【作者】 罗兵；

【导师】 朱利泉；

【作者基本信息】 西南大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 自交不亲和性(Self-incompatibility, SI)是植物防止自体受精,促进远缘杂交的一种遗传机制。甘蓝自交不亲和性中,花粉和柱头相互识别特异性受一个带有复等位基因的多态性S位点(S-locus)所控制。在S位点有3个高度多态性基因,即S-受体激酶(S receptor kinase, SRK)编码基因、S-富含半胱氨酸蛋白(S cystine rich,SCR)编码基因和S-糖蛋白(S glycoprotein, SLG)编码基因。其中,SRK和SCR分别为自交不亲和的雌雄决定因子,SLG是SRK识别SCR的辅助受体。受粉时,同一S单倍型的SRK与SCR相互结合导致自交不亲和性的发生。SRK胞外S结构域编码区(eSRK)负责与SCR相互识别,但eSRK与SCR的之间识别强度、识别模体及在此基础上的分子作用机制等目前还不清楚。对此,本研究以高度自交不亲和性结球甘蓝D3、B3、E1、A1、H1、230、240、243、N1和G1为材料,克隆了全部雌决定因子和部分雄性决定因子,利用巢式RT-PCR、酵母双杂交技术深入研究了SRK与SCR相互作用,主要研究内容和结果如下。1.SRK胞外域的克隆及其生物信息学分析采用巢式RT-PCR,以甘蓝D3、B3、E1、A1、H1、230、240、243、N1和G1的柱头和花粉cDNA为模板扩增了甘蓝的SRK胞外域序列(eSRK)。序列分析表明：eSRK D3和eSRK240的cDNA序列皆为1 303bp,分别编码434个氨基酸的蛋白；eSRK230, eSRK243, eSRKE1, eSRKGl和eSRKNl的cDNA序列都为1 309bp,分别编码436个氨基酸的蛋白；eSRK A1, eSRK B3和eSRK H1的cDNA序列皆为1312bp,分别编码437个氨基酸的蛋白。甘蓝eSRK蛋白氨基酸序列呈高度多态性,有三个高变区,但其中12个半胱氨酸高度保守。生物信息学分析表明,不同材料eSRK的二级结构富含p-折叠和无规则卷曲,其三级结构皆相似。克隆的eSRK皆含有信号肽,并且都含三个明显的保守结构域,分别为B-Lectin结构域,SLG结构域以及PAN_APPLE结构域。甘蓝eSRK的进化速度快,不同S单倍型的eSRK遗传分支时间差异大。2.SCR基因的克隆及其生物信息学分析通过SCR信号肽保守氨基酸和mRNA的ploy A区设计引物,利用巢式PCR,以甘蓝cDNA为模板,克隆了3种甘蓝D3、E1和G1的SCR基因的部分cDNA序列,长度分别为319bp、311bp和377bp,包含3’-UTR区。三者分别编码1个58,58和55个氨基酸的SCR蛋白。D3和E1的SCR分别与SCR3和SCR7的序列一致,而甘蓝G1的SCR为一个新的S单倍型基因。三者皆是SCR的成熟肽编码区。三种蛋白的三维结构近似,都有一个α螺旋和3个β折叠,高变区富含碱性氨基酸。三者都有潜在的磷酸化位点。在核苷酸和氨基酸水平上,SCRG1与SCRD3的相似性分别为69.5%和63.8%,与SCRE1的相似性分别为53.4%和46.6%。SCRD3与SCRE1间分别有55.7%和48.3%的相似性。G1与D3的SCR在演化过程中分支时间较晚,而二者与E1的SCR在进化上分支时间较早。3.S受体激酶识别SCR的生物信息学分析以S3、S7、S8、S12、S13、S24、S29、S39和S64单倍型eSRK和SCR为材料,利用蛋白质遗传进化图和作用位点预测等方法分析了SRK中SCR识别位点。结果表明,eSRK与相应S单倍型SCR的遗传进化图很相似；HVⅠ、HVII和HVIII作为整体的遗传进化图与eSRK的遗传进化图高度相似。单独的高变区的遗传进化图与eSRK的遗传进化图相差比较大,其中HVC相差最大。表明eSRK的高变区(HVⅠHVⅡ和HVⅢ)是SCR的识别位点。在线预测表明,所有的eSRK的HVⅠ皆有蛋白质作用位点,而N端亚结构域(B-lectin结构域)没有任何蛋白质作用位点。进一步说明高变区是SCR的识别区域。不同S单倍型蛋白作用位点不同,氨基酸种类也不同,但其中Glu、Arg和Trp出现的概率最大。预测结果为实验研究提供了理论基础。4.利用酵母双杂交法检测甘蓝SCR与SRK之间的相互作用以具有典型自交不亲和性的甘蓝D3和E1为材料,利用巢式PCR分别扩增SRK胞外域(eSRK)和SCR,通过酵母双杂交检测二者之间的相互作用。并利用同源重组技术分别将eSRK与GAL4报告基因DNA活化域融合(pGADT7eSRK)以及SCR分别与GAL4报告基因DNA结合域融合(pGBKT7SCR)。重组质粒(pGADT7eSRKD3)和(pGADT7eSRKE1)分别转化酵母Y187,重组质粒(pGBKT7SCR D3)和(pGBKT7SCR E1)分别转化酵母Y2HGold。转化酵母皆未出现自激活现象。融合的二倍体酵母(pGADT7eSRKD3xpGBKT7SCR D3)和(pGADT7eSRK E1xpGBKT7SCR E1)均能在选择性固体培养基(SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA)上生长,并且菌落呈蓝色。但融合的二倍体酵母(pGADT7eSRKD3xpGBKT7SCR E1)和(pGADT7eSRK E1xpGBKT7SCR D3)均不能在SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA培养基上生长。结果表明,eSRK D3与SCR D3蛋白之间以及eSRK E1与SCR E1蛋白之间能够相互结合,但eSRK E1与SCR D3蛋白之间以及eSRK D3与SCR El蛋白之间不相互结合,为深入研究eSRK-SCR作用位点成功建立了一个技术平台。5.S受体激酶SCR识别模体的研究以甘蓝D3为材料,系统地克隆了eSRK的不同亚结构域,并利用同源重组技术分别将eSRK的不同亚结构域与酵母表达载体pGADT7连接,记为pGADT7eSRKn(n=1,2......15),重组质粒pGADT7eSRKn分别分别转化酵母Y187,转化酵母皆未出现自激活现象。所有的二倍体酵母(pGADT7eSRKnxpGBKT7SCRD3)均能在SD/-Ade/-His/-Leu/-Trp固体培养基上生长,但以X-gal为底物检测时,二倍体酵母(pGADT7eSRK5xpGBKT7SCR D3)、(pGADT7eSRK6xpGBKT7SCR D3)、(pGADT7eSRK7xpGBKT7SCR D3)和(pGADT7eSRK10xpGBKT7SCR D3)不显蓝色。结果表明,SCR的结合域是eSRK的高变II区和III区。为了验证酵母双杂交的结果,以甘蓝D3为材料,克隆了eSRK的高变区(HVII+HVIII)亚结构域(eSRKv)和SCR,并分别与原核表达载体pGEX-6P-3和pET-22b (+)连接。IPTG能够诱导融合蛋白eSRKv-GST和SCR-HIS的表达,并且eSRKv-GST与SCR-HIS在体外能够相互作用。进一步证实了HVII和HVIII参与结合SCR, HVII和HVIII为相互作用的模体。6.蛋白质作用强度与酵母生长的关系以二倍体酵母：(eSRKD3×SCRD3)、(eSRK2×SCRD3)、(eSRK3×SCRD3)、(eSRK4×SCRD3)、(eSRK8×SCRD3)、(eSRK9×SCRD3)、(eSRK11×SCRD3)、(eSRK12×SCRD3)、(eSRK13×SCRD3)、(eSRK14×SCRD3)、(eSRK15×SCRD3)和(eSRKE1×SCRE1)为材料,分析了酵母生长与eSRK-SCR作用强度的关系。构建了3种SRK-SCR作用强度与酵母生长的关系模型。第一种模型为：SRK-SCR作用强度与酵母生长之间呈正向显著相关。该模型为构建二者的定量关系模型奠定了基础。第二种模型为：以SRK-SCR作用强度为自变量时,模型为：Y=0.0358+0.011X更为合理。该模型为以后实验提供了依据。第三种模型为：以酵母生长量为自变量时,模型为：Y=-11.929+60.387X更为合理。该模型构建不但可以降低实验成本,而且建立了芸苔属自交不亲和性的全新测定方法。更多还原

【Abstract】 Self-incompatibility (SI) is a general concept for several genetic mechanisms in angiosperms, which prevent self-fertilization and encourage outcrossing. Self-incompatibility in Brassica species is controlled by a single S locus with multiple alleles, and it involves three highly polymorphic genes, S receptor kinase(SRK), S cystine rich(SCR) and S glycoprotein (SLG). The SRK and the SCR are the female determinant and the male determinant of the self incompatibility respectively.Binding of SCR to SRK then would elicit a signaling cascade in the papillar cell, leading to the rejection of self-pollen. The extracellular domain of SRK(eSRK) is responsible for SCR binding. However, The molecular mechanism of SRK-SCR action. is not well known up to now.For further study on the issue, the eSRK were amplified by nested PCR using Brassica oleracea L. D3, B3, E1, A1, H1,230,240,243, N1 and G1 with the trait of typical SI, while SCR were amplified by nested PCR from Brassica oleracea L. D3, E1 and G1. The interactions between eSRK and SCR were detected by the yeast two-hybrid system.The main contents are as the followings:1. Cloning and bioinformatics analysis of eSRKThe cDNA from stigma and pollen of Brassica oleracea L. D3, B3, E1, A1, H1, 230,240,243, N1 and G1 were used as templates for PCR amplification of eSRK. Sequence analysis showed that eSRKs of D3 and 240 are both 1303 nucleotides long and encodes a peptides of 434 amino acids respectively.The eSRKs of 230,243, E1, G1 and N1 all contain 1309 bp, which encodes a protein 436 amino acid respectively. The determined nucleotide and deduced amino acid sequences of eSRK A1, eSRK B3 and eSRK H1 are both 1312 bp and 437 amino acids, respectively. All eSRK proteins contain B-Lectin, SLG domain and PAN_APPLE domain. The a-helix and B-sheets of eSRK’s Secondary Structure are rich. The eSRK is highly polymorphic and composed of three hyper-variable regions, but contain the twelve conserved cysteine residues. The eSRK is quick evolution and genetic branching period of the eSRK had great difference in different S haplotypes.2. Cloning and bioinformatics analysis of SCRTotal RNA was extracted from D3, E1 and G1, and their corresponding cDNAs were obtained through reverse transcription. Primers were designed to amplify SCR using 5’-end conserved amino acids and 3’-ploy(A). These cDNA sequences were 319 bp,311 bp and 377 bp in length respectively, containing 3’UTR, and could be translated into protein with 58,58, and 55 amino acids in the reading frame. SCRs of D3 and E1 have same sequences as SCR3 and SCR7. SCR of G1 is a new S-Haplotype. The gene and protein sequences of SCR-G1 and SCR-D3 have 69.5% and 63.8% similarity, respectively. SCRG1 and SCRE1 show 53.4% nucleotide similarity and 46.6% protein similarity. SCRD3 and SCRE1 show 55.7% nucleotide similarity and 48.3% protein similarity. The results showed that SCRGland SCRD3 are evolutionarily closely linked, whereas the SCR of G1, D3 and the SCR of E1 are more distant relatives. All three proteins have similiar structure and potential phosphorylation sites, contain one a-helix and three B-sheets, and are full of basic amino acids in hypervariable region.3.Bioinformatics analysis of recognition site of SCR in SRKThe eSRK and SCR sequences from Brassica. S3, S7, S8, S12, S13, S24, S29, S39 and S64 were used for domain analysis and binding recognition site analyses. The results showed that the phylogenetic tree of eSRK was similar to that of SCR. The HVⅠ, HVⅡand HVⅢof eSRK as a whole, whose phylogenetic tree was high similarity to that of eSRK. But as far as individual hypervariable region, the similarity ends there, especially HVC. The results showed that the hypervariable region of eSRK is the ecognition site of SCR. All HVI of eSRKs have binding sites of SCR, but no binding site in theβ-lectin domain.Different S-haplotypes eSRK have different binding site and amino acids, but Glu, Arg and Trp have the maximum probability in existence.4.Detection of Interactions between SCR and SRK in Brassica oleracea L. by Yeast Two-Hybrid System.For further study on mechanism of the mutual recognition between SRK and SCR in Brassica, the eSRK and SCR were amplified by nested PCR using Brassica oleracea L.’D3’and’El’with the trait of typical self-incompatibility (SI), and the interactions between eSRK and SCR was detected by the yeast two-hybrid system. Then the full-length eSRK and the SCR were fused to the Ga14 DNA activation domain (designated pGADT7eSRKD3 and pGADT7eSRKDE1) and the Ga14 DNA binding domain (designated pGBKT7SCRD3 and pGBKT7SCRE1) by the gene homologous recombination technique respectively. Then the pGADT7eSRKD3 and pGADT7eSRKDE1 plasmid were transformed into the Y187 yeast strain respectively, whereas pGBKT7SCRD3 and pGBKT7SCRE1 were transformed into the Y2HGold yeast strain respectively. The four transformed yeast strains did not exhibit autoactivation. The diploids pGADT7eSRKD3xpGBKT7SCR D3 and pGADT7eSRK E1xpGBKT7SCR E1, which were fused by the transformed Y2HGold yeast strain and the transformed Y187 yeast strain, grew on selective agar plates (SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA), and the resulting colonies were blue, which strongly indicated that eSRK and SCR could combine with each other. Whereas the diploids pGADT7eSRKD3xpGBKT7SCR E1 and pGADT7eSRK E1xpGBKT7SCR D3 could not grow on the same selective agar plates. The results showed that the SCR could bind to the ectodomain of its cognate "self" SRK and not bind nor activate "non-self" SRK.5. Study on the recognizing motif of SCR in SRKMany short eSRKs from different subdomains were amplified using Brassica oleracea L.’D3’. The multiple short eSRKs were fused to the Ga14 DNA activation domain (designated as pGADT7eSRKn(n=1,2......15)), then be transformed into the Y187 yeast strain respectively. All transformed yeast strains did not exhibit autoactivation. The diploids pGADT7eSRKnxpGBKT7SCRD3, which were fused by the transformed Y2HGold yeast strain and the transformed Y187 yeast strain, grew on selective agar plates (SD/-Ade/-His/-Leu/-Trp). When theβ-galactosidase were assayed by colony-lift filter using X-gal, the colonies pGADT7eSRK5xpGBKT7SCRD3,pGADT7eSRK6xpGBKT7SCRD3,pGADT7eSRK7 xpGBKT7SCRD3 and pGADT7eSRK10xpGBKT7SCRD3 were white. The results showed that HVⅡand HVⅢof eSRK have direct interaction with SCR. In order to test the result, HVⅡand HVⅢof eSRK was expressed in E. coli (BL21) using the expression vector pGEX-6P-3 (including a GST-tag), SCRD3 was expressed using vector pET-22b(+) (including a His-tag). Result by Western blot showed that SCR-His could combine with SRK-HVⅡ-HVⅢ-GST in vitro.6.Connection of interaction intensity between eSRK and SCR with yeast growth Three modules were estabolished about connection of SRK-SCR interaction with yeast growth using the diploids eSRKD3×eSCRD3, eSRK2×eSCRD3, eSRK3×eSCRD3, eSRK4×eSCRD3, eSRK8×eSCRD3, eSRK9×eSCRD3, eSRK11×eSCRD3, eSRK12×eSCRD3, eSRK13×eSCRD3, eSRK14×eSCRD3, eSRK15×eSCRD3 and eSRKE1×eSCRE1. Module 1:There was a significantly positive correlation between SRK-SCR interaction with yeast growth. Module 2:The module Y=0.0358+0.011X was very reasonable with SRK-SCR interaction as the independent variable. Module 3:The moduleY=-11.929+60.387X was very reasonable with yeast growth as the independent variable. The third model could reduce the cost of research, moreover, it might establish a new measuring method for Self-incompatibility of Brassica.更多还原