活动中承担着多个角色。文章简述了SERKs的典型结构域特征,重点介绍该类蛋白在体细胞胚发生、生殖发育、激素感应和病理反应方面发挥的功能,同时对该蛋白激酶的研究价值和应用前景进行了探讨。     

植物体细胞胚胎发生受体类蛋白激酶的生物学功能

体细胞胚胎发生受体类蛋白激酶(Somatic embryogenesis receptor-like kinases, SERKs)属于膜富亮氨酸重复序列受体类蛋白激酶(Leucine-rich repeat sequence receptor-like kinase, LRR-RLK)家族的第二亚类。SERK具有典型的胞外信号受体结构域、跨膜结构域和胞内激酶活性结构域, 研究发现SERKs在植物生命活动中承担着多个角色。文章简述了SERKs的典型结构域特征, 重点介绍该类蛋白在体细胞胚发生、生殖发育、激素感应和病理反应方面发挥的功能, 同时对该蛋白激酶的研究价值和应用前景进行了探讨。     

植物凝集素类受体蛋白激酶研究进展

自然界中植物的生长发育受到各种环境变化的影响。为了响应外界各种环境条件,植物演化出一系列识别和传递环境信号的蛋白分子,其中比较典型的是植物细胞质膜上的类受体蛋白激酶(RLKs)。凝集素类受体蛋白激酶(LecRLKs)是类受体蛋白激酶家族中的一个亚族,它主要包含3个结构域:细胞外凝集素结构域、跨膜结构域和细胞内激酶结构域。根据细胞外凝集素结构域的不同,LecRLKs可分为3种不同类型:L、G和C型。近年来,研究表明LecRLKs在植物生物/非生物胁迫和发育调控中发挥非常重要的作用。该文综述了植物凝集素类受体蛋白激酶的研究历史、结构特点、分类以及生物学功能,并重点阐述凝集素类受体蛋白激酶在植物生物/非生物胁迫响应和调控发育方面的功能。对不同类型和不同功能的植物凝集素类受体蛋白激酶进行阐述将有利于对该类蛋白开展功能研究,并为作物改良提供有益借鉴。     

植物类受体激酶的功能与底物鉴定

受体激酶是一类具有信号肽序列、胞外受体结构区、跨膜区和保守的胞内激酶区的蛋白质,普遍存在于各种生物体内．植物类受体激酶是植物体内信号分子的重要受体,几乎参与了所有的细胞代谢过程,包括生长发育、表达调控和逆境反应等．鉴于激酶的重要性和特殊性,其底物筛选已经成为该领域的研究热点．目前,筛选植物激酶底物的方法主要有酵母双杂交技术和蛋白质芯片技术等,但是,已经进行体内鉴定的底物极其有限．本文总结了植物类受体激酶的功能及其底物与筛选鉴定方法,并介绍了一些激酶的底物．     

植物类受体蛋白激酶的研究进展

植物类受体蛋白激酶(receptor-like protein kinase,RLKs)通过胞外结构域识别病原信号分子,发生磷酸化、去磷酸化反应而开启或关闭下游靶蛋白,调节植物固有免疫反应,诱导抗病防御反应.目前对植物类受体蛋白激酶的功能、信号传导和配体识别等方面的研究已成为该领域的重点.本文对近年来国内外有关植物类受体蛋白激酶的结构、功能及其在植物抗病防御反应中的作用研究进行综述,为今后进一步深入研究植物类受体蛋白激酶的生理生化功能及应用提供参考.     

类受体激酶FER调节植物与病原菌相互作用的分子机制

植物细胞依赖细胞质膜上的受体感知并传递环境信号, 而受体通过与配体特异结合启动一系列下游信号转导途径, 维持植物正常的生命活动及其对外界环境变化的适应。类受体激酶是其中一类重要受体, 通常由胞外结合结构域、跨膜结构域和胞内激酶结构域3部分组成, 是植物适应外界环境变化的重要调节枢纽。FER属于CrRLK1L类受体蛋白激酶家族, 最早被发现在高等植物雌雄配子体识别过程中发挥作用。随后, 众多研究表明, FER在植物生长发育、激素间交互作用、植物与病原菌互作和逆境响应等多种生物学过程中扮演重要角色, 是近年来植物信号通路研究领域的“明星蛋白”。随着植物病理学研究的不断深入, FER在植物与病原菌互作过程中的功能备受关注。该文主要综述FER调节植物与病原菌互作的研究进展, 旨在为进一步解析类受体蛋白激酶在植物细胞响应病原菌侵染过程中的信号转导机制提供参考。     

类受体激酶——植物环境适应性的调节枢纽

“莫听穿林打叶声,何妨吟啸且徐行。”大雨滂沱,苏轼拄着竹杖穿着蓑衣寻找避雨之所……树林竹叶虽不能避雨,却也不怕大雨的击打,这全都依赖于植物进化出了一套高度精密的信号响应机制来“趋利避害”,实现对环境变化的适应。植物感受环境信号需要类受体激酶(Receptor-like kinases,RLKs)。类受体激酶是一类定位在细胞膜上的单次跨膜蛋白,包括一个感受外界信号的胞外受体结构域,一个跨膜结构域和一个胞内激酶结构域。常见的类受体激酶信号通路中,首先由胞外受体结构域感受和识别细胞外界信号,将信号传递到细胞质一侧,胞质激酶结构域与下游蛋白相互作用,并启动其生化反应(如磷酸化),最终通过细胞核-细胞质穿梭信使将信号传递到细胞核内,调控下游基因表达进行信号输出,从而实现对环境快速变化的适应。     

普通小麦中一个类受体激酶基因的克隆、鉴定和表达分析

为研究抗白粉病小麦（Triticum aestivum L．）品系在小麦白粉病菌（Blumeria graminis f. sp．tritici）侵染后有无LRK10同源基因表达,依据小麦蛋白激酶LRK10和其它植物蛋白激酶第6亚结构域设计了一个5’-RACE兼并性引物。以接种小麦白粉病菌后的小麦抗白粉病品系“99—2439”幼苗叶片cDNA为模板进行5’-RACE扩增,获得了一个1551bp长的蛋白激酶基因cDNA片段（S1125,GenBank登录号：AY584533）。此后,通过RACE技术成功地获得了该基因的全长cDNA克隆。该克隆编码637个氨基酸组成的多肽。同源性查寻表明,该基因属于先前命名为wfrk（wheat leaf rust kinase）的小麦类受体蛋白激酶基因家族。与LRK10相似,这个新的小麦类受体蛋白激酶有5个明显的功能域：位于氨基端的疏水信号序列、推测的胞外结构域、跨膜域、高荷电序列和位于羧基端的丝氨酸／苏氨酸激酶域,因此被命名为TaLRK（Triticum aestivum LRK）。以小麦肌动蛋白基因为对照,通过半定量反转录PCR（semi—QRT—PCR）技术对叶片中TaLRK基因在小麦白粉病菌接种后的转录水平表达谱进行了研究。结果表明,小麦白粉病菌的侵染使TaLRK基因的转录显著增强。组织特异性表达分析证明,这一基因仅在小麦的绿色部分表达。研究结果提示TaLRK可能参与了小麦的抗白粉病反应。     

大豆类受体蛋白激酶基因(rlpk2)RNAi双元表达载体的构建及其转基因

植物类受体蛋白激酶(plant receptor-like kinases RLKs)以其特有的结构在植物的生长、发育和防御等多种生理生化过程中发挥着重要的作用。利用RNA干扰技术(RNA interference RNAi)来研究RLKs的功能已日趋成熟。本文根据植物中hpRNA(hairpin RNA)的原理,以大豆类受体蛋白激酶基因rlpk2为靶基因,在rlpk2-cDNA序列3'端选择312bp作为构建RNAi的序列,借助中间克隆载体,经过三次亚克隆,最后形成含rlpk2-RNAi表达盒的双元表达载体pART27-R2,并转入农杆菌LBA4404。采用农杆菌介导大豆子叶节转化方法,共获得了三株转基因植株。转基因植株 RT-PCR分析表明rlpk2基因已被成功敲减(knock-down),并且发现敲减大豆叶片中的rlpk2基因表达明显改善大豆叶片的光合能力,结合前期研究结果,表明rlpk2基因可能在维持叶绿体的结构及保护叶绿体膜系统的完整性方面起负调节作用。     

Abiotic stress-inducible receptor-like kinases negatively control ABA signaling in Arabidopsis

Membrane-anchored receptor-like protein kinases (RLKs) recognize extracellular signals at the cell surface and activate the downstream signaling pathway by phosphorylating specific target proteins. We analyzed a receptor-like cytosolic kinase (RLCK) gene, ARCK1, whose expression was induced by abiotic stress. ARCK1 belongs to the cysteine-rich repeat (CRR) RLK sub-family and encodes a cytosolic protein kinase. The arck1 mutant showed higher sensitivity than the wild-type to ABA and osmotic stress during the post-germinative growth phase. CRK36, an abiotic stress-inducible RLK belonging to the CRR RLK sub-family, was screened as a potential interacting factor with ARCK1 by co-expression analyses and a yeast two-hybrid system. CRK36 physically interacted with ARCK1 in plant cells, and the kinase domain of CRK36 phosphorylated ARCK1 in vitro. We generated CRK36 RNAi transgenic plants, and found that transgenic plants with suppressed CRK36 expression showed higher sensitivity than arck1-2 to ABA and osmotic stress during the post-germinative growth phase. Microarray analysis using CRK36 RNAi plants revealed that suppression of CRK36 up-regulates several ABA-responsive genes, such as LEA genes, oleosin, ABI4 and ABI5. These results suggest that CRK36 and ARCK1 form a complex and negatively control ABA and osmotic stress signal transduction.     

Identification of genes encoding receptor-like protein kinases as possible targets of pathogen- and salicylic acid-induced WRKY DNA-binding proteins in Arabidopsis

To understand how plant host genes are regulated during the activation of plant defence responses, we are studying a group of pathogen- and salicylic acid (SA)-induced DNA-binding proteins containing the novel WRKY domain. To identify downstream target genes of these WRKY proteins, we have searched the Arabidopsis genome and identified four closely linked genes on chromosome IV that contain an unusually large number of the W-box sequences [(T)TGAC(C/T)] recognized by WRKY proteins within a few hundred base pairs upstream of their coding regions. All four genes encode proteins characteristic of receptor-like protein kinases (RLK), each consisting of an N-terminal signal sequence, an extracellular receptor domain, a single transmembrane domain and a C-terminal cytoplasmic serine/threonine protein kinase domain. All four RLK genes were induced by treatment with SA or infection by a bacterial pathogen. Studies with one of the RLK genes (RLK4) indicated that a cluster of W-box elements in its promoter region were recognized by both purified WRKY proteins and SA-induced W-box binding activities from SA-treated Arabidopsis plants. Further analysis using the RLK4 gene promoter fused to a reporter gene in transgenic Arabidopsis indicated that the consensus WRKY protein-binding sites in the RLK4 gene promoter were important for the inducible expression of the reporter gene. These results indicate that pathogen- and SA-induced W-box binding proteins regulate not only genes encoding defence proteins with direct or indirect anti-microbial activities, but also genes encoding proteins with regulatory functions.     

Two members of the thioredoxin-h family interact with the kinase domain of a Brassica S locus receptor kinase.

To determine potential targets of the S locus receptor kinase (SRK) during the Brassica self-incompatibility response, a yeast two-hybrid library was screened with the SRK-910 protein kinase domain. Two thioredoxin-h-like clones, THL-1 and THL-2, were found to interact specifically with the SRK-910 protein kinase domain and not to interact with the protein kinase domains from the Arabidopsis receptor-like protein kinases (RLK) RLK4 and RLK5. The interaction between THL-1 and the SRK-910 protein kinase domain was confirmed using coimmunoprecipitation experiments with fusion proteins produced in Escherichia coli. THL-1 has thioredoxin activity based on an insulin reduction assay, and THL-1 is weakly phosphorylated by the SRK-910 protein kinase domain. THL-1 and THL-2 are both expressed in a variety of tissues but show some differences in steady state mRNA levels, with THL-2 being preferentially expressed in floral tissues. This indicates a more general biological function for these thioredoxins in addition to a potential role as effector molecules in the self-incompatibility signal cascade.     

Interaction of the maize and Arabidopsis kinase interaction domains with a subset of receptor-like protein kinases: implications for transmembrane signaling in plants

The kinase interaction (KI) domain of kinase-associated protein phosphatase (KAPP) interacts with the phosphorylated form of an Arabidopsis thaliana receptor-like protein kinase (RLK). The KI domain may recruit KAPP into an RLK-initiated signaling complex. To examine additional roles that this domain may play in plant signal transduction, a search was conducted for other KI domain-containing proteins. One gene was isolated which encodes a KI domain, the maize homolog of KAPP. To test whether the maize KI domain associates with other maize proteins, it was used as a probe in a protein–protein interaction cloning strategy. A new maize RLK, K I domain i nteracting k inase 1 (KIK1), was identified by its interaction with the maize KI domain. The maize KI domain and the KIK1 kinase domain association required phosphorylation of the kinase. This work establishes that the KI domain phosphorylation-dependent signaling mechanism is present in both monocots and dicots. Additionally, it was determined that both the maize and Arabidopsis KI domains interact with several but not all of the active RLKs assayed. These multiple associations imply that KAPP may function in a number of RLK-initiated signaling pathways.     

菜用大豆CIPK基因对逆境胁迫及激素的响应特征

CIPK(calcineurin B-like-interacting protein kinase)是一类丝氨酸/苏氨酸蛋白激酶,在植物响应逆境胁迫和激素信号转导中发挥重要作用。本研究利用大豆基因组数据库,在全基因组水平鉴定获得52个CIPK蛋白激酶。蛋白比对分析发现所有Gm CIPK含有高度保守特征性的N端激酶区、连接区和C端调控区。系统进化树分析发现大豆Gm CIPK与拟南芥、水稻CIPK分类一致,分为4个亚家族,且每个亚家族含有3个不同物种的成员,表明Gm CIPK基因的分化早于物种的分化。启动子分析表明,多数Gm CIPK基因的启动子区含有逆境和激素应答元件。组织表达分析发现,Gm CIPK基因呈现多样化的组织表达特性。进一步选取组织表达量相对较高的14个Gm CIPK进行荧光定量PCR分析,结果表明这些菜用大豆CIPK基因在不同程度上均受高温、干旱、高盐胁迫以及ABA、ACC、SA、Me JA激素的诱导表达。采用蛋白同源比对和蛋白互作在线数据库对拟南芥及大豆同源CIPK蛋白激酶与其他蛋白的互作关系进行了预测分析,发现17对同源CIPK与其他蛋白(激酶、磷酸酶、转录因子等)存在互作。本研究为菜用大豆CIPK基因的功能研究与利用奠定了基础。