【作者】 杨红；

【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， 硕士

【摘要】 自交不亲和性(self-incompatibility, SI)在许多显花植物中普遍存在,是阻止自体受精、预防近亲繁殖和促进远系杂交、保持遗传多样性的一种重要机制。倍受关注的甘蓝等芸苔属植物的SI反应,涉及从授粉到受精过程中发生的花粉配体和雌蕊受体之间的相互作用,在经典遗传学中受假设的孟德尔位点——S位点控制。柱头对花粉的识别取决于S位点呈现紧密连锁以及高度多态性的两个基因：①雄性专一性决定因子——S-位点富含半胱氨酸蛋白(S-locus cystein-rich protein,SCR)、亦称为S位点蛋白11 (S-locus protein 11, SP11),该基因编码开放阅读框内含高频率的半胱氨酸残基,在花药中积累并特异表达,为PCP家族中具有亲水性的小分子碱性分泌蛋白。②雌性决定因子——S受体激酶基因(S receptor kinase gene, SRK),编码一种跨膜的蛋白激酶,由N端胞外域(S域)、跨膜域和一个具有丝氨酸/苏氨酸激酶活性的C端胞内域组成；该基因有7个外显子,其中胞外S结构域由第一个外显子编码,是花粉配体结合位点,决定自交不亲和性的特异识别反应,从而引起柱头乳突细胞SI信号转导,最终导致自交不亲和反应。本研究利用酵母双杂交系统鉴定甘蓝自交不亲和决定因子S位点富含半胱氨酸蛋/S位点蛋白11(SCR/SP11)与S位点受体激酶(SRK)胞外域(eSRK)片段间可能的相互作用区域。以结球甘蓝B3为材料,利用分子克隆技术,分别构建以pGBKT7为载体的全长SCRB3、SCRB3-1、SCRB3-2的重组诱饵质粒；以pGADT7为载体的全长采用RT-PCR扩增不同片段长度的、eSRKB3-1、eSRKB3-2的重组激活域(AD)质粒。建立了适用于SCR与SRK相互作用的酵母双杂交检测体系,初步确定了SCR与eSRK存在相互作用,SRK跨膜域的存在与否对其相互作用的研究没有影响。为我们更深入的研究SCR与SRK相互作用机理,提供了理论基础。主要研究结果总结如下：1不同截短长度S-位点富含半胱氨酸蛋白(S-locus cystein-rich protein, SCR)基因(SCRB3-5)的克隆及其结构特征以结球甘蓝B3幼叶的gDNA和开花1-2d的花蕾总cDNA为模板,采用PCR扩增不同片段长度的SCRB3-5基因。经1%(w/v)琼脂糖凝胶电泳检测,SCRB3-5的目的片段大小均与引物的理论扩增值大小相符,SCRB3中含有302bp的内含子。利用分子克隆技术构建酵母双杂交诱饵表达载体,DNA测序结果分析表明,pGBKT7-SCRB3-5载体中包含有SCRB3-5片段且插入的SCRB3-5片段序列与SP1128序列完全一致。采用Vector NTI Advance 10.0对不同S单倍型的6个SCR/SP11 (S6、S8、S13、S28、S54、Sf2)进行序列比对,不同S单倍型SCR/SP11蛋白之间较大的“一致性和相似性”差异揭示SCR/SP11较高的多态性。SCRB3共编码85个氨基酸,包含完整的信号肽和成熟肽；SCRB3-1包含了SCR蛋白信号肽C端的3个氨基酸VEA和完整的成熟肽；SCRB3-1包含成熟肽部分的58个氨基酸。2不同截短长度的S位点受体激酶(SRK)胞外域(eSRK)片段(eSRKB3-5)的克隆及其结构特征采用PCR和RT-PCR扩增不同片段长度的eSRKB3-5,经1%(w/v)琼脂糖凝胶电泳检测,目的片段大小均与引物的理论扩增值大小相符；且分别以gDNA和cDNA扩增的目的片段大小一致,表明eSRKB3-5中没有内含子。利用分子克隆技术成功构建酵母双杂交AD表达载体pGADT7-eSRKB3-5,DNA测序结果分析表明,pGBKT7-eSRKB3-5载体中包含的eSRKB3-5片段序列与eSRK28序列完全一致。BLAST在线分析结果表明,eSRKB3编码426个氨基酸,包含了SRK蛋白B-Lectin结构域、SLG结构域和PAN_APPLE结构域,是SRK胞外域的全长；eSRKB3-1是去除信号肽区域的eSRK序列,编码第30位至第426位氨基酸；eSRKB3-2包含了eSRK序列的高变区域,编码第140位至第426位氨基酸。3酵母双杂交重组诱饵质粒的毒性及自激活检测Y2HGold [pGBKT7-SCRB3-5]和Y2HGold [pGBKT7]转化株在缺陷型SD/-Trp平板上生长状态良好,并且菌斑大小无明显差异；由此表明诱饵质粒pGBKT7-SCRB3-5的表达对酵母细胞无毒性作用。另外,Y2HGold [pGBKT7-SCRB3-5]在缺陷型SD/-Trp、SD/-Trp/x-a-gal平板上均能生长、在SD/-Trp/x-a-gal/AbA平板上不生长,其中在SD/-Trp/x-a-gal平板上生长的菌落无明显蓝色出现,说明pGBKT7-SCRB3-5在酵母细胞中没有激活报告基因AUR1-C和MEL1。4酵母双杂交重组AD质粒的毒性及自激活检测将pGADT7空载和pGADT7-eSRKB3-5转化Y187,观察得到Y187 [pGADT7和Y187[pGADT7-eSRKB3-5]在SD/-Leu平板上生长状态良好,由此表明重组表达质粒pGADT7-eSRKB3-5成功转入酵母细胞Y187且无毒性作用。此外,Y187 [pGADT7-eSRKB3-5]在SD/-Leu平板上呈现生长状态良好的白色菌斑；在SD/-Leu/x-a-gal平板上没有明显蓝色克隆出现。由此可说明pGADT7-eSRKB3-5重组载体在酵母细胞中没有激活报告基因MEL1,无自身的转录激活作用发生5 SCRB3-5与eSRKB3-5片段间相互作用检测利用生物信息学分析软件对SCRB3与eSRKB3进行相互作用分析,结果表明SCRB3与eSRKB3在空间能够相互识别形成融合蛋白。SCRB3-5与eSRKB3-5片段间两两组合的9个试验组均能在DDO平板上出现生长状态良好的菌斑,其中有Y2HGold [pGBKT7-SCRB3-2]xY187 [pGADT7-eSRKB3-2]、Y2HGold[pGBKT7-SCRB3-2]xY187[pGADT7-eSRKB3-1]、Y2HGold[pGBKT7-SCRB3-1]xY187[pGADT7-eSRKB3-2]和Y2HGold[pGBKT7-SCRB3-1]xY187[pGADT7-eSRKB3-1]4个试验组能在QDO/x/A平板上出现蓝色克隆,激活报告基因4UR1-C、MEL1、ADE2和HIS3的表达。初步确定了SCR与eSRI存在相互作用,SRK跨膜域的存在与否对其相互作用的研究没有影响。更多还原

【Abstract】 Ever since Darwin’s pioneering research, the evolution of self-incompatibility has been regarded as one of the most prevalent evolutionary transitions in many flowering plants. The response of self-incompatibility is a major mechanism to prevent inbreeding and promote much-line hybrid, maintaining genetic diversity, which consists of male and female specificity genes at the S locus. In the self-incompatibility (SI) response of crucifers, the recognition of self-related pollen with the stigma epidermis which is effected through the activity of two tightly linked and highly polymorphic genes encoded by the S locus.①The S locus cysteine-rich protein gene SCR [also designated SP11] encodes a small hydrophilic and positively charged peptide that is localized to the pollen coat.②The S locus receptor kinase gene SRK has 7 exons encoding a single-pass transmembrane serine threonine kinase, which contains the N terminal extracellular domain (S domain), transmembrane Domain and a serine/threonine kinase activity of the intracellular C-terminal domains. The extracellular S domain was encoded by the first exon which is pollen ligand binding site, determined the specific self-incompatibility Identification response, led to the stigma papilla cells SI signal transduction, eventually leading self-incompatibility response.In this study a yeast two-hybrid system was adopted to investigate their potential interaction domains between SCR/SP11 and the extracellular fragment of SRK (eSRK). The full length SCRB3, SCRB3-1 and SCRB3-2 amplified from Brassica oleracea L.’B3’were cloned into the pGBKT7 to construct the recombinant bait plasmids which were then transformed into yeast Y2HGold cells by PEG/LiAc method. The DNA fragments including eSRKB3, eSRKB3-1 and eSRKB3-2 were cloned into the pGADT7 to construct the recombinant activation domain (AD) plasmids which were transformed into yeast Y187 cells. And these recombinant plasmids are no toxic and without autonomous activation effect to yeast cells. The yeast two-hybrid system could be used to study the interaction domain between SCR and eSRK. The transmembrane domain of SRK is presence or not which will have no effect on studying the interaction between SCR and SRK. Our result will be used to further study the the interaction mechanism between SCR and SRK. Our results showed that:1 The amplification of different truncated fragments SCRB3-s and Sequence AnalysisThe different Truncated fragments SCRB3-s were amplified from leaf genomic DNA and bud RNA in Brassica oleracea L.’B3’by PCR and RT-PCR. By using 1%(w/v) agarose gel electrophoresis, the SCRB3-s fragments length are identical with the theory value of primer extension, and we find that SCRB3 contains a 302bp intron. Then subcloned into pGBKT7 and constructed the recombinant bait plasmids. The DNA sequencing analysis showed that pGBKT7-SCRB3-s recombinant bait plasmids contain SCRB3-S fragments, and the insertion site and the reading frame are correct, we finded that the sequences SCRB3-S are identical with SP1128 sequences. Doing sequence alignment of 6 different S haplotypes (S6, S8, S13, S28, S54, Sf2) by Vector NTI Advance 10.0, the consistency and similarity during different S haplotypes revealed high polymorphism of SCR gene. The SCRB3 sequence encodes 85 amino acids which include a complete signal peptide and a mature peptide; SCRB3-1 protein contains 3 amino acids (VEA) of C terminal peptide and complete mature peptide; SCRB3-2 contains 58 amino acids.2 The amplification of different Truncated fragments eSRKB3-s and Sequence AnalysisThe different Truncated fragments eSRKB3-s were amplified from leaf genomic DNA and bud RNA in Brassica oleracea L.’B3’by PCR and RT-PCR. By using 1%(w/v) agarose gel electrophoresis,the eSRKB3-s s fragments length are identical with the theory value of primer extension, and there is no intron in the eSRKB3 gene. Then subcloned into pGADT7 and constructed the recombinant plasmids. The DNA sequencing analysis showed that pGADT7-eSRKB3-s recombinant plasmids contain eSRKB3-s fragments, and the insertion site and the reading frame are correct, we finded that the sequences eSRKB3-s are identical with SRK28 sequences. The analysis of BLAS online showed that.the eSRKB3 encodes 426 amino acids, contains B-Lectin domain, SLG domain and PAN_APPLE domain. The eSRKB3-1 did not include the signal peptide sequence, which encoding the 30 to 426 amino acids; eSRKB3-2 encodes the amino acids between 140 and 426, which contains the hypervariable region of eSRK sequence.3 The toxicity and Autoactivation detection of recombinant bait plasmidsThe transformants of Y2HGold [pGBKT7-SCRB3-s] and Y2HGold [pGBKT7] were cultured on SD/-Trp plates at 30℃. After three days, we can find white well clones in the plates. The result showed that the recombinant bait plasmids pGBKT7-SCRB3-s were no toxic to yeast cell. In addition, Y2HGold [pGBKT7-SCRB3.S] could grow on SD/-Trp,SD/-Trp/x-a-gal plates and could not grow on SD/-Trp/x-a-gal/AbA plates, and the colonies did not turn blue on SD/-Trp/x-a-gal plates which indicates that pGBKT7-SCR B3-5 do not activate the reporter gene AUR1-C, and MEL1 in yeast cells.4 The toxicity and Autoactivation detection of recombinant AD plasmid pGADT7-eSRKB3-sThe empty vector pGADT7 and pGADT7-eSRKB3-s were transformed into Y187,the ransformants of Y187 [pGADT7] and Y187 [pGADT7-eSRKB3-s] grew well on the SD/-Leu plates, while the yeast strain Y187 which was not transformed recombinant plasmid did not grow on SD /-Leu medium. It is suggested that recombinant plasmid pGADT7-eSRKB3-s was successfully transformed into Y187 yeast cells and was not toxic to yeast cell Y187. In addition, the white clones of Y187 [pGADT7-eSRKB3-s] grew well in SD/-Leu plates and the colonies did not turn blue on SD/-Leu/x-a-gal plates. The results indicate that recombinant plasmid pGADT7-eSRKB3-s do not activate the reporter gene MEL1.5 The interaction detection between different truncated fragments of SCRB3.S and eSRKB3-sThe analysis of bioinformatics software shows that SCRB3 can mutually recognize with SRKB3 in the space to form a fusion protein.The recombinant plasmids were successfully transformed into the diploid yeast mating cell, the four group of Y2HGold[pGBKT7-SCRB3-2]xY187[pGADT7-eSRKB3-2], Y2HGold[pGBKT7-SCRB3-2]xY187[pGADT7-eSRKB3-1],Y2HGold[pGBKT7-SCRB3-1]xY187[pG ADT7-eSRKB3-2]andY2HGold[pGBKT7-SCRB3-1]xY187[pGADT7-eSRKB3-1] could emerge significantly blue clones, then remove blue clones on DDO/x/A plates to QDO/x/A plate, after four days, we can find the blue clones on the plate, which may indicate that activate the reporter gene AUR1-C, MEL1,ADE2 and HIS3. The resoults show that the yeast two-hybrid system could be used to study the interaction domain between SCR and eSRK.The transmembrane domain of SRK is presence or not which will have no effect on studying the interaction between SCR and SRK. Key words:Brassica oleracea L. var capitata L.; self-incompatibility; S-locus receptor kinase (SRK); S-locus cysteine-rich protein (SCR); yeast two-hybrid更多还原