甘蓝自交不亲和细胞信号转导的功能基因研究进展

自交不亲和性是开花植物防止近亲繁殖而广泛存在的一种遗传屏障,在育种工作中意义重大,它在遗传上受一个带有复等位基因的多态性S基因座所控制。本文综述了近年来在甘蓝自交不亲和信号转导途径中相关功能基因的研究进展。SRK基因和SP11/SCR基因是控制甘蓝自交不亲和性的两个关键因子。SRK基因是雌蕊柱头中自交不亲和反应的专一决定因子,编码SP11/SCR基因的蛋白在花药壁和花粉中特异表达。编码SLG基因的蛋白在自交不亲和反应中起增强SRK活性的作用。另外也对信号传导途径中下游蛋白质的作用模式作了展望。     

芸薹属自交不亲和功能因子及分子机制研究进展

芸薹属的自交不亲和性受S位点基因控制,其上的SRK基因和SCR/SP11基因是自交不亲和反应中的两个关键因子,另外所发现的其他因子也对自交不亲和发挥了重要作用。文章介绍了影响自交不亲和的功能因子及其分子结构特征和功能,对自交不亲和反应的分子机制作了进一步阐述,同时对这些功能基因在芸薹属植物甘蓝染色体组上的定位研究作出展望。     

A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants

Self-incompatibility, the rejection of self pollen, is the most widespread mechanism by which flowering plants prevent inbreeding. In Brassica, the S receptor kinase (SRK) has been implicated in the self-incompatibility response, but the molecular mechanisms involving SRK are unknown. One putative downstream effector for SRK is ARC1, a protein that binds to the SRK kinase domain. Here it is shown that suppression of ARC1 messenger RNA levels in the self-incompatible Brassica napus W1 line is correlated with a partial breakdown of self-incompatibility, resulting in seed production. This provides strong evidence that ARC1 is a positive effector of the Brassica self-incompatibility response.     

Phylogenetic Analysis on SLG and SRK Genes in Brassica and Raphanus

S-locus genes were cloned from three Brassica napus and three B. campestris lines by using PCR walking and homologue sequence methods. A phylogenetic gene tree was constructed based on the six cloned genes and fifty-one previously reported SLG/SRK genes of Brassica and Raphanus. The SLGs from R. sativus were dispersed in the phylogenetic tree intermingling with SLG/SRKs from B. oleracea, B. napus and B. campestris. The SLG/SRK genes of class Ⅱ clustered independently in one group. The SLG/SRK genes of class Ⅰ showed to be more divergent than class Ⅱ genes. These results suggested that the divergence of class Ⅰ and class Ⅱ should have occurred before the differentiation of the genera Brassica and Raphanus. In addition, SLG and SRK of the same S haplotypes belonged to the same class. It suggested that class-Ⅰ and class-Ⅱ group divergence occurred first, and then SLG and SRK diverged. The three SC SRK genes from B. napus and B. campestris were grouped into one cluster, displaying difference from the SC SLG of B.oleracea. These three SC SRK genes were close to SI SRK of SI1300, SI271 and guanyou in phylogenetic relationships.These results indicated that SC and SI genes diverged more recently. It is not clear yet whether the differentiation of SC and SI genes was earlier than the differentiation of Brassica and Raphanus. Studies based on more genes are necessary for a comprehensive elucidation of the phylogenetic relationships in Brassicaceae.     

不结球白菜S位点受体激酶基因片段的克隆与表达分析

以不结球白菜自交不亲和系03的基因组DNA和柱头cDNA为模板,利用引物SRKF/SRKR扩增获得长度为964 bp和646 bp的SRK基因片段。序列比较分析表明,克隆的基因片段属于SRK基因的激酶域,该序列包含4个外显子和3个内含子,编码215个氨基酸,与芜菁、甘蓝、芥蓝等SRK基因有90%以上的相似性。荧光定量PCR分析结果表明:自交不亲和系03和自交亲和系105不同组织中SRK基因的表达水平存在显著的差异,SRK基因主要在自交不亲和系的柱头中高度表达,自交亲和系中该基因的表达主要分布于叶片、花蕾和柱头组织中。     

芸苔属植物自交不亲和性遗传机理研究及应用进展

芸苔属作物S位点上有三个紧密连锁的基因SLG,SRK,SCR/SP11。其中SRK和SCR/SP11蛋白分别为柱头和花粉SI信号识别决定因子。复等位基因显隐性关系与SRK基因的表达水平并不相关,可能是由于在绒毡层中SCR基因受到强烈的下游调控导致杂合体中隐性SCR基因在小孢子细胞中表达受到抑制。分子进化研究表明十字花科S位点基因的多态性来自于共同的祖先,并在不同种分化之前已经形成。利用芸苔属植物自交不亲和性遗传机理发展而来的PCR-RFLP方法提供了在芸苔属作物育种中切实可行的精确鉴定植株的S基因型和自交不亲和性的有效方法。     

植物自交不亲和性的分子生物学进展

自交不亲和性是植物特异性地识别并拒绝白花或亲缘关系很近的花粉的一种遗传机制，该特异性受S基因座控制。S-RNase介导的配子体型自交不亲和性在被子植物中分布最广泛，在茄科、蔷薇科和玄参科植物上研究比较深入。研究表明，雌雄双方的特异性决定因子分别是S基因座中具有多态性的核酸酶和含有F-box结构域蛋白基因，但其他修饰基因也参与其中。本文概括了该类自交不亲和性的研究现状，并根据最新研究进展就其工作模型作进一步探讨，同时简要提出今后的研究方向。     

eSRKs酵母双杂交诱饵载体的构建及其自激活作用的检测

为研究甘蓝自交不亲和决定因子S位点富含半胱氨酸蛋白/S位点蛋白11(SCR/SP11)与S位点受体激酶(SRK)胞外域高变区(eSRKs)的相互作用,构建了eSRKs基因的酵母双杂交诱饵载体,检测其自激活作用,并验证是否适用于后续的相互作用研究.以甘蓝B3为材料,通过RT-PCR技术获得eSRKs目的基因片段,将其克隆到pGBKT7载体中,构建酵母双杂交诱饵载体pGBKT7-eSRKs;测序正确后,将重组质粒转入Y2HGold,检测其表达产物对酵母细胞有无毒性以及对报告基因有无自激活作用,结果获得了正确的eSRKs基因片段,并成功克隆到pGBKT7诱饵载体中,且转化在有诱饵载体pGBKT7-eSRKs的Y2HGold在SD/-Trp营养缺陷平板上生长良好,表明表达产物对酵母细胞无毒性;显色反应结果表明对报告基因也无自激活作用,为下一步利用酵母双杂交系统检测SCR蛋白与SRK蛋白胞外域高变区的相互作用奠定基础.     

Allele-specific receptor-ligand interactions in Brassica self-incompatibility

Genetic self-incompatibility in Brassica is determined by alleles of the transmembrane serine-threonine kinase SRK, which functions in the stigma epidermis, and of the cysteine-rich peptide SCR, which functions in pollen. Using tagged versions of SRK and SCR as well as endogenous stigma and pollen proteins, we show that SCR binds the SRK ectodomain and that this binding is allele specific. Thus, SRK and SCR function as a receptor-ligand pair in the recognition of self pollen. Specificity in the self-incompatibility response derives from allele-specific formation of SRK-SCR complexes at the pollen-stigma interface.     

The male determinant of self-incompatibility in Brassica

In the S locus-controlled self-incompatibility system of Brassica, recognition of self-related pollen at the surface of stigma epidermal cells leads to inhibition of pollen tube development. The female (stigmatic) determinant of this recognition reaction is a polymorphic transmembrane receptor protein kinase encoded at the S locus. Another highly polymorphic, anther-expressed gene, SCR, also encoded at the S locus, fulfills the requirements for the hypothesized pollen determinant. Loss-of-function and gain-of-function studies prove that the SCR gene product is necessary and sufficient for determining pollen self-incompatibility specificity, possibly by acting as a ligand for the stigmatic receptor.     

甘蓝SRK底物蛋白基因的克隆与序列分析

采用PCR、3’RACE以及其他分子生物学方法,以甘蓝基因组DNA和柱头cDNA为模板对SSP基因进行扩增克隆,得到长度分别为778 bp和825 bp的基因片段。序列分析表明,甘蓝SSP基因不包含内含子,SSP核苷酸序列与拟南芥肽酶M28家族蛋白有同源性,序列相似性达到67.4%,两者的氨基酸序列相似性达到62.7%,而且都含有一个TFR_dimer结构域,推测SSP蛋白极有可能与在自交不亲和过程中受ARC1作用而泛素化了的某一未知蛋白的降解相关。     

MAP kinase phosphatase as a locus of flexibility in a mitogen-activated protein kinase signaling network

Intracellular signaling networks receive and process information to control cellular machines. The mitogen-activated protein kinase (MAPK) 1,2/protein kinase C (PKC) system is one such network that regulates many cellular machines, including the cell cycle machinery and autocrine/paracrine factor synthesizing machinery. We used a combination of computational analysis and experiments in mouse NIH-3T3 fibroblasts to understand the design principles of this controller network. We find that the growth factor-stimulated signaling network containing MAPK 1, 2/PKC can operate with one (monostable) or two (bistable) stable states. At low concentrations of MAPK phosphatase, the system exhibits bistable behavior, such that brief stimulus results in sustained MAPK activation. The MAPK-induced increase in the amounts of MAPK phosphatase eliminates the prolonged response capability and moves the network to a monostable state, in which it behaves as a proportional response system responding acutely to stimulus. Thus, the MAPK 1, 2/PKC controller network is flexibly designed, and MAPK phosphatase may be critical for this flexible response.     

Signaling the arrest of pollen tube development in self-incompatible plants

Self-incompatibility (SI), the cellular recognition system that limits inbreeding, has served as a paradigm for the study of cell-to-cell communication in plants since the phenomenon was first described by Darwin. Recent studies indicate that SI is achieved by diverse molecular mechanisms in different plant species. In the mustard family, the mechanism of SI shows parallels to the signaling systems found in animals that are mediated by cell-surface receptors with signal-transducing protein kinase activity.     

A pollen factor linking inter- and intraspecific pollen rejection in tomato

Self-incompatibility (SI)--intraspecific pollen recognition systems that allow plants to avoid inbreeding--in the Solanaceae (the nightshade family) is controlled by a polymorphic S locus where "self" pollen is rejected on pistils with matching S alleles. In contrast, unilateral interspecific incompatibility (UI) prevents hybridization between related species, most commonly when the pollen donor is self-compatible (SC) and the recipient is SI. We observed that in Solanum, a pollen-expressed Cullin1 gene with high similarity to Petunia SI factors interacts genetically with a gene at or near the S locus to control UI. Cultivated tomato and related red- or orange-fruited species (all SC) exhibit the same loss-of-function mutation in this gene, whereas the green-fruited species (mostly SI) contain a functional allele; hence, similar biochemical mechanisms underlie the rejection of both "self" and interspecific pollen.     

新牧1号杂花苜蓿交配系统的等位酶分析

采用不连续系统垂直板聚丙烯酰胺凝胶电泳技术,对新牧1号杂花苜蓿的交配系统进行研究,旨在分析苜蓿的自花粉早熟及克隆生长习性对居群遗传结构的影响.结果显示:(1)两种群多态位点的百分率均为50;, 其中,子代平均每位点的等位基因数为1.58,平均每位点的有效等位基因数为2.07,亲代平均每位点的等位基因数为1.6,平均每位点的有效等位基因数为2.1;(2)子代和亲代居群的固定系数F平均值分别为-0.006 7,-0.15,都偏离了F=0下的平衡状态,子代和亲代都属于高异交率的外繁育类型,但从固定指数的大小看,子代的异交率与亲代相比有明显的下降趋势.(3)苜蓿的自花粉早熟、克隆生长习性和昆虫传粉造成的自花粉滞留导致了自交率的提高,因其严格的自交不亲和系统在保证较低的自交率方面起到了有效作用,使得种群的遗传结构仍处于一种良好的状态,保持着较高的异交比例.     

Photoreceptor deactivation and retinal degeneration mediated by a photoreceptor-specific protein kinase C

The protein kinase C (PKC) family of serine-threonine kinases has been implicated in the regulation of a variety of signaling cascades. One member of this family, eye-PKC, is expressed exclusively in the Drosophila visual system. The inaC (inactivation-no-afterpotential C) locus was shown to be the structural gene for eye-PKC. Analysis of the light response from inaC mutants showed that this kinase is required for the deactivation and rapid desensitization of the visual cascade. Light adaptation was also defective in inaC mutant flies. In flies carrying the retinal degeneration mutation rdgB, absence of eye-PKC suppressed photoreceptor cell degeneration. These results indicate that eye-PKC functions in the light-dependent regulation of the phototransduction cascade in Drosophila.     

基于SRK和SCR序列的甘蓝自交不亲和系单倍型分析

采用RT-PCR和其他分子生物学方法,以甘蓝自交不亲和系E1和F1柱头cDNA为模板扩增SRKE1和SRKF1基因,以花药cDNA为模板扩增SCRE1和SCRF1基因.经序列比对首次证明E1和F1材料分别为S28和S7单倍型.经三维结构分析,预测SCRE1上C5-C6和C7-C8之间氨基酸序列决定SCRE1的单倍型特异性,SRKE1上TNNSFYSRLKVS序列决定了SRKE1的单倍型特异性,并对两者相对作用的关键氨基酸位点进行了分析.     

Generation of self-incompatible Arabidopsis thaliana by transfer of two S locus genes from A. lyrata

Transitions from cross-fertilizing to self-fertilizing mating systems have occurred frequently in natural and domesticated plant populations, but the underlying genetic causes are unknown. We show that gene transfer of the stigma receptor kinase SRK and its pollen-borne ligand SCR from one S-locus haplotype of the self-incompatible and cross-fertilizing Arabidopsis lyrata is sufficient to impart self-incompatibility phenotype in self-fertile Arabidopsis thaliana, which lacks functional orthologs of these genes. This successful complementation demonstrates that the signaling cascade leading to inhibition of self-related pollen was maintained in A. thaliana. Analysis of self-incompatibility will be facilitated by the tools available in this species.     

芸芥果胶酸裂解酶基因的克隆及序列分析

采用RACE技术从芸芥自交亲和系花药中克隆出果胶酸裂解酶(Pectate lyases,PL)基因,全长1 657bp,其完整开放阅读框(Open Read Frame,ORF)为1 371bp,编码456个氨基酸,分子质量51.179ku,等电点9.42。序列比对结果表明,该蛋白与其他植物的PL蛋白具有较高的同源性,因此命名为EsPL。进化树分析表明,芸芥EsPL基因与十字花科芸薹属植物甘蓝型油菜、白菜型油菜的PL基因亲缘关系较近。实时荧光定量PCR结果显示,EsPL在芸芥开花后自交亲和系花药中的表达量显著高于自交不亲和系花药中表达量,该基因可能在芸芥自交亲和性状调控过程中发挥重要作用。