甘蓝eSRK重组体、突变体的构建及其与SCR相互作用单倍型的研究
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。
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 material
E 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 genes
We 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 SCRs
3.1Detection of the interaction between chimeric eSRKs and SCRs by Yeast Two-Hybrid system
To 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 vitro
pET43.1(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 system
To 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.
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。
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 material
E 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 genes
We 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 SCRs
3.1Detection of the interaction between chimeric eSRKs and SCRs by Yeast Two-Hybrid system
To 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 vitro
pET43.1(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 system
To 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.
引文
[1]Holsinger KE, Steinbachs JE:Mating systems and evolution in flowering plants. In:Evolution and Diversification of Land Plants. Edited by Iwatsuki K, Raven PH. Berlin:Springer-Verlag; 1997:223-248.
[2]Barrett SCH:The evolution of matingstrategies in floweringplants. Trans in Plant Sci 1998,3(9):335-341.
[3]Charlesworth B, Charlesworth D:Darwin and Genetics. Genetics 2009, 183(3):757-766.
[4]Barrett SCH:The evolution of plant sexual diversity. Nature Reviews Genetics 2002,3(4):274-284.
[5]Pandey KK:Evolution of Incompatibility Systems in Plants:Origin of 'Independent' and 'Complementary' Control of Incompatibility in Angiosperms. New Phytol 1980,84:381-400.
[6]de Nettancourt D:Incompatibility and incongruity in wild and cultivated plants. In.,2nd edn. Berlin:Springer-Verlag; 2001.
[7]Stein JC, Howlett B, Boyes DC, Nasrallah ME, Nasrallah JB:Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proceedings of the National Academy of Sciences of the United States of A merica 1991,88(19):8816-8820.
[8]Schopfer CR, Nasrallah ME, Nasrallah JB:The male determinant of self-incompatibility in Brassica. Science (New York, NY) 1999, 286(5445):1697-1700.
[9]Shiba H, Takayama S, Iwano M, Shimosato H, Funato M, Nakagawa T, Che FS, Suzuki G, Watanabe M, Hinata K et al: A pollen coat protein, SP11/SCR, determines the pollen S-specificity in the self-incompatibility of Brassica species. Plant Physiol 2001,125(4):2095-2103.
[10]Takasaki T, Hatakeyama K, Suzuki G, Watanabe M, Isogai A, Hinata K:The S receptor kinase determines self-incompatibility in Brassica stigma. Nature 2000, 403(6772):913-916.
[11]Takayama S, Shimosato H, Shiba H, Funato M, Che FS, Watanabe M, Iwano M, Isogai A:Direct ligand-receptor complex interaction controls Brassica self-incompatibility. Nature 2001,413(6855):534-538.
[12]Kachroo A, Schopfer CR, Nasrallah ME, Nasrallah JB:Allele-specific receptor-ligand interactions in Brassica self-incompatibility. Science (New York, NY) 2001,293(5536):1824-1826.
[13]Gu T, Mazzurco M, Sulaman W, Matias DD, Goring DR:Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase. Proceedings of the National Academy of Sciences of the United States of America 1998, 95(1):382-387.
[14]Stone SL, Arnoldo M, Goring DR:A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants. Science (New York, NY) 1999, 286(5445):1729-1731.
[15]Murase K, Shiba H, Iwano M, Che FS, Watanabe M, Isogai A, Takayama S:A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling. Science 2004,303(5663):1516-1519.
[16]Bower MS, Matias DD, Fernandes-Carvalho E, Mazzurco M, Gu T, Rothstein SJ, Goring DR:Two members of the thioredoxin-h family interact with the kinase domain of a Brassica S locus receptor kinase. The Plant cell 1996, 8(9):1641-1650.
[17]Cabrillac D, Cock JM, Dumas C, Gaude T:The S-locus receptor kinase is inhibited by thioredoxins and activated by pollen coat proteins. Nature 2001, 410(6825):220-223.
[18]Haffani YZ, Gaude T, Cock JM, Goring DR:Antisense suppression of thioredoxin h mRNA in Brassica napus cv. Westar pistils causes a low level constitutive pollen rejection response. Plant MolBiol 2004,55(5):619-630.
[19]Vanoosthuyse V, Tichtinsky G, Dumas C, Gaude T, Cock JM:Interaction of calmodulin, a sorting nexin and kinase-associated protein phosphatase with the Brassica oleracea S locus receptor kinase. Plant Physiol 2003,133(2):919-929.
[20]Naithani S, Chookajorn T, Ripoll DR, Nasrallah JB:Structural modules for receptor dimerization in the S-locus receptor kinase extracellular domain. Proceedings of the National Academy of Sciences of the United States of America 2007,104(29):12211-12216.
[21]Ivanov R, Gaude T:Endocytosis and Endosomal Regulation of the S-Receptor Kinase during the Self-Incompatibility Response in Brassica oleracea. Plant Cell 2009,21(7):2107-2117.
[22]Kemp BP, Doughty J:S cysteine-rich (SCR) binding domain analysis of the Brassica self-incompatibility S-locus receptor kinase. New Phytol 2007, 175(4):619-629.
[23]Boggs NA, Dwyer KG, Nasrallah ME, Nasrallah JB:In Vivo Detection of Residues Required for Ligand-Selective Activation of the S-Locus Receptor in Arabidopsis. Curr Biol 2009,19(9):786-791.
[24]Miege C, Ruffio-Chable V, Schierup MH, Cabrillac D, Dumas C, Gaude T, Cock JM:Intrahaplotype polymorphism at the Brassica S locus. Genetics 2001, 159(2):811-822.
[25]Germain H, Houde J, Gray-Mitsumune M, Sawasaki T, Endo Y, Rivoal J, Matton DP:Characterization of ScORK28, a transmembrane functional protein receptor kinase predominantly expressed in ovaries from the wild potato species Solanum chacoense. FEBS Lett 2007,581(26):5137-5142.
[26]Goring DR, Rothstein SJ:The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase. The Plant cell 1992,4(10):1273-1281.
[27]Giranton JL, Dumas C, Cock JM, Gaude T:The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta. Proceedings of the National Academy of Sciences of the United States of America 2000, 97(7):3759-3764.
[28]Stein JC, Dixit R, Nasrallah ME, Nasrallah JB:SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco. The Plant cell 1996,8(3):429-445.
[29]Kusaba M, Dwyer K, Hendershot J, Vrebalov J, Nasrallah JB, Nasrallah ME: Self-incompatibility in the genus Arabidopsis:characterization of the S locus in the outcrossing A. lyrata and its autogamous relative A. thaliana. The Plant cell 2001,13(3):627-643.
[30]Delorme V, Giranton JL, Hatzfeld Y, Friry A, Heizmann P, Ariza MJ, Dumas C, Gaude T, Cock JM:Characterization of the S locus genes, SLG and SRK, of the Brassica S3 haplotype:identification of a membrane-localized protein encoded by the S locus receptor kinase gene. The Plant journal:for cell and molecular biology 1995,7(3):429-440.
[31]张爱芬,王立,侯喜林,刘同坤,李英：不结球白菜S位点受体激酶基因片段的克隆与表达分析.南京农业大学学报2011,34(3)：25-30.
[32]张莉,朱利泉,贾华,李明,吴玮铷,唐章林,王小佳：甘蓝S位点受体激酶基因(SRK)编码区的甲基化分析.农业生物技术学报2010,18(3)：482-488.
[33]Cock JM, Swarup R, Dumas C:Natural antisense transcripts of the S locus receptor kinase gene and related sequences in Brassica oleracea. Molecular & general genetics:MGG 1997,255(5):514-524.
[34]Liu P, Sherman-Broyles S, Nasrallah ME, Nasrallah JB:A cryptic modifier causing transient self-incompatibility in Arabidopsis thaliana. Curr Biol 2007, 17(8):734-740.
[35]Tantikanjana T, Rizvi N, Nasrallah ME, Nasrallah JB:A Dual Role for the S-Locus Receptor Kinase in Self-Incompatibility and Pistil Development Revealed by an Arabidopsis rdr6 Mutation. Plant Cell 2009,21(9):2642-2654.
[36]Nasrallah JB, Liu P, Sherman-Broyles S, Schmidt R, Nasrallah ME:Epigenetic mechanisms for breakdown of self-incompatibility in interspecific hybrids. Genetics 2007,175(4):1965-1973.
[37]Giranton JL, Ariza MJ, Dumas C, Cock JM, Gaude T:The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SKR extracellular domain in Brassica oleracea. The Plant journal:for cell and molecular biology 1995,8(6):827-834.
[38]Shimosato H, Yokota N, Shiba H, Iwano M, Entani T, Che FS, Watanabe M, Isogai A, Takayama S:Characterization of the SP11/SCR high-affinity binding site involved in self/nonself recognition in Brassica self-incompatibility. Plant Cell 2001,19(1):107-117.
[39]Iwano M, Shiba H, Funato M, Shimosato H, Takayama S, Isogai A: Immunohistochemical studies on translocation of pollen S-haplotype determinant in self-incompatibility of Brassica rapa. Plant Cell Physiol 2003, 44(4):428-436.
[40]Cabrillac D, Delorme V, Garin J, Ruffio-Chable V, Giranton JL, Dumas C, Gaude T, Cock JM:The S15 self-incompatibility haplotype in Brassica oleracea includes three S gene family members expressed in stigmas. The Plant cell 1999, 11(5):971-986.
[41]Kakita M, Murase K, Iwano M, Matsumoto T, Watanabe M, Shiba H, Isogai A, Takayama S:Two distinct forms of M-locus protein kinase localize to the plasma membrane and interact directly with S-Locus receptor kinase to transduce self-incompatibility signaling in Brassica rapa. Plant Cell 2007, 19(12):3961-3973.
[42]Ivanov R, Gaude T:Brassica self-incompatibility:a glimpse below the surface. Plant signaling & behavior 2009,4(10):996-998.
[43]Hatakeyama K, Takasaki T, Suzuki G, Nishio T, Watanabe M, Isogai A, Hinata K: The S receptor kinase gene determines dominance relationships in stigma expression of self-incompatibility in Brassica. The Plant journal:for cell and molecular biology 2001,26(1):69-76.
[44]Suzuki G, Watanabe M, Kai N, Matsuda N, Toriyama K, Takayama S, Isogai A, Hinata K:Three members of the S multigene family are linked to the S locus of Brassica. Molecular & general genetics:MGG 1997,256(3):257-264.
[45]蓝兴国,杨佳,李玉花：羽衣甘蓝花粉SCR 13-b基因的分离及其功能分析.园艺学报2009,36(4)：539-544.
[46]Watanabe M, Suzuki G, Takayama S, Isogai A, Hinata K:Genomic Organization of the SLG/SRK Region of the S Locus in Brassica Species Annals of Botany 1999,85:155-160.
[47]Shiba H, Kimura N, Takayama S, Hinata K, Suzuki A, Isogai A:Alteration of the self-incompatibility phenotype in Brassica by transformation of the antisense SLG gene. Bioscience, biotechnology, and biochemistry 2000,64(5):1016-1024.
[48]Hinata K, Watanabe M, Yamakawa S, Satta Y, Isogai A:Evolutionary aspects of the S-related genes of the Brassica self-incompatibility system:synonymous and nonsynonymous base substitutions. Genetics 1995,140(3):1099-1104.
[49]Takayama S, Shiba H, Iwano M, Shimosato H, Che FS, Kai N, Watanabe M, Suzuki G, Hinata K, Isogai A:The pollen determinant of self-incompatibility in Brassica campestris. Proceedings of the National Academy of Sciences of the United States of America 2000,97(4):1920-1925.
[50]Sato Y, Fujimoto R, Toriyama K, Nishio T:Commonality of self-recognition specificity of S haplotypes between Brassica oleracea and Brassica rapa. Plant MolBiol 2003,52(3):617-626.
[51]Chookajorn T, Kachroo A, Ripoll DR, Clark AG, Nasrallah JB:Specificity determinants and diversification of the Brassica self-incompatibility pollen ligand. Proceedings of the National Academy of Sciences of the United States of America 2004,101(4):911-917.
[52]Mishima M, Takayama S, Sasaki K, Jee J, Kojima C, Isogai A, Shirakawa M: Structure of the male determinant factor for Brassica self-incompatibility. Journal of Biological Chemistry 2003,278(38):36389-36395.
[53]Schopfer CR, Nasrallah JB:Self-incompatibility. Prospects for a novel putative peptide-signaling molecule. Plant Physiol 2000,124(3):935-940.
[54. Shiba H, Iwano M, Entani T, Ishimoto K, Shimosato H, Che FS, Satta Y, Ito A, Takada Y, Watanabe M et al: The dominance of alleles controlling self-incompatibility in Brassica pollen is regulated at the RNA level. Plant Cell 2002,14(2):491-504.
[55]Shiba H, Kakizaki T, Iwano M, Tarutani Y, Watanabe M, Isogai A, Takayama S: Dominance relationships between self-incompatibility alleles controlled by DNA methylation. Nature Genet 2006,38(3):297-299.
[56]Stone SL, Anderson EM, Mullen RT, Goring DR:ARC1 is an E3 ubiquitin ligase and promotes the ubiquitination of proteins during the rejection of self-incompatible Brassica pollen. Plant Cell 2003,15(4):885-898.
[57]Kakita M, Shimosato H, Murasea K, Isogai A, Takayama S:Direct interaction between S-locus receptor kinase and M-locus protein kinase involved in Brassica self-incompatibility signaling.pdf>. Plant Biotechnology 2007, 24:185-190.
[58]Samuel MA, Mudgil Y, Salt JN, Delmas F, Ramachandran S, Chilelli A, Goring DR:Interactions between the s- domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in arabidopsis. Plant Physiol 2008,147(4):2084-2095.
[59]牛义,王志敏,高启国,宋明,王小佳,朱利泉：甘蓝自交不亲和信号传导元件ARC1的体外表达及其与SRK相互作用验证.作物学报2009,35(7)：1202-1208.
[60]蓝兴国,杨佳,赵昕,于凯,李玉花：羽衣甘蓝授粉过程中柱头蛋白质的泛素化.植物生理学通讯2010,46(3)：228-230.
[61]Geldner N, Robatzek S:Plant receptors go endosomal:A moving view on signal transduction. Plant Physiol 2008,147(4):1565-1574.
[62]杨昆,张贺翠,Converse R,朱利泉,杨永军,薛丽琰,罗兵,常登龙,高启国,王小佳：甘蓝自交不亲和信号传导元件ARC1与EXO70A1的相互作用.作物学报2012,37(12)：2136-2144.
[63]Samuel MA, Chong YT, Haasen KE, Aldea-Brydges MG, Stone SL, Goring DR: Cellular Pathways Regulating Responses to Compatible and Self-Incompatible Pollen in Brassica and Arabidopsis Stigmas Intersect at Exo70A1, a Putative Component of the Exocyst Complex. Plant Cell 2009,21(9):2655-2671.
[64]杨佳,蓝兴国,郝艾馨,李玉花：羽衣甘蓝Exo70A1基因的分离及表达分析.园艺学报2012,39(1)：127-134.
[65]杨昆,周永祥,张贺翠,赵永斌,杨永军,陆俊杏,朱利泉,薛丽琰,吕俊,高启国：甘蓝、大白菜和甘蓝型油菜EXO70A1基因的克隆与表达特性.作物学报2012,38(4)：578-588.
[66]Synek L, Schlager N, Elias M, Quentin M, Hauser MT, Zarsky V:AtEXO70A1, a member of a family of putative exocyst subunits specifically expanded in land plants, is important for polar growth and plant development. Plant J 2006, 48(1):54-72.
[67]Nasrallah ME, Liu P, Sherman-Broyles S, Boggs NA, Nasrallah JB:Natural variation in expression of self-incompatibility in Arabidopsis thaliana: Implications for the evolution of selfing. Proceedings of the National Academy of Sciences of the United States of America 2004,101(45):16070-16074.
[68]Iwano M, Shiba H, Matoba K, Miwa T, Funato M, Entani T, Nakayama P, Shimosato H, Takaoka A, Isogai A et al: Actin dynamics in papilla cells of Brassica rapa during self-and cross-pollination. Plant Physiol 2007, 144(1):72-81.
[69]Elleman CJ, Dickinson HG:Identification of Pollen Components Regulating Pollination-Specific Responses in the Stigmatic Papillae of Brassica oleracea. New Phytol 1996,133:197-205.
[70]Wang H-J, Wan A-R, Jauh G-Y:An actin-binding protein, L1LIM1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes. Plant Physiol 2008,147(4):1619-1636.
[71]孙梓健,韦静宜,王小佳,宋明,汤青林,王志敏,任雪松：结球甘蓝花粉钙调素基因的克隆与表达分析.园艺学报2012,39(4)：677-686.
[72]Updegraff EP, Zhao F, Preuss D:The extracellular lipase EXL4 is required for efficient hydration of Arabidopsis pollen. Sexual Plant Reproduction 2009, 22(3):197-204.
[73]Schiott M, Romanowsky SM, Baekgaard L, Jakobsen MK, Palmgren MG, Harper JF:A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization. Proceedings of the National Academy of Sciences of the United States of America 2004,101(25):9502-9507.
[74]Iwano M, Entani T, Shiba H, Kakita M, Nagai T, Mizuno H, Miyawaki A, Shoji T, Kubo K, Isogai A et al: Fine-Tuning of the Cytoplasmic Ca(2+) Concentration Is Essential for Pollen Tube Growth. Plant Physiol 2009, 150(3):1322-1334.
[75]Iwano M, Shiba H, Miwa T, Che FS, Takayama S, Nagai T, Miyawaki A, Isogai A: Ca2+ dynamics in a pollen grain and papilla cell during pollination of Arabidopsis. Plant Physiol 2004, 136(3):3562-3571.
[76]Ivanov R, Fobis-Loisy I, Gaude T: When no means no: guide to Brassicaceae self-incompatibility. Trends Plant Sci 2010, 15(7):387-394.
[77]Sarker RH, Elleman CJ, Dickinson HG: Control of pollen hydration in Brassica requires continued protein synthesis, and glycosylation in necessary for intraspecific incompatibility. Proceedings of the National Academy of Sciences of the United States of America 1988, 85(12):4340-4344.
[78]Okamoto S, Odashima M, Fujimoto R, Sato Y, Kitashiba H, Nishio T: Self-compatibility in Brassica napus is caused by independent mutations in S-locus genes. Plant J 2001, 50(3):391-400.
[79]Nasrallah ME, Liu P, Nasrallah JB: Generation of self-incompatible Arabidopsis thaliana by transfer of two S locus genes from A-lyrata. Science 2002, 297(5579):247-249.
[80]杨洋,高启国,宋明,牛义,汤青林,朱利泉,王小佳：甘蓝自交不亲和决定因子的体外表达和相互作用的检测.园艺学报2009,36(3)：355-362.
[81]罗兵,薛丽琰,朱利泉,张贺翠,彭一波,陈松,杨红,杨昆,李成琼,王小佳：利用酵母双杂交法检测甘蓝SCR与SRK之间的相互作用.作物学报2011,37(4)：579-586.
[82]杨红,朱利泉,张贺翠,杨永军,薛丽琰,杨昆,余浩,彭一波,罗兵,吴志刚et al：利用酵母双杂交系统鉴定甘蓝SCR与SRK胞外域片段的相互作用.中国农业科学2011,44(9)：1953-1962.
[83]Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H: Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor symposia on quantitative biology 1986, 51 Pt 1:263-273.
[84]Higuchi R, Krummel B, Saiki RK: A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic acids research 1988, 16(15):7351-7367.
[85]Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR: Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989, 77(1):51-59.
[86]Horton RM, Cai ZL, Ho SN, Pease LR:Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. BioTechniques 1990, 8(5):528-535.
[87]Vallejo AN, Pogulis RJ, Pease LR:PCR Mutagenesis by Overlap Extension and Gene SOE. CSHprotocols 2008,2008.
[88]王维鹏,夏剑波,李磊,郝友华,杨东亮：重叠延伸PCR法构建VPS4B基因定点突变真核表达载体.临床检验杂志2011,29(1)：57-59.
[89]李江,马永平,李世彬,贺志良,姜柯安：重叠延伸PCR法合成EV71VP1-LTB融合基因及其原核表达.中国微生态学杂志2011,23(5)：385-389.
[90]Hunt HD, Pullen JK, Cai Z, Horton RM, Ho SN, Pease LR:novel MHC variants spliced by overlap exetension. In:Transgenic mice and mutants in MHC research. Edited by Egorov IK, David CS. berlin:Springer-Verlag; 1990:47-55.
[91]Daugherty BL, DeMartino JA, Law MF, Kawka DW, Singer Ⅱ, Mark GE: Polymerase chain reaction facilitates the cloning, CDR-grafting, and rapid expression of a murine monoclonal antibody directed against the CD18 component of leukocyte integrins. Nucleic acids research 1991, 19(9):2471-2476.
[92]Davis GT, Bedzyk WD, Voss EW, Jacobs TW:Single chain antibody (SCA) encoding genes:one-step construction and expression in eukaryotic cells. Bio/technology (Nature Publishing Company) 1991,9(2):165-169.
[93]Pullen JK, Hunt HD, Pease LR:Peptide interactions with the Kb antigen recognition site. Journal of immunology (Baltimore, Md:1950) 1991, 146(7):2145-2151.
[94]Cai Z, Pease LR:Structural and functional analysis of three D/L-like class Ⅰ molecules from H-2v:indications of an ancestral family of D/L genes. The Journal of experimental medicine 1992,175(2):583-596.
[95]Gobius KS, Rowlinson SW, Barnard R, Mattick JS, Waters MJ:The first disulphide loop of the rabbit growth hormone receptor is required for binding to the hormone. Journal of molecular endocrinology 1992,9(3):213-220.
[96]Bobek LA, Tsai H, Levine MJ:Expression of human salivary histatin and cystatin/histatin chimeric cDNAs in Escherichia coli. Critical reviews in oral biology and medicine:an official publication of the American Association of Oral Biologists 1993,4(3-4):581-590.
[97]Kirchhoff F, Desrosiers RC:A PCR-derived library of random point mutations within the V3 region of simian immunodeficiency virus. PCR methods and applications 1993,2(4):301-304.
[98]Pease LR, Horton RM, Pullen JK, Hunt HD, Yun TJ, Rohren EM, Prescott JL, Jobe SM, Allen KS:Amino acid changes in the peptide binding site have structural consequences at the surface of class Ⅰ glycoproteins. Journal of immunology (Baltimore, Md:1950) 1993,150(8 Pt 1):3375-3381.
[99]Yun TJ, Tallquist MD, Rohren EM, Sheil JM, Pease LR:Minor pocket B influences peptide binding, peptide presentation and alloantigenicity of H-2Kb. International immunology 1994,6(7):1037-1047.
[100]Heckman KL, Pease LR:Gene splicing and mutagenesis by PCR-driven overlap extension. Nature Protocols 2007,2(4):924-932.
[101]王丽,杨文理,龚舒,梅志强,何涛：用重叠延伸PCR技术纠正人工合成乙型肝炎病毒核心抗原基因中的突变.泸州医学院学报2010,33(1)：25-27.
[102]horton RM, Pease LR:recombination and mutagenesis of DNA sequence using PCR. In:Direct mutagenesis:A pratical approach. Edited by J.Mcpherson M. unite kingdom:Oxford Univercity; 1991:217-247.
[103]Rychlik W:Selection of primers for polymerase chain reaction. Methods in molecular biology (Clifton, NJ) 1993,15:31-40.
[104]Fields S, Song O:A novel genetic system to detect protein-protein interactions. Nature 1989,340(6230):245-246.
[105]Fields S, Sternglanz R:The two-hybrid system:an assay for protein-protein interactions. Trends in genetics:TIG 1994,10(8):286-292.
[106]Vidal M, Brachmann RK, Fattaey A, Harlow E, Boeke JD:Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proceedings of the National Academy of Sciences of the United States of America 1996,93(19):10315-10320.
[107]Vidal M, Braun P, Chen E, Boeke JD, Harlow E:Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system. Proceedings of the National Academy of Sciences of the United States of America 1996,93(19):10321-10326.
[108]Stagljar I, Korostensky C, Johnsson N, te Heesen S:A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proceedings of the National Academy of Sciences of the United States of America 1998,95(9):5187-5192.
[109]Johnsson N, Varshavsky A:Split ubiquitin as a sensor of protein interactions in vivo. Proceedings of the National Academy of Sciences of the United States of America 1994,91(22):10340-10344.
[110]Li JJ, Herskowitz I:Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science (New York, NY) 1993, 262(5141):1870-1874.
[111]Liu J, Wilson TE, Milbrandt J, Johnston M:Identifying DNA-Binding Sites and Analyzing DNA-Binding Domains Using a Yeast Selection System. methods 1993,5(2):125-137.
[112]Alexander MK, Bourns BD, Zakian VA:One-hybrid systems for detecting protein-DNA interactions. Methods in molecular biology (Clifton, NJ) 2001, 177:241-259.
[113]Chien CT, Bartel PL, Sternglanz R, Fields S:The two-hybrid system:a method to identify and clone genes for proteins that interact with a protein of interest. Proceedings of the National Academy of Sciences of the United States of America 1991,88(21):9578-9582.
[114]Chevray PM, Nathans D:Protein interaction cloning in yeast:identification of mammalian proteins that react with the leucine zipper of Jun. Proceedings of the National Academy of Sciences of the United States of America 1992, 89(13):5789-5793.
[115]Dalton S, Treisman R:Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element. Cell 1992,68(3):597-612.
[116]SenGupta DJ, Zhang B, Kraemer B, Pochart P, Fields S, Wickens M:A three-hybrid system to detect RNA-protein interactions in vivo. Proceedings of the National Academy of Sciences of the United States of America 1996, 93(16):8496-8501.
[117]Vojtek AB, Hollenberg SM, Cooper JA:Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 1993,74(1):205-214.
[118]Kitson J, Raven T, Jiang YP, Goeddel DV, Giles KM, Pun KT, Grinham CJ, Brown R, Farrow SN:A death-domain-containing receptor that mediates apoptosis. Nature 1996,384(6607):372-375.
[119]Engelender S, Kaminsky Z, Guo X, Sharp AH, Amaravi RK, Kleiderlein JJ, Margolis RL, Troncoso JC, Lanahan AA, Worley PF et al: Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions. Nature Genet 1999,22(1):110-114.
[120]Hua SB, Luo Y, Qiu M, Chan E, Zhou H, Zhu L:Construction of a modular yeast two-hybrid cDNA library from human EST clones for the human genome protein linkage map. Gene 1998,215(1):143-152.
[121]Dortay H, Mehnert N, Buerkle L, Schmuelling T, Heyl A:Analysis of protein interactions within the cytokinin-signaling pathway of Arabidopsis thaliana. Febs Journal 2006,273(20):4631-4644.
[122]Kachroo A, Nasrallah ME, Nasrallah JB:Self-incompatibility in the Brassicaceae:Receptor-ligand signaling and cell-to-cell communication. Plant Cell 2002,14:S227-S238.
[123]Mazzurco M, Sulaman W, Elina H, Cock JM, Goring DR:Further analysis of the interactions between the Brassica S receptor kinase and three interacting proteins (ARC1, THL1 and THL2) in the yeast two-hybrid system. Plant MolBiol 2001, 45(3):365-376.
[124]Schierup MH, Mable BK, Awadalla P, Charlesworth D:Identification and characterization of a polymorphic receptor kinase gene linked to the self-incompatibility locus of Arabidopsis lyrata. Genetics 2001,158(1):387-399.
[125]Kusaba M, Nishio T, Satta Y, Hinata K, Ockendon D:Striking sequence similarity in inter-and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris:implications for the evolution and recognition mechanism. Proceedings of the National Academy of Sciences of the United States of America 1997,94(14):7673-7678.
[126]Sato K, Nishio T, Kimura R, Kusaba M, Suzuki T, Hatakeyama K, Ockendon DJ, Satta Y:Coevolution of the S-locus genes SRK, SLG and SP11/SCR in Brassica oleracea and B-rapa. Genetics 2002,162(2):931-940.
[127]Awadalla P, Charlesworth D:Recombination and selection at Brassica self-incompatibility loci. Genetics 1999,152(1):413-425.
[128]Sainudiin R, Wong WSW, Yogeeswaran K, Nasrallah JB, Yang ZH, Nielsen R: Detecting site-specific physicochemical selective pressures:Applications to the class IHLA of the human major histocompatibility complex and the SRK of the plant sporophytic self-incompatibility system. J Mol Evol 2005,60(3):315-326.
[129]Nordborg M, Innan H:The genealogy of sequences containing multiple sites subject to strong selection in a subdivided population. Genetics 2003, 163(3):1201-1213.
[130]Lu Y, Chanroj S, Zulkifli L, Johnson MA, Uozumi N, Cheung A, Sze H:Pollen Tubes Lacking a Pair of K(+) Transporters Fail to Target Ovules in Arabidopsis. Plant Cell 2011,23(1):81-93.
[131]何启伟,郭素英：十字花科蔬菜优势育种.北京：农业出版社；1993.
[132]Martin FW:Staining and observing pollen tubes in the style by means of fluorescence. Stain technology 1959,34(3):125-128.
[133]Voss Stern GA, Ockendon DJ, Gabrielson RL, Maguire JD:Self-incompatibility alleles in broccoli. Hort science 1982,17:748-749.
[134]F.M.奥斯伯,R.布伦特,R.E.金斯顿,D.D.穆尔,J.G.塞德曼,J.A.史密斯,K.斯特拉尔：精编分子生物学实验指南.北京：科学出版社;2008.
[135]J.E.科林根：精编蛋白质科学实验指南.北京：科学出版社；2007.
[136]Fowler TJ, Mitton MF, Vaillancourt LJ, Raper CA:Changes in mate recognition through alterations of pheromones and receptors in the multisexual mushroom fungus Schizophyllum commune. Genetics 2001,158(4):1491-1503.
[137]Gola S, Hegner J, Kothe E:Chimeric pheromone receptors in the basidiomycete Schizophyllum commune. Fungal genetics and biology:FG & B 2000, 30(3):191-196.
[138]Gola S, Kothe E:The little difference:in vivo analysis of pheromone discrimination in Schizophyllum commune. Curr Genet 2003,42(5):276-283.
[139]Dixit R, Nasrallah ME, Nasrallah JB:Post-transcriptional maturation of the S receptor kinase of Brassica correlates with co-expression of the S-locus glycoprotein in the stigmas of two Brassica strains and in transgenic tobacco plants. Plant Physiol 2000,124(1):297-311.
[2]Barrett SCH:The evolution of matingstrategies in floweringplants. Trans in Plant Sci 1998,3(9):335-341.
[3]Charlesworth B, Charlesworth D:Darwin and Genetics. Genetics 2009, 183(3):757-766.
[4]Barrett SCH:The evolution of plant sexual diversity. Nature Reviews Genetics 2002,3(4):274-284.
[5]Pandey KK:Evolution of Incompatibility Systems in Plants:Origin of 'Independent' and 'Complementary' Control of Incompatibility in Angiosperms. New Phytol 1980,84:381-400.
[6]de Nettancourt D:Incompatibility and incongruity in wild and cultivated plants. In.,2nd edn. Berlin:Springer-Verlag; 2001.
[7]Stein JC, Howlett B, Boyes DC, Nasrallah ME, Nasrallah JB:Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proceedings of the National Academy of Sciences of the United States of A merica 1991,88(19):8816-8820.
[8]Schopfer CR, Nasrallah ME, Nasrallah JB:The male determinant of self-incompatibility in Brassica. Science (New York, NY) 1999, 286(5445):1697-1700.
[9]Shiba H, Takayama S, Iwano M, Shimosato H, Funato M, Nakagawa T, Che FS, Suzuki G, Watanabe M, Hinata K et al: A pollen coat protein, SP11/SCR, determines the pollen S-specificity in the self-incompatibility of Brassica species. Plant Physiol 2001,125(4):2095-2103.
[10]Takasaki T, Hatakeyama K, Suzuki G, Watanabe M, Isogai A, Hinata K:The S receptor kinase determines self-incompatibility in Brassica stigma. Nature 2000, 403(6772):913-916.
[11]Takayama S, Shimosato H, Shiba H, Funato M, Che FS, Watanabe M, Iwano M, Isogai A:Direct ligand-receptor complex interaction controls Brassica self-incompatibility. Nature 2001,413(6855):534-538.
[12]Kachroo A, Schopfer CR, Nasrallah ME, Nasrallah JB:Allele-specific receptor-ligand interactions in Brassica self-incompatibility. Science (New York, NY) 2001,293(5536):1824-1826.
[13]Gu T, Mazzurco M, Sulaman W, Matias DD, Goring DR:Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase. Proceedings of the National Academy of Sciences of the United States of America 1998, 95(1):382-387.
[14]Stone SL, Arnoldo M, Goring DR:A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants. Science (New York, NY) 1999, 286(5445):1729-1731.
[15]Murase K, Shiba H, Iwano M, Che FS, Watanabe M, Isogai A, Takayama S:A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling. Science 2004,303(5663):1516-1519.
[16]Bower MS, Matias DD, Fernandes-Carvalho E, Mazzurco M, Gu T, Rothstein SJ, Goring DR:Two members of the thioredoxin-h family interact with the kinase domain of a Brassica S locus receptor kinase. The Plant cell 1996, 8(9):1641-1650.
[17]Cabrillac D, Cock JM, Dumas C, Gaude T:The S-locus receptor kinase is inhibited by thioredoxins and activated by pollen coat proteins. Nature 2001, 410(6825):220-223.
[18]Haffani YZ, Gaude T, Cock JM, Goring DR:Antisense suppression of thioredoxin h mRNA in Brassica napus cv. Westar pistils causes a low level constitutive pollen rejection response. Plant MolBiol 2004,55(5):619-630.
[19]Vanoosthuyse V, Tichtinsky G, Dumas C, Gaude T, Cock JM:Interaction of calmodulin, a sorting nexin and kinase-associated protein phosphatase with the Brassica oleracea S locus receptor kinase. Plant Physiol 2003,133(2):919-929.
[20]Naithani S, Chookajorn T, Ripoll DR, Nasrallah JB:Structural modules for receptor dimerization in the S-locus receptor kinase extracellular domain. Proceedings of the National Academy of Sciences of the United States of America 2007,104(29):12211-12216.
[21]Ivanov R, Gaude T:Endocytosis and Endosomal Regulation of the S-Receptor Kinase during the Self-Incompatibility Response in Brassica oleracea. Plant Cell 2009,21(7):2107-2117.
[22]Kemp BP, Doughty J:S cysteine-rich (SCR) binding domain analysis of the Brassica self-incompatibility S-locus receptor kinase. New Phytol 2007, 175(4):619-629.
[23]Boggs NA, Dwyer KG, Nasrallah ME, Nasrallah JB:In Vivo Detection of Residues Required for Ligand-Selective Activation of the S-Locus Receptor in Arabidopsis. Curr Biol 2009,19(9):786-791.
[24]Miege C, Ruffio-Chable V, Schierup MH, Cabrillac D, Dumas C, Gaude T, Cock JM:Intrahaplotype polymorphism at the Brassica S locus. Genetics 2001, 159(2):811-822.
[25]Germain H, Houde J, Gray-Mitsumune M, Sawasaki T, Endo Y, Rivoal J, Matton DP:Characterization of ScORK28, a transmembrane functional protein receptor kinase predominantly expressed in ovaries from the wild potato species Solanum chacoense. FEBS Lett 2007,581(26):5137-5142.
[26]Goring DR, Rothstein SJ:The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase. The Plant cell 1992,4(10):1273-1281.
[27]Giranton JL, Dumas C, Cock JM, Gaude T:The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta. Proceedings of the National Academy of Sciences of the United States of America 2000, 97(7):3759-3764.
[28]Stein JC, Dixit R, Nasrallah ME, Nasrallah JB:SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco. The Plant cell 1996,8(3):429-445.
[29]Kusaba M, Dwyer K, Hendershot J, Vrebalov J, Nasrallah JB, Nasrallah ME: Self-incompatibility in the genus Arabidopsis:characterization of the S locus in the outcrossing A. lyrata and its autogamous relative A. thaliana. The Plant cell 2001,13(3):627-643.
[30]Delorme V, Giranton JL, Hatzfeld Y, Friry A, Heizmann P, Ariza MJ, Dumas C, Gaude T, Cock JM:Characterization of the S locus genes, SLG and SRK, of the Brassica S3 haplotype:identification of a membrane-localized protein encoded by the S locus receptor kinase gene. The Plant journal:for cell and molecular biology 1995,7(3):429-440.
[31]张爱芬,王立,侯喜林,刘同坤,李英：不结球白菜S位点受体激酶基因片段的克隆与表达分析.南京农业大学学报2011,34(3)：25-30.
[32]张莉,朱利泉,贾华,李明,吴玮铷,唐章林,王小佳：甘蓝S位点受体激酶基因(SRK)编码区的甲基化分析.农业生物技术学报2010,18(3)：482-488.
[33]Cock JM, Swarup R, Dumas C:Natural antisense transcripts of the S locus receptor kinase gene and related sequences in Brassica oleracea. Molecular & general genetics:MGG 1997,255(5):514-524.
[34]Liu P, Sherman-Broyles S, Nasrallah ME, Nasrallah JB:A cryptic modifier causing transient self-incompatibility in Arabidopsis thaliana. Curr Biol 2007, 17(8):734-740.
[35]Tantikanjana T, Rizvi N, Nasrallah ME, Nasrallah JB:A Dual Role for the S-Locus Receptor Kinase in Self-Incompatibility and Pistil Development Revealed by an Arabidopsis rdr6 Mutation. Plant Cell 2009,21(9):2642-2654.
[36]Nasrallah JB, Liu P, Sherman-Broyles S, Schmidt R, Nasrallah ME:Epigenetic mechanisms for breakdown of self-incompatibility in interspecific hybrids. Genetics 2007,175(4):1965-1973.
[37]Giranton JL, Ariza MJ, Dumas C, Cock JM, Gaude T:The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SKR extracellular domain in Brassica oleracea. The Plant journal:for cell and molecular biology 1995,8(6):827-834.
[38]Shimosato H, Yokota N, Shiba H, Iwano M, Entani T, Che FS, Watanabe M, Isogai A, Takayama S:Characterization of the SP11/SCR high-affinity binding site involved in self/nonself recognition in Brassica self-incompatibility. Plant Cell 2001,19(1):107-117.
[39]Iwano M, Shiba H, Funato M, Shimosato H, Takayama S, Isogai A: Immunohistochemical studies on translocation of pollen S-haplotype determinant in self-incompatibility of Brassica rapa. Plant Cell Physiol 2003, 44(4):428-436.
[40]Cabrillac D, Delorme V, Garin J, Ruffio-Chable V, Giranton JL, Dumas C, Gaude T, Cock JM:The S15 self-incompatibility haplotype in Brassica oleracea includes three S gene family members expressed in stigmas. The Plant cell 1999, 11(5):971-986.
[41]Kakita M, Murase K, Iwano M, Matsumoto T, Watanabe M, Shiba H, Isogai A, Takayama S:Two distinct forms of M-locus protein kinase localize to the plasma membrane and interact directly with S-Locus receptor kinase to transduce self-incompatibility signaling in Brassica rapa. Plant Cell 2007, 19(12):3961-3973.
[42]Ivanov R, Gaude T:Brassica self-incompatibility:a glimpse below the surface. Plant signaling & behavior 2009,4(10):996-998.
[43]Hatakeyama K, Takasaki T, Suzuki G, Nishio T, Watanabe M, Isogai A, Hinata K: The S receptor kinase gene determines dominance relationships in stigma expression of self-incompatibility in Brassica. The Plant journal:for cell and molecular biology 2001,26(1):69-76.
[44]Suzuki G, Watanabe M, Kai N, Matsuda N, Toriyama K, Takayama S, Isogai A, Hinata K:Three members of the S multigene family are linked to the S locus of Brassica. Molecular & general genetics:MGG 1997,256(3):257-264.
[45]蓝兴国,杨佳,李玉花：羽衣甘蓝花粉SCR 13-b基因的分离及其功能分析.园艺学报2009,36(4)：539-544.
[46]Watanabe M, Suzuki G, Takayama S, Isogai A, Hinata K:Genomic Organization of the SLG/SRK Region of the S Locus in Brassica Species Annals of Botany 1999,85:155-160.
[47]Shiba H, Kimura N, Takayama S, Hinata K, Suzuki A, Isogai A:Alteration of the self-incompatibility phenotype in Brassica by transformation of the antisense SLG gene. Bioscience, biotechnology, and biochemistry 2000,64(5):1016-1024.
[48]Hinata K, Watanabe M, Yamakawa S, Satta Y, Isogai A:Evolutionary aspects of the S-related genes of the Brassica self-incompatibility system:synonymous and nonsynonymous base substitutions. Genetics 1995,140(3):1099-1104.
[49]Takayama S, Shiba H, Iwano M, Shimosato H, Che FS, Kai N, Watanabe M, Suzuki G, Hinata K, Isogai A:The pollen determinant of self-incompatibility in Brassica campestris. Proceedings of the National Academy of Sciences of the United States of America 2000,97(4):1920-1925.
[50]Sato Y, Fujimoto R, Toriyama K, Nishio T:Commonality of self-recognition specificity of S haplotypes between Brassica oleracea and Brassica rapa. Plant MolBiol 2003,52(3):617-626.
[51]Chookajorn T, Kachroo A, Ripoll DR, Clark AG, Nasrallah JB:Specificity determinants and diversification of the Brassica self-incompatibility pollen ligand. Proceedings of the National Academy of Sciences of the United States of America 2004,101(4):911-917.
[52]Mishima M, Takayama S, Sasaki K, Jee J, Kojima C, Isogai A, Shirakawa M: Structure of the male determinant factor for Brassica self-incompatibility. Journal of Biological Chemistry 2003,278(38):36389-36395.
[53]Schopfer CR, Nasrallah JB:Self-incompatibility. Prospects for a novel putative peptide-signaling molecule. Plant Physiol 2000,124(3):935-940.
[54. Shiba H, Iwano M, Entani T, Ishimoto K, Shimosato H, Che FS, Satta Y, Ito A, Takada Y, Watanabe M et al: The dominance of alleles controlling self-incompatibility in Brassica pollen is regulated at the RNA level. Plant Cell 2002,14(2):491-504.
[55]Shiba H, Kakizaki T, Iwano M, Tarutani Y, Watanabe M, Isogai A, Takayama S: Dominance relationships between self-incompatibility alleles controlled by DNA methylation. Nature Genet 2006,38(3):297-299.
[56]Stone SL, Anderson EM, Mullen RT, Goring DR:ARC1 is an E3 ubiquitin ligase and promotes the ubiquitination of proteins during the rejection of self-incompatible Brassica pollen. Plant Cell 2003,15(4):885-898.
[57]Kakita M, Shimosato H, Murasea K, Isogai A, Takayama S:Direct interaction between S-locus receptor kinase and M-locus protein kinase involved in Brassica self-incompatibility signaling.pdf>. Plant Biotechnology 2007, 24:185-190.
[58]Samuel MA, Mudgil Y, Salt JN, Delmas F, Ramachandran S, Chilelli A, Goring DR:Interactions between the s- domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in arabidopsis. Plant Physiol 2008,147(4):2084-2095.
[59]牛义,王志敏,高启国,宋明,王小佳,朱利泉：甘蓝自交不亲和信号传导元件ARC1的体外表达及其与SRK相互作用验证.作物学报2009,35(7)：1202-1208.
[60]蓝兴国,杨佳,赵昕,于凯,李玉花：羽衣甘蓝授粉过程中柱头蛋白质的泛素化.植物生理学通讯2010,46(3)：228-230.
[61]Geldner N, Robatzek S:Plant receptors go endosomal:A moving view on signal transduction. Plant Physiol 2008,147(4):1565-1574.
[62]杨昆,张贺翠,Converse R,朱利泉,杨永军,薛丽琰,罗兵,常登龙,高启国,王小佳：甘蓝自交不亲和信号传导元件ARC1与EXO70A1的相互作用.作物学报2012,37(12)：2136-2144.
[63]Samuel MA, Chong YT, Haasen KE, Aldea-Brydges MG, Stone SL, Goring DR: Cellular Pathways Regulating Responses to Compatible and Self-Incompatible Pollen in Brassica and Arabidopsis Stigmas Intersect at Exo70A1, a Putative Component of the Exocyst Complex. Plant Cell 2009,21(9):2655-2671.
[64]杨佳,蓝兴国,郝艾馨,李玉花：羽衣甘蓝Exo70A1基因的分离及表达分析.园艺学报2012,39(1)：127-134.
[65]杨昆,周永祥,张贺翠,赵永斌,杨永军,陆俊杏,朱利泉,薛丽琰,吕俊,高启国：甘蓝、大白菜和甘蓝型油菜EXO70A1基因的克隆与表达特性.作物学报2012,38(4)：578-588.
[66]Synek L, Schlager N, Elias M, Quentin M, Hauser MT, Zarsky V:AtEXO70A1, a member of a family of putative exocyst subunits specifically expanded in land plants, is important for polar growth and plant development. Plant J 2006, 48(1):54-72.
[67]Nasrallah ME, Liu P, Sherman-Broyles S, Boggs NA, Nasrallah JB:Natural variation in expression of self-incompatibility in Arabidopsis thaliana: Implications for the evolution of selfing. Proceedings of the National Academy of Sciences of the United States of America 2004,101(45):16070-16074.
[68]Iwano M, Shiba H, Matoba K, Miwa T, Funato M, Entani T, Nakayama P, Shimosato H, Takaoka A, Isogai A et al: Actin dynamics in papilla cells of Brassica rapa during self-and cross-pollination. Plant Physiol 2007, 144(1):72-81.
[69]Elleman CJ, Dickinson HG:Identification of Pollen Components Regulating Pollination-Specific Responses in the Stigmatic Papillae of Brassica oleracea. New Phytol 1996,133:197-205.
[70]Wang H-J, Wan A-R, Jauh G-Y:An actin-binding protein, L1LIM1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes. Plant Physiol 2008,147(4):1619-1636.
[71]孙梓健,韦静宜,王小佳,宋明,汤青林,王志敏,任雪松：结球甘蓝花粉钙调素基因的克隆与表达分析.园艺学报2012,39(4)：677-686.
[72]Updegraff EP, Zhao F, Preuss D:The extracellular lipase EXL4 is required for efficient hydration of Arabidopsis pollen. Sexual Plant Reproduction 2009, 22(3):197-204.
[73]Schiott M, Romanowsky SM, Baekgaard L, Jakobsen MK, Palmgren MG, Harper JF:A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization. Proceedings of the National Academy of Sciences of the United States of America 2004,101(25):9502-9507.
[74]Iwano M, Entani T, Shiba H, Kakita M, Nagai T, Mizuno H, Miyawaki A, Shoji T, Kubo K, Isogai A et al: Fine-Tuning of the Cytoplasmic Ca(2+) Concentration Is Essential for Pollen Tube Growth. Plant Physiol 2009, 150(3):1322-1334.
[75]Iwano M, Shiba H, Miwa T, Che FS, Takayama S, Nagai T, Miyawaki A, Isogai A: Ca2+ dynamics in a pollen grain and papilla cell during pollination of Arabidopsis. Plant Physiol 2004, 136(3):3562-3571.
[76]Ivanov R, Fobis-Loisy I, Gaude T: When no means no: guide to Brassicaceae self-incompatibility. Trends Plant Sci 2010, 15(7):387-394.
[77]Sarker RH, Elleman CJ, Dickinson HG: Control of pollen hydration in Brassica requires continued protein synthesis, and glycosylation in necessary for intraspecific incompatibility. Proceedings of the National Academy of Sciences of the United States of America 1988, 85(12):4340-4344.
[78]Okamoto S, Odashima M, Fujimoto R, Sato Y, Kitashiba H, Nishio T: Self-compatibility in Brassica napus is caused by independent mutations in S-locus genes. Plant J 2001, 50(3):391-400.
[79]Nasrallah ME, Liu P, Nasrallah JB: Generation of self-incompatible Arabidopsis thaliana by transfer of two S locus genes from A-lyrata. Science 2002, 297(5579):247-249.
[80]杨洋,高启国,宋明,牛义,汤青林,朱利泉,王小佳：甘蓝自交不亲和决定因子的体外表达和相互作用的检测.园艺学报2009,36(3)：355-362.
[81]罗兵,薛丽琰,朱利泉,张贺翠,彭一波,陈松,杨红,杨昆,李成琼,王小佳：利用酵母双杂交法检测甘蓝SCR与SRK之间的相互作用.作物学报2011,37(4)：579-586.
[82]杨红,朱利泉,张贺翠,杨永军,薛丽琰,杨昆,余浩,彭一波,罗兵,吴志刚et al：利用酵母双杂交系统鉴定甘蓝SCR与SRK胞外域片段的相互作用.中国农业科学2011,44(9)：1953-1962.
[83]Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H: Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor symposia on quantitative biology 1986, 51 Pt 1:263-273.
[84]Higuchi R, Krummel B, Saiki RK: A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic acids research 1988, 16(15):7351-7367.
[85]Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR: Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989, 77(1):51-59.
[86]Horton RM, Cai ZL, Ho SN, Pease LR:Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. BioTechniques 1990, 8(5):528-535.
[87]Vallejo AN, Pogulis RJ, Pease LR:PCR Mutagenesis by Overlap Extension and Gene SOE. CSHprotocols 2008,2008.
[88]王维鹏,夏剑波,李磊,郝友华,杨东亮：重叠延伸PCR法构建VPS4B基因定点突变真核表达载体.临床检验杂志2011,29(1)：57-59.
[89]李江,马永平,李世彬,贺志良,姜柯安：重叠延伸PCR法合成EV71VP1-LTB融合基因及其原核表达.中国微生态学杂志2011,23(5)：385-389.
[90]Hunt HD, Pullen JK, Cai Z, Horton RM, Ho SN, Pease LR:novel MHC variants spliced by overlap exetension. In:Transgenic mice and mutants in MHC research. Edited by Egorov IK, David CS. berlin:Springer-Verlag; 1990:47-55.
[91]Daugherty BL, DeMartino JA, Law MF, Kawka DW, Singer Ⅱ, Mark GE: Polymerase chain reaction facilitates the cloning, CDR-grafting, and rapid expression of a murine monoclonal antibody directed against the CD18 component of leukocyte integrins. Nucleic acids research 1991, 19(9):2471-2476.
[92]Davis GT, Bedzyk WD, Voss EW, Jacobs TW:Single chain antibody (SCA) encoding genes:one-step construction and expression in eukaryotic cells. Bio/technology (Nature Publishing Company) 1991,9(2):165-169.
[93]Pullen JK, Hunt HD, Pease LR:Peptide interactions with the Kb antigen recognition site. Journal of immunology (Baltimore, Md:1950) 1991, 146(7):2145-2151.
[94]Cai Z, Pease LR:Structural and functional analysis of three D/L-like class Ⅰ molecules from H-2v:indications of an ancestral family of D/L genes. The Journal of experimental medicine 1992,175(2):583-596.
[95]Gobius KS, Rowlinson SW, Barnard R, Mattick JS, Waters MJ:The first disulphide loop of the rabbit growth hormone receptor is required for binding to the hormone. Journal of molecular endocrinology 1992,9(3):213-220.
[96]Bobek LA, Tsai H, Levine MJ:Expression of human salivary histatin and cystatin/histatin chimeric cDNAs in Escherichia coli. Critical reviews in oral biology and medicine:an official publication of the American Association of Oral Biologists 1993,4(3-4):581-590.
[97]Kirchhoff F, Desrosiers RC:A PCR-derived library of random point mutations within the V3 region of simian immunodeficiency virus. PCR methods and applications 1993,2(4):301-304.
[98]Pease LR, Horton RM, Pullen JK, Hunt HD, Yun TJ, Rohren EM, Prescott JL, Jobe SM, Allen KS:Amino acid changes in the peptide binding site have structural consequences at the surface of class Ⅰ glycoproteins. Journal of immunology (Baltimore, Md:1950) 1993,150(8 Pt 1):3375-3381.
[99]Yun TJ, Tallquist MD, Rohren EM, Sheil JM, Pease LR:Minor pocket B influences peptide binding, peptide presentation and alloantigenicity of H-2Kb. International immunology 1994,6(7):1037-1047.
[100]Heckman KL, Pease LR:Gene splicing and mutagenesis by PCR-driven overlap extension. Nature Protocols 2007,2(4):924-932.
[101]王丽,杨文理,龚舒,梅志强,何涛：用重叠延伸PCR技术纠正人工合成乙型肝炎病毒核心抗原基因中的突变.泸州医学院学报2010,33(1)：25-27.
[102]horton RM, Pease LR:recombination and mutagenesis of DNA sequence using PCR. In:Direct mutagenesis:A pratical approach. Edited by J.Mcpherson M. unite kingdom:Oxford Univercity; 1991:217-247.
[103]Rychlik W:Selection of primers for polymerase chain reaction. Methods in molecular biology (Clifton, NJ) 1993,15:31-40.
[104]Fields S, Song O:A novel genetic system to detect protein-protein interactions. Nature 1989,340(6230):245-246.
[105]Fields S, Sternglanz R:The two-hybrid system:an assay for protein-protein interactions. Trends in genetics:TIG 1994,10(8):286-292.
[106]Vidal M, Brachmann RK, Fattaey A, Harlow E, Boeke JD:Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proceedings of the National Academy of Sciences of the United States of America 1996,93(19):10315-10320.
[107]Vidal M, Braun P, Chen E, Boeke JD, Harlow E:Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system. Proceedings of the National Academy of Sciences of the United States of America 1996,93(19):10321-10326.
[108]Stagljar I, Korostensky C, Johnsson N, te Heesen S:A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proceedings of the National Academy of Sciences of the United States of America 1998,95(9):5187-5192.
[109]Johnsson N, Varshavsky A:Split ubiquitin as a sensor of protein interactions in vivo. Proceedings of the National Academy of Sciences of the United States of America 1994,91(22):10340-10344.
[110]Li JJ, Herskowitz I:Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science (New York, NY) 1993, 262(5141):1870-1874.
[111]Liu J, Wilson TE, Milbrandt J, Johnston M:Identifying DNA-Binding Sites and Analyzing DNA-Binding Domains Using a Yeast Selection System. methods 1993,5(2):125-137.
[112]Alexander MK, Bourns BD, Zakian VA:One-hybrid systems for detecting protein-DNA interactions. Methods in molecular biology (Clifton, NJ) 2001, 177:241-259.
[113]Chien CT, Bartel PL, Sternglanz R, Fields S:The two-hybrid system:a method to identify and clone genes for proteins that interact with a protein of interest. Proceedings of the National Academy of Sciences of the United States of America 1991,88(21):9578-9582.
[114]Chevray PM, Nathans D:Protein interaction cloning in yeast:identification of mammalian proteins that react with the leucine zipper of Jun. Proceedings of the National Academy of Sciences of the United States of America 1992, 89(13):5789-5793.
[115]Dalton S, Treisman R:Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element. Cell 1992,68(3):597-612.
[116]SenGupta DJ, Zhang B, Kraemer B, Pochart P, Fields S, Wickens M:A three-hybrid system to detect RNA-protein interactions in vivo. Proceedings of the National Academy of Sciences of the United States of America 1996, 93(16):8496-8501.
[117]Vojtek AB, Hollenberg SM, Cooper JA:Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 1993,74(1):205-214.
[118]Kitson J, Raven T, Jiang YP, Goeddel DV, Giles KM, Pun KT, Grinham CJ, Brown R, Farrow SN:A death-domain-containing receptor that mediates apoptosis. Nature 1996,384(6607):372-375.
[119]Engelender S, Kaminsky Z, Guo X, Sharp AH, Amaravi RK, Kleiderlein JJ, Margolis RL, Troncoso JC, Lanahan AA, Worley PF et al: Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions. Nature Genet 1999,22(1):110-114.
[120]Hua SB, Luo Y, Qiu M, Chan E, Zhou H, Zhu L:Construction of a modular yeast two-hybrid cDNA library from human EST clones for the human genome protein linkage map. Gene 1998,215(1):143-152.
[121]Dortay H, Mehnert N, Buerkle L, Schmuelling T, Heyl A:Analysis of protein interactions within the cytokinin-signaling pathway of Arabidopsis thaliana. Febs Journal 2006,273(20):4631-4644.
[122]Kachroo A, Nasrallah ME, Nasrallah JB:Self-incompatibility in the Brassicaceae:Receptor-ligand signaling and cell-to-cell communication. Plant Cell 2002,14:S227-S238.
[123]Mazzurco M, Sulaman W, Elina H, Cock JM, Goring DR:Further analysis of the interactions between the Brassica S receptor kinase and three interacting proteins (ARC1, THL1 and THL2) in the yeast two-hybrid system. Plant MolBiol 2001, 45(3):365-376.
[124]Schierup MH, Mable BK, Awadalla P, Charlesworth D:Identification and characterization of a polymorphic receptor kinase gene linked to the self-incompatibility locus of Arabidopsis lyrata. Genetics 2001,158(1):387-399.
[125]Kusaba M, Nishio T, Satta Y, Hinata K, Ockendon D:Striking sequence similarity in inter-and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris:implications for the evolution and recognition mechanism. Proceedings of the National Academy of Sciences of the United States of America 1997,94(14):7673-7678.
[126]Sato K, Nishio T, Kimura R, Kusaba M, Suzuki T, Hatakeyama K, Ockendon DJ, Satta Y:Coevolution of the S-locus genes SRK, SLG and SP11/SCR in Brassica oleracea and B-rapa. Genetics 2002,162(2):931-940.
[127]Awadalla P, Charlesworth D:Recombination and selection at Brassica self-incompatibility loci. Genetics 1999,152(1):413-425.
[128]Sainudiin R, Wong WSW, Yogeeswaran K, Nasrallah JB, Yang ZH, Nielsen R: Detecting site-specific physicochemical selective pressures:Applications to the class IHLA of the human major histocompatibility complex and the SRK of the plant sporophytic self-incompatibility system. J Mol Evol 2005,60(3):315-326.
[129]Nordborg M, Innan H:The genealogy of sequences containing multiple sites subject to strong selection in a subdivided population. Genetics 2003, 163(3):1201-1213.
[130]Lu Y, Chanroj S, Zulkifli L, Johnson MA, Uozumi N, Cheung A, Sze H:Pollen Tubes Lacking a Pair of K(+) Transporters Fail to Target Ovules in Arabidopsis. Plant Cell 2011,23(1):81-93.
[131]何启伟,郭素英：十字花科蔬菜优势育种.北京：农业出版社；1993.
[132]Martin FW:Staining and observing pollen tubes in the style by means of fluorescence. Stain technology 1959,34(3):125-128.
[133]Voss Stern GA, Ockendon DJ, Gabrielson RL, Maguire JD:Self-incompatibility alleles in broccoli. Hort science 1982,17:748-749.
[134]F.M.奥斯伯,R.布伦特,R.E.金斯顿,D.D.穆尔,J.G.塞德曼,J.A.史密斯,K.斯特拉尔：精编分子生物学实验指南.北京：科学出版社;2008.
[135]J.E.科林根：精编蛋白质科学实验指南.北京：科学出版社；2007.
[136]Fowler TJ, Mitton MF, Vaillancourt LJ, Raper CA:Changes in mate recognition through alterations of pheromones and receptors in the multisexual mushroom fungus Schizophyllum commune. Genetics 2001,158(4):1491-1503.
[137]Gola S, Hegner J, Kothe E:Chimeric pheromone receptors in the basidiomycete Schizophyllum commune. Fungal genetics and biology:FG & B 2000, 30(3):191-196.
[138]Gola S, Kothe E:The little difference:in vivo analysis of pheromone discrimination in Schizophyllum commune. Curr Genet 2003,42(5):276-283.
[139]Dixit R, Nasrallah ME, Nasrallah JB:Post-transcriptional maturation of the S receptor kinase of Brassica correlates with co-expression of the S-locus glycoprotein in the stigmas of two Brassica strains and in transgenic tobacco plants. Plant Physiol 2000,124(1):297-311.