【作者】 韦静宜；

【导师】 王小佳；

【作者基本信息】 西南大学 ， 蔬菜学， 2012， 博士

【摘要】 自交不亲和(Self-incompatibility, SI)是显花植物在自然选择下进化出的反自交机制,而自交不亲和信号传导是研究植物胞间信号传导的模式系统。SⅠ反应在十字花科中表现为自我或自我相关的花粉在柱头表面的萌发抑制。这个反应是由发生在柱头表面的受体-配体相互作用所激发的。该受体是一种跨膜的丝氨酸／苏氨酸受体激酶称为S-位点受体激酶(S-locus receptor kinase, SRK)。SRK位于覆盖整个柱头表面的细胞膜乳突细胞中。SRK胞外域(eSRK)主要是负责配体结合,在单倍型间呈现高度多态性,是个S特异性决定因子。SRK配体被称为S位点富含半胱氨酸(S cysteine-rich, SCR),或者SP11,位于富含脂质的花粉壁中,是一个富含半胱氨酸的小分子蛋白质。2001年Kachroo等人的研究表明,SCR与SRK单倍型特异性结合导致自我花粉被拒绝。迄今为止,还没有实验研究出SRK的单倍型特异性的决定因素,生物信息学的预测为这一命题带了各种各样的假说。eSRK (SRK胞外域)负责配体结合,根据序列相似度可以将eSRK分成三个子域。N-端类似于甘露糖结合凝集素(B-Lection Domain),中间的高变区包含了与单倍型特异性相关的大量变异,C-端是相似最高的PAN或APPLE域,参与蛋白质-蛋白质或蛋白质-碳水化合物的相互作用。研究人员最感兴趣的就是高变区,目前已确定有三个高变区(HVI的,HVⅡ和和HVⅢ),这些区域被认为是平衡选择的产物。据推测,高变区中一些多态氨基酸是在自然选择和植物理化性质发生改变的情况下获得,这或许可以解释新S单倍型是如何演变而来。序列分析表明,SCR就是结合在SRK高变区上。在育种实践中,自交不亲和系已经成为重要育种材料,因此开展自交不亲和性相关研究具有重要的理论价值和现实意义。为探索eSRK序列上参与配体结合及相互作用的区域,我们以结球甘蓝高代自交系E、F为材料,构建了3个甘蓝不同单倍型的eSRK基因重组体,利用酵母双杂交系统检测各个重组体与SCR的相互作用。我们进一步分析了SRK高变区上的多态性位点,筛选出9个多态性较高的位点,对其进行突变,希望获得在SRK-SCR单倍型特异性相互作用中起决定作用的氨基酸位点。1植物材料自交不亲和性的分析为了鉴定本试验所使用的植物材料——结球甘蓝材料E、F的自交不亲和性,我们采用荧光显微法观察了两种材料花期自交及杂交后花粉管萌发情况,并测定了二者花期自交和杂交的亲和指数。结果显示,结球甘蓝材料E在花期自交时仅有少数(<10条)甚至没有花粉管进入柱头,说明结球甘蓝材料E具有自交不亲和特性。同样的,结球甘蓝材料F在授上自身的花粉后,在柱头中只能观察到少数(<10条)进入柱头组织中的花粉管。结球甘蓝材料E的花期自交亲和指数为0.23,F花期自交亲和指数为0.19,与多年来的统计数据(未列出)相一致。通过荧光显微可观察到E、F花期相互杂交后柱头中有大量(>25)的花粉管,二者花期正反交的亲和指数均大于5。表明结球甘蓝材料E、F为强自交不亲和系,并且杂交亲和。2甘蓝材料eSRK和SCR基因的克隆及分析针对eSRK N端和C端相对保守的序列设计特异性引物eSRKs和eSRKAS,从柱头cDNA中克隆E、F的eSRK基因,分别命名为eSRKE、eSRKF。序列分析表明,eSRKE、eSRKF核酸序列分别与BoSRK28、BoSRK7高度同源,由此判定,E属于甘蓝S28单倍型,F属于甘蓝S7单倍型。两个eSRK的氨基酸序列都包含了12个保守的半胱氨酸、B-lectin、S-locus glycop以及PAN-APPLE区域等SRK胞外域绝大部分区域,除了信号肽。根据所获得的单倍型信息设计了克隆SCR基因的引物,并顺利从结球甘蓝材料E、F花蕾gDNA中扩增出分别与eSRKE、eSRKF同源的SCR基因,命名为SCRE和SCRF。所获得的SCRF基因编码53个氨基酸,SCRE编码61个氨基酸,两段氨基酸序列均包含了SCR成熟肽的全部氨基酸序列,其中包含了在所有SCR氨基酸序列中高度保守的1个甘氨酸和8个半胱氨酸。3甘蓝eSRK中决定其单倍型特异性的肽段的研究3.1酵母双杂交系统检测SCR与各个eSRK间的相互作用为了探讨HV Ⅰ/Ⅱ区域在SRK单倍型特异性及其与SCR互作中的作用,本研究采用重组技术构建甘蓝不同单倍型eSRK(SRKE与SRKF)基因间的重组体：eSRKE-1、eSRKE-2和eSRKE-3,用酵母双杂交系统3检测各eSRK重组体与SCR之间的相互作用。结果显示,pGBKT7-SCRE×pGADT7-eSRKE、 PGBKT7-SCRF×pGADT7-eSRKF口阳性对照组pGBKT7-p53×pGADT7-T这三对组合能在SD/-His/-Trp/-Leu/-Ade/X-α-Gal/25mM3-AT平板上生长,其余8个组合在SD/-His/-Trp/-Leu/-Ade/X-α-Gal/25mM3-AT平板上不能生长,表明：1)SCRE能与eSRKE作用,而不能与eSRKF作用,说明eSRKE、eSRKF属于不同单倍型；2)SCRE与重组体eSRKE-1、eSRKE-2、eSRKE-3均不发生作用,HVⅠ和HVⅡ区域内差异的氨基酸位点共同参与了与SCR的作用；3)SCRF不能与eSRKE-1、eSRKE-2、eSRKE-3作用,替换HV Ⅰ/HVⅡ区域后并不能改变SRK的单倍型。3.2SCR与eSRK及其重组体相互作用的体外检测构建了pGEX-SCRE、pGEX-SCRF、PET43.1(a)-eSRKE、PET43.1(a)-eSRKF、 PET43.1(a)-eSRKE-1、PET43.1(a)-eSRKE-2和PET43.1(a)-eSRKE-37个原核表达载体。在16℃下用0.1mM IPTG过夜诱导pGEX-SCRE、pGEX-SCRF重组质粒表达,获得了带有GST标签的可溶的SCRE和SCRF蛋白,分子量大小分别为33.9kD、33.0kD。在25℃下用0.1mM IPTG过夜诱导PET43.1(a)-eSRKF、PET43.1(a)-eSRKF、 PET43.1(a)-eSRKE-1、PET43.1(a)-eSRKE-2和PET43.1(a)-eSRKE-3重组质粒表达,获得了带有His标签的可溶的eSRKE、eSRKF、eSRKE-1、eSRKE2和eSRKE-3融合蛋白,分子量大小均为107.7kD。将纯化后的SCRE、SCRF分别与所有的eSRK融合蛋白(eSRKE、eSRKF、eSRKE-1、eSRKE-2和eSRKE-3)进行孵育,混合物经GST磁珠纯化后进行SDS-聚丙烯酰胺电泳,结果显示每道泳道上都出现eSRK和SCR的条带,说明无论是SCRE还是SCRF,其体外表达产物均能与eSRKE、eSRKF、 eSRKE-1、eSRKE-2、eSRKE-3蛋白相互结合,不存在单倍型特异性,但各个eSRK对配体的结合能力稍有差异。这一结果表明重组SRK可溶蛋白在体外对配体有着普遍性结合,失去了它在细胞膜上的配体特异性结合能力。4甘蓝eSRK突变体的构建及其与SCR相互作用的研究为了更一步探讨eSRK上决定其配体特异性结合及单倍型特异性的氨基酸位点,本次实验在eSRKE氨基酸骨架上用重叠延伸PCR技术构建了9个点突变,即M1、M2……M9。将eSRKE和9个突变体与酵母双杂交载体pGADT7进行连接,分别与pGBKT7-SCRE共转化酵母AH109感受态细胞,观察转化子在SD/-His/-Trp/-Leu和SD/-His/-Trp/-Leu/-Ade/X-a-Gal/25mM3-AT平板上的生长状态,以此检测eSRKE及其9个突变体与SCRE相互作用的情况。结果显示,M1、M3、M5、M6、M9和阴性对照组在SD/-His/-Trp/-Leu/-Ade/X-a-Gal/25mM3-AT平板上呈现白色,而且不能生长；eSRKE、M2、M4、M7、M8和阳性对照组SD/-His/-Trp/-Leu/-Ade/X-a-Gal/25mM3-AT平板上生长良好且呈现出蓝色。结果表明,M2、M4、M7与SCRE的相互作用最强烈,M8与SCRE的相互作用力与eSRKE相当,M1、M3、M5、M6、M9不能与SCRE相互作用。说明将L179、G182、I248、I252、F269突变成丙氨酸后,eSRKE失去了与SCRE特异性结合的能力；将S181、Q184、V253、1264突变成丙氨酸后并不能消除eSRKE与SCRE特异性结合的功能,但其与SCRE相互作用的强度稍有差异,表明单个位点的变化并不一定能消除SRK的单倍型特异性及其配体结合的特异性。更多还原

【Abstract】 The selective pressure on hermaphrodite flowering plants to prevent inbreeding has been a powerful evolutionary driving force, resulting in the evolution of numerous self-incompatibility (SI) systems. SI is a model system to study plant cell signaling. Amongst members of the Brassicaceae, SI is characterized by the inhibition of self-or self-related pollen at the stigmatic surface. The molecular events of Brassica SI have been elucidated in some detail and depend on a receptor:ligand interaction that occurs at the stigmatic surface. The receptor is a membrane-spanning serine/threonine receptor kinase termed S-locus receptor kinase (SRK). SRK is located in the plasma membrane of the papillar cells that cover the stigmatic surface. The extracellular domain (eSRK), which is responsible for ligand binding, is highly polymorphic between haplotypes, as one would expect for a specificity determining molecule. The ligand for SRK is termed S cysteine-rich (SCR) and has also been designated SP11. SCR is a member of one family of small, cysteine-rich proteins. The haplotype specific binding of SCR to SRK result in rejection of self-pollen.To date, the specificity determinants of SRK have not been examined experimentally; however, bioinformatic speculation has resulted in a number of hypotheses as to which region(s) of the molecule is involved. It is known that eSRK is responsible for ligand binding and eSRK can be split into three subdomains based on sequence similarity. The N-terminal region is similar to that of mannose-binding lectins, the middle hyper variable region contains most of the variability seen among haplotypes, and the final C-terminal region is most similar to a PAN or apple domain involved in protein-protein or protein-carbohydrate interactions. Of most interest to researchers has been the hyper variable region, where three distinct regions of variability (HVI, HVII and HVIII) have been identified and are thought to be under balancing selection. In addition, a number of specific amino acids are speculated to be under selection to change physiochemical properties, which may explain the evolution of new S haplotypes. Data are presented demonstrating that the majority of SCR binding is focused in the hyper variable subdomain.Researching on self-incompatibility will impact not only on the understanding of SRK and receptor kinase signaling in plants, but will also provide a good tool to enhance forth putting of SI accessions in Brassicas breeding. To identify amino acid fragments within the SRK extracellular domain (eSRK) that are required for ligand-selective activation, we assayed chimeric eSRK between two S-locus haplotype (S7and S28) in Brassica oleracea (inbred lines E and F), and identified the interaction between eSRK chimeras and SCRs by Yeast Two-Hybrid System. To identify residues within the hvⅠ-hvⅡ region that determine SI specificity, we focused on polymorpHic sites that differ between the hvl-hvⅢ regions of eSRKs. The eSRKE were modified by site-directed mutagenesis to generate9mutants containing single-site substitutions at hvⅠ-hvⅢ regions. The interaction between eSRKE mutants and SCRE will be detected by Yeast Two-Hybrid System.1Affinity analysis of plant materialE and F are inbred lines of Brassica oleracea L. var.capitata L. To identify the.compatibility of E、F, we assayed affinity index determination and fluorescence microscopy of pollen germination in situ. Germination assay showed that there were few pollens came from E can germination on E’s pistil, and only a few (<10pollen tubes) pollen tubes got through the stigma, as well as F, indicated that E and F wre self-incompatibility lines. when self-fertilization they got a low compatibility index as0.23and0.19, respectively. When cross-pollination, compatibility index of E and F were higher than5, there are more than25pollen tubes could get through the stigma. All those results above suggested that E and F belonged to different S haplotype. 2Cloning of eSRK and SCR genesWe designed specific primers eSRKS/eSRKAS used to clone eSRK cDNA of E, F by RT-PCR base on the relatively conservative regions of eSRK amino acid residue, which named as eSRKE and eSRKF respectively. eSRKE nucleic acid sequence was highly homologous to BoSRK28, and eSRKF nucleic acid sequence was highly homologous to BoSRK7,both of which were determined by BLAST analysis in NCBI. BLAST results suggested that E was S28haplotype, and F was S7haplotype. Two eSRK amino acid sequences containd three subdomains of eSRK proteins, mannose-binding lectins like domain, the middle hyper variable region and PAN-Apple domain, signal peptide of the N-terminal was not included. There were12conserved cysteine residues in eSRK-E and eSRKF amino acid sequences. Primers used to clone SCR gene were based on the nucletides information. SCRE and SCRFwere homologous to BoSP11-28and BoSP11-7, respectively, which were amplified from gDNA of E and F. The SCRF gene encodes53amino acids, the SCRE encoding61amino acids. The two amino acid sequence contained all the amino acids of the mature peptide of SCR. They also contained one glycine residue and eight cysteine residues which were conserved with all other SCR alleles.3Detection of the interaction between chimeric eSRKs and SCRs3.1Detection of the interaction between chimeric eSRKs and SCRs by Yeast Two-Hybrid systemTo study the role of the HV Ⅰ/Ⅱ region in SRK ligand-selective activation, we performed Yeast Two-Hybrid System assay on chimeric eSRK between eSRKE and eSRK-F, and identified the interaction between eSRK chimeras and SCRs. The clones contained pGBKT7-SCRE×pGADT7-eSRKE, pGBKT7-SCRF×pGADT7-eSRKF or positive control pGBKT7-p53×pGADT7-T grown on SD/-His/-Trp/-Leu/-Ade/X-a-Gal/25mM3-AT agar plate, and clones contained the others did not grow on SD/-His/-Trp/-Leu/-Ade/X-a-Gal/25mM3-AT agar plate. This pHenomenon showed that SRKE (not chimeras) could interact with SCRE, the same as SRKF-SCRF. All of eSRK chimeras could not interact with SCRs. The results demonstrated that HV Ⅰ and HV Ⅱ region were essential for specificity in the SRK-SCR interaction. However, eSRK chimeras could not interact with SCRF, should be due to the overall sequence or3D conformation of the segments which determine SI specificity, although they contained hypervariable regions came from eSRKF.3.2Expression of the chimeric eSRKs and SCRs in E.coli, and detection of the interaction between chimeric eSRKs and SCRs in vitropET43.1(a)+was used as prokaryotic expression plasmid to express His6eSRK fused proteins and chimeric His6eSRKs fused proteins. SCR genes were ligand with pGEX-6p-1expression plasmid, which can express GST fused protein. The E.coli BL21(DE3) contained pET43.1(a)-eSRK (eSRK、eSRKF、eSRKE-1、eSRKE-2and eSRKE-3) plasmid were induced overnight by0.1mM IPTG under25℃. The expreesion products were purified by MagHis M Protein Purification Systerm and analyzed by SDS-PAGE. SDS-PAGE showed that the soluble protein SRKE、eSRKF、eSRKE-1、 eSRKE-2and eSRKE-3were expressed at107.7kD. The soluble recombinant SCRE、 SCRF were overnight expressed by0.1mM IPTG under16℃. The expression products were purified by MagGSTTM Protein Purification System and analyzed by SDS-PAGE. SDS-PAGE showed that recombinant SCRE and SCRF was expressed at33.9kD and33.0kD receptively.To test the binding specificities of recombinant eSRKs and their chimeras, eSRKE、 eSRKF、eSRKE-1、eSRKE-2and eSRKE-3were incubated with an equal amount of SCRE receptively. Bound SCRE was purified by MagGSTTM Protein Purification System and analyzed by SDS-PAGE. SDS-PAGE showed that all of receptors bound SCRE, showed no obvious haplotype specificity. SCRF was able to pull down eSRKE、eSRKF eSRKE-1、eSRKE-2and eSRKE-3, like SCRE. These results indicate that, for these haplotypes at least, eSRKs and their chimeras in isolation retain the ability to bind SCR but do not display S specificity in vitro, although SCRE and SCRF showed unequal efficiency in binding with each receptors.4Detection of the interaction between eSRKE mutants and SCRE by Yeast Two-Hybrid systemTo identify residues determines SI specificity within the hvl-hvⅡ region, we replaced L179、S181、G182、Q184、1248、1252、V253、1264and F269in eSRKE amino acid backbone with Alanine residue receptively. Those mutants were named as M1, M2, M3, M4, M5, M6, M7, M8, M9receptively. To test the ligand-specific activation of eSRKE mutants, each of mutants and SCRE was cotransformed into AH109yeast stain. Transformants were screened on SD/-His/-Trp/-Leu agar plates, then on SD/-His/-Trp/-Leu/-Ade/X-a-Gal/25mM3-AT agar plates. All of transformants can grow on SD/-His/-Trp/-Leu agar plates. Except for M1、M3、M5、M6、M9and negative control, all of the other transformants grow on SD/-His/-Trp/-Leu/-Ade/X-a-Gal/25mM3-AT agar plates, and showed different colors from pale blue to dark blue. This result indicated that S181A, Q184A, V253A, I264A mutants showed different efficiency in ligand-specific activation; L179A, G182A, I248A, I252A, F269A mutants did not interact with SCRE.更多还原