芸薹属植物自交不亲和分子机制的研究进展

综述了近年来在芸薹属植物自交不亲和信号转导途径中相关基因的研究进展,同时对其自交不亲和信号转导途径中的分子机制作一阐述。 Abstract:In recent years certain progess in Brassica signaling was reviewed about some self-compatibility-related genes such as SRK,SLG,SCR,ARC1,THL1 and THL2.Meanwhile,molecular mechanism in Brassica self-compatibility signaling was reviewed,including its action models.     

芸芥自交亲和性变异的初步研究

为了判断芸芥(Eruca sativa)自交亲和性的变异情况, 采用套袋自交、剥蕾自交和开放授粉3种方法, 对来源于中国、伊朗和巴基斯坦的52份芸芥的自交亲和指数及相对亲和指数进行了统计分析。结果表明: (1) 芸芥为高度自交不亲和植物, 其不同品种(系)中存在自交亲和基因; (2) 芸芥不同品种间自交亲和性存在广泛的变异, 品种间自交亲和指数介于0-4.98之间, 品种内不同个体间自交亲和性也存在广泛的变异; (3) 参试材料分为4种类型, 即高自交亲和、自交亲和、自交不亲和(0<自交亲和指数<1.00)及高自交不亲和; (4) 芸芥自交亲和性因生态类型而异, 西南地区的材料自交亲和性最高, 西北地区次之, 华北地区最低。总之, 芸芥为一种高度自交不亲和植物, 其自交亲和性状存在广泛的变异, 共有4种变异类型。     

芸苔属自交不亲和细胞信号转导的研究进展

在芸苔属植物的自交不亲和细胞信号转导过程中,信号分子-SCR配体是由花粉粒产生的,被柱头乳突细胞SRK受体识别后,进行细胞内信号转导.这对受体-配体是两个由S位点编码的且高度多态的蛋白质,它们决定着自交不亲和反应.配体是位于花粉粒表面的一个小的胞被蛋白,由SCR基因编码;受体是位于柱头乳突细胞原生质膜上的跨膜的蛋白质激酶,由SRK基因编码.在自交授粉过程中,配体SCR和受体SRK的相互作用激活了受体SRK,被激活的SRK通过其下游组分ARC1介导底物的泛肽化,然后泛肽化的底物在蛋白酶体/CSN中被降解,从而导致了自交不亲和性反应.这些降解的底物可能是促进花粉水合、萌发和花粉管生长的雌蕊亲和因子.主要针对芸苔属自交不亲和细胞信号转导作一综述.     

植物类受体胞质激酶的结构和功能

本文介绍植物类受体胞质激酶的结构及其在植物的抗病、抗逆、生长发育、自交不亲和、油菜素内酯信号转导等方面的功能。     

配子体自交不亲和植物花粉S基因研究进展

配子体自交不亲和植物的自交不亲和性是由雌蕊自交不亲和因子和花粉自交不亲和因子相互作用的结果。目前已经分离和鉴定了雌蕊自交不亲和基因及其表达产物。最近从金鱼草、Prumusdulcis、梅等植物中分离的F-box基因,它具有花粉S基因特点,即在花药、成熟的花粉和花粉管中特异表达;在基因位置上,与S-RNase基因紧密连锁;不同物种或同一物种不同品种F-box基因间核苷酸和氨基酸序列上存在高度多态性。通过分子生物学方法和杂交授粉试验证明所分离的F-box基因是花粉自交不亲和基因,但目前尚未分离出该类基因相应的表达蛋白。主要综述了配子体自交不亲和植物花粉自交不亲和基因的发现、基因的结构、雌蕊自交不亲和因子和花粉自交不亲和因子相互作用的模型。     

芸苔属自交不亲和细胞信号转导的研究进展

在芸苔属植物的自交不亲和细胞信号转导过程中,信号分子-SCR配体是由花粉粒产生的,被柱头乳突细胞SRK受体识别后,进行细胞内信号转导。这对受体-配体是两个由S位点编码的且高度多态的蛋白质,它们决定着自交不亲和反应。配体是位于花粉粒表面的一个小的胞被蛋白,由SCR基因编码;受体是位于柱头乳突细胞原生质膜上的跨膜的蛋白质激酶,由SRK基因编码。在自交授粉过程中,配体SCR和受体SRK的相互作用激活了受体SRK,被激活的SRK通过其下游组分ARC1介导底物的泛肽化,然后泛肽化的底物在蛋白酶体/CSN中被降解,从而导致了自交不亲和性反应。这些降解的底物可能是促进花粉水合、萌发和花粉管生长的雌蕊亲和因子。主要针对芸苔属自交不亲和细胞信号转导作一综述。     

无籽沙糖桔WUBE1基因的功能验证

为了解泛素活化酶E1基因(UBE1)在无籽沙糖桔自交不亲和反应中的作用,通过根癌农杆菌介导法将来源于自交不亲和无籽沙糖桔(Citrus reticulata ‘Wuzishatangju’) WUBE1基因转化烟草(Nicotiana tabacum)。结果表明,外源基因WUBE1已导入烟草基因组中并得到表达。转WUBE1基因的自交授粉组合花粉管在生长过程中,部分花粉管出现停止生长的现象,到达花柱基部的花粉管数量少于异交授粉和野生型自交组合。转WUBE1基因烟草的花粉生活力、发芽率、自交和异交后每个果荚中的种子数与野生型烟草无显著差异。这表明单一的WUBE1基因不能调控无籽沙糖桔自交不亲和反应,很可能是通过Ub/26S途径参与了无籽沙糖桔自交不亲和反应。     

高等植物自交不亲和性的分子生物学

自交不亲和性是大多数高等植物防止近亲繁殖的一种遗传屏障。它涉及受精时雄配子（花粉）和雌蕊之间的相互作用。目前,已经分离获得了编码控制雌蕊自交不亲和性的Ｓ基因。在孢子体型自交不亲和的芸苔属中,雌蕊Ｓ基因编码Ｓ位点糖蛋白（ＳＬＧ）和Ｓ受体激酶（ＳＲＫ）。它们可能与磷酸化和去磷酸化参与了的某种信号传递有关,最后导致自交花粉生长的抑制。在配子分配体型自交不亲和的茄科中,雌蕊Ｓ位点糖蛋白为一种核糖核酸酶,称为Ｓ－核酸酶（Ｓ－ＲＮａｓｅ）。自交不亲和反应与Ｓ－核酸酶引起的花粉管ＲＮＡ降解有关,并且可能通过花粉管特异性地摄入Ｓ－核酸酶或者花粉管内存在的特异性的核酸酶抑制剂的作用,达到对自交花粉生长的抑制。另外,从配子体型自交不亲和的罂粟中,分离到了与芸台属和茄科不同的雌蕊Ｓ基因,其作用机理可能与Ｃａ＋＋参与的信号传递有关。     

白菜自交不亲和性的荧光测定

通过亲和指数法及荧光显微观察对白菜的自交不亲和性进行了测定。结果表明,白菜自交不亲和的反应部位在柱头,自花授粉后柱头表面产生明显的胼胝质反应。两种观测法的结果相吻合,荧光显微镜观察法准确、方便,可应用于白菜自交不亲和系的育种实践。     

萝卜自交不亲和基因SLG6的CAPS标记的开发

萝卜是我国的主要蔬菜之一,其杂种优势十分明显,培育自交不亲和系是萝卜杂种优势育种的主要途径之一.本研究根据萝卜自交不亲和基因SLG6序列设计特异引物,以8个自交系为材料,其中自交不亲和系和自交亲和系各4个,扩增SLG6基因第232～711bp之间的单拷贝片段,8个材料均获得了一条480bp的特异片段.用限制性内切酶TaqⅠ对该片段进行酶切,自交不亲和系均产生约125bp和244bp的片段,其中,244bp的片段为自交不亲和系所特有,可作为SLG6基因的CAPS标记用于萝卜自交不亲和基因SLG6的检测;而自交亲和系则具有与自交不亲和系相同的125bp的片段和不同的多态性片段.     

Molecular mechanism of self-recognition in Brassica self-incompatibility

In most self-incompatible plant species, recognition of self-pollen is controlled by a single locus, termed the S-locus. In Brassica, genetic dissection of the S-locus has revealed the presence of three highly-polymorphic genes: S-receptor kinase (SRK), S-locus protein 11 (SP11) (also known as S-locus cysteine-rich protein; SCR) and S-locus glycoprotein (SLG). SRK encodes a membrane-spanning serine/threonine kinase that determines the S-haplotype specificity of the stigma. SP11 encodes a small cysteine-rich protein that determines the S-haplotype specificity of pollen. SLG encodes a secreted form of stigma protein similar to the extracellular domain of SRK. Recent biochemical studies have revealed that SP11 functions as the sole ligand for its cognate SRK receptor complex. Their interaction induces the autophosphorylation of SRK, which is expected to trigger the signalling cascade that results in the rejection of self-pollen. This so-called ligand-receptor complex interaction and receptor activation occur in an S-haplotype-specific manner, and this specificity is almost certainly the basis for self-pollen recognition.     

Characterization of the SP11/SCR high-affinity binding site involved in self/nonself recognition in brassica self-incompatibility

In Brassica self-incompatibility, the recognition of self/nonself pollen grains, is controlled by the S-locus, which encodes three highly polymorphic proteins: S-locus receptor kinase (SRK), S-locus protein 11 (SP11; also designated S-locus Cys-rich protein), and S-locus glycoprotein (SLG). SP11, located in the pollen coat, determines pollen S-haplotype specificity, whereas SRK, located on the plasma membrane of stigmatic papilla cells, determines stigmatic S-haplotype specificity. SLG shares significant sequence similarity with the extracellular domain of SRK and is abundant in the stigmatic cell wall, but its function is controversial. We previously showed that SP11 binds directly to its cognate SRK with high affinity (K(d) = 0.7 nM) and induces its autophosphorylation. We also found that an SLG-like, 60-kD protein on the stigmatic membrane forms a high-affinity binding site for SP11. Here, we show that the 60-kD stigmatic membrane protein is a truncated form of SRK containing the extracellular domain, transmembrane domain, and part of the juxtamembrane domain. A transiently expressed, membrane-anchored form of SRK exhibits high-affinity binding to SP11, whereas the soluble SRK (eSRK) lacking the transmembrane domain exhibits no high-affinity binding, as is the case with SLG. The different binding affinities of the membrane-anchored SRK and soluble eSRK or SLG will be significant for the specific perception of SP11 by SRK.     

Immunohistochemical studies on translocation of pollen S-haplotype determinant in self-incompatibility of Brassica rapa

The self-incompatibility system in Brassica is controlled by the S-locus, which contains S-receptor kinase (SRK) and S-locus protein 11 (SP11). SRK and SP11 control stigma and pollen S-haplotype specificity, respectively. SP11 binding to SRK induces the autophosphorylation of SRK, which triggers the signaling cascade that results in the rejection of self-pollen. The localization of SP11 protein during pollen development and pollination, however, have never been demonstrated. In this study, we examined the localization of S(8)-SP11 protein in the anther or pollinated stigma by immuno-electron microscopy. The immunostaining suggested that S(8)-SP11 was secreted from the tapetal cell into the anther locule as a cluster and translocated to the pollen surface at the early developmental stage of the anther. During the pollination process, SP11 was translocated from the pollen surface to the papilla cell, and then penetrated the cuticle layer of the papilla cell to diffuse across the pectin cellulose layer. Furthermore, SP11 protein could only penetrate the cuticle layer of the papilla cell in the presence of pollen grains, and could not penetrate on its own. This suggests that another factor from the pollen grain is needed for SP11 protein to penetrate the papilla cell wall.     

A pollen coat protein, SP11/SCR, determines the pollen S-specificity in the self-incompatibility of Brassica species

Many flowering plants have evolved self-incompatibility (SI) systems to prevent inbreeding. In the Brassicaceae, SI is genetically controlled by a single polymorphic locus, termed the S-locus. Pollen rejection occurs when stigma and pollen share the same S-haplotype. Recognition of S-haplotype specificity has recently been shown to involve at least two S-locus genes, S-receptor kinase (SRK) and S-locus protein 11 or S-locus Cys-rich (SP11/SCR). SRK encodes a polymorphic membrane-spanning protein kinase, which is the sole female determinant of the S-haplotype specificity. SP11/SCR encodes a highly polymorphic Cys-rich small basic protein specifically expressed in the anther tapetum and in pollen. In cauliflower (B. oleracea), the gain-of-function approach has demonstrated that an allele of SP11/SCR encodes the male determinant of S-specificity. Here we examined the function of two alleles of SP11/SCR of B. rapa by the same approach and further established that SP11/SCR is the sole male determinant of SI in the genus Brassica sp. Our results also suggested that the 522-bp 5'-upstream region of the S9-SP11 gene used to drive the transgene contained all the regulatory elements required for the unique sporophytic/gametophytic expression observed for the native SP11 gene. Promoter deletion analyses suggested that the highly conserved 192-bp upstream region was sufficient for driving this unique expression. Furthermore, immunohistochemical analyses revealed that the protein product of the SP11 transgene was present in the tapetum and pollen, and that in pollen of late developmental stages, the SP11 protein was mainly localized in the pollen coat, a finding consistent with its expected biological role.     

In vivo imaging of the S-locus receptor kinase,the female specificity determinant of self-incompatibility,in transgenic self-incompatible Arabidopsis thaliana

Background and Aims The S-locus receptor kinase (SRK), which is expressed in stigma epidermal cells, is responsible for the recognition and inhibition of ‘self’ pollen in the self-incompatibility (SI) response of the Brassicaceae. The allele-specific interaction of SRK with its cognate pollen coat-localized ligand, the S-locus cysteine-rich (SCR) protein, is thought to trigger a signalling cascade within the stigma epidermal cell that leads to the arrest of ‘self’ pollen at the stigma surface. In addition to the full-length signalling SRK receptor, stigma epidermal cells express two other SRK protein species that lack the kinase domain and whose role in the SI response is not understood: a soluble version of the SRK ectodomain designated eSRK and a membrane-tethered form designated tSRK. The goal of this study was to describe the sub-cellular distribution of the various SRK protein species in stigma epidermal cells as a prelude to visualizing receptor dynamics in response to SCR binding.Methods The Arabidopsis lyrata SRKb variant was tagged with the Citrine variant of yellow fluorescent protein (cYFP) and expressed in A. thaliana plants of the C24 accession, which had been shown to exhibit a robust SI response upon transformation with the SRKb–SCRb gene pair. The transgenes used in this study were designed for differential production and visualization of the three SRK protein species in stigma epidermal cells. Transgenic stigmas were analysed by pollination assays and confocal microscopy.Key Results and Conclusions Pollination assays demonstrated that the cYFP-tagged SRK proteins are functional and that the eSRK is not required for SI. Confocal microscopic analysis of cYFP-tagged SRK proteins in live stigma epidermal cells revealed the differential sub-cellular localization of the three SRK protein species but showed no evidence for redistribution of these proteins subsequent to incompatible pollination.     

A plant receptor-like gene, the S-locus receptor kinase of Brassica oleracea L., encodes a functional serine/threonine kinase.

To investigate the catalytic properties of the Brassica oleracea S-locus receptor kinase (SRK), we have expressed the domain that is homologous to protein kinases as a fusion protein in Escherichia coli. Following in vivo labeling of cultures with 32P-labeled inorganic phosphate, we observed phosphorylation of the fusion protein on serine and threonine, but not on tyrosine. In contrast, labeling was not observed when lysine-524, a residue conserved among all protein kinases, was mutated to arginine, thus confirming that SRK phosphorylation was the result of intrinsic serine/threonine kinase activity.     

Evolution of the self-incompatibility system in the Brassicaceae: identification of S-locus receptor kinase (SRK) in self-incompatible Capsella grandiflora

Self-incompatibility (SI) has been well studied in the genera Brassica and Arabidopsis, which have become models for investigation into the SI system. To understand the evolution of the SI system in the Brassicaceae, comparative analyses of the S-locus in genera other than Brassica and Arabidopsis are necessary. We report the identification of six putative S-locus receptor kinase genes (SRK) in natural populations of Capsella grandiflora, an SI species from a genus which is closely related to Arabidopsis. These S-alleles display striking similarities to the Arabidopsis lyrata SRK alleles in sequence and structure. Our phylogenetic analysis supports the scenario of differing SI evolution along the two lineages (The Brassica lineage and Arabidopsis/Capsella lineage). Our results also argue that the ancestral S-locus lacked the SLG gene (S-locus glycoprotein) and that the diversification of S-alleles predates the separation of Arabidopsis and Capsella.     

Molecular mechanisms of self-incompatibility in Brassica

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region several closely linked genes have been identified. One of them, S-locus receptor kinase (SRK), determines S haplotype specificity of the stigma and it's the key protein for SI reaction. The role of the S locus glycoprotein (SLG) gene remains unclear. In the last decade approximately 15 additional genes linked to S-locus have been found. Recently, a gene has been identified (SCR) that encodes a small cysteine-rich protein which is a candidate for the pollen ligand. In addition to S locus linked genes there are unlinked SLRgenes (S-locus related genes). In this review, we discuss the role of these genes and the current view on the self-incompatibility mechanism in Brassica.     

Evolution of the S-locus region in Arabidopsis relatives

The S locus, a single polymorphic locus, is responsible for self-incompatibility (SI) in the Brassicaceae family and many related plant families. Despite its importance, our knowledge of S-locus evolution is largely restricted to the causal genes encoding the S-locus receptor kinase (SRK) receptor and S-locus cysteine-rich protein (SCR) ligand of the SI system. Here, we present high-quality sequences of the genomic region of six S-locus haplotypes: Arabidopsis (Arabidopsis thaliana; one haplotype), Arabidopsis lyrata (four haplotypes), and Capsella rubella (one haplotype). We compared these with reference S-locus haplotypes of the self-compatible Arabidopsis and its SI congener A. lyrata. We subsequently reconstructed the likely genomic organization of the S locus in the most recent common ancestor of Arabidopsis and Capsella. As previously reported, the two SI-determining genes, SCR and SRK, showed a pattern of coevolution. In addition, consistent with previous studies, we found that duplication, gene conversion, and positive selection have been important factors in the evolution of these two genes and appear to contribute to the generation of new recognition specificities. Intriguingly, the inactive pseudo-S-locus haplotype in the self-compatible species C. rubella is likely to be an old S-locus haplotype that only very recently became fixed when C. rubella split off from its SI ancestor, Capsella grandiflora.