【作者】 杨昆；

【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， 2012， 博士

【摘要】 包括甘蓝在内的芸薹属植物是孢子体自交不亲和性的典型代表,其自交不亲和反应起始于S基因座的两个基因SCR (S locus cysteine-rich protein)与SRK (S locus protein kinase)的编译产物的相互识别,同配型花粉的信号由柱头乳突细胞外被传至胞内,由SRK的激酶域激活ARC1(ARM repeat containing),进一步将信号传至EX070A1,再历经后续的级联反应,最终导致自交不亲和。本文以三种自交不亲和相关元件(SRK、ARC1和EX070A1)及编码基因为研究对象,对三种基因的序列、染色体定位及其编码蛋白的互作特性进行了研究。简要阐述如下：1.运用PCR技术从甘蓝、大白菜与甘蓝型油菜中分离出EX070A1基因,并对所克隆的序列进行生物信息学分析；通过转化酵母Y187和半定量RT-PCR技术检测BoEXO70A1基因的表达特性。结果发现：①三种芸薹属植物EX070A1的编码框均(coding sequences, CDS)为1917bp,且呈现出97.1%的序列一致性；它们的gDNA序列均为单一序列,长度分别为3797bp、3752bp和3770bp,其序列一致度高达91.0%,且均由12个外显子及11个内含子组成,除了第4、5、6、8个内含子外,其余内含子的保守性均低于外显子；推导的三种蛋白质序列(BnEXO70A1、 BrEXO70A1和BoEXO70A1)的相似度与序列一致性分别高达99.8%与98.1%,其二级结构、三维结构及理化特性也高度相似。②所克隆的EXO70A1基因的11个内含子的剪切位点均符合"GU-AG"法则,剪切受体(AG)的前20～50个碱基的位置存在一段保守的序列"CU(A/G)A(C/U)";③三种芸薹属植物与拟南芥EXO70A1基因的12个外显子的对应序列长度完全相同,所构成的编码区的序列一致性高达90.1%,推导的蛋白质序列之间的相似度与一致性分别高达99.8%与93.7%；④分子进化分析发现不同植物间的EXO70A1在整个EXO70蛋白家族中表现出较高的保守性；⑤BoEXO70A1在酵母细胞Y187呈现弱表达；EXO70A1在甘蓝的雄蕊、幼茎、幼嫩花瓣、雌蕊、幼根及叶片中均能表达,可能属于组成型表达基因,但是其表达量在不同发育时期的不同器官中呈现出差异,授粉前雌蕊中最高,雄蕊中最低。2.运用PCR技术从甘蓝、大白菜与甘蓝型油菜中同源克隆出ARC1基因的DNA,并对所克隆的序列进行生物信息学分析。①ARC1基因在3种植物中均不包含内含子序列,BoARC1蛋白编码序列由1992bp(包含终止密码子)构成,而BrARC1和BnARC1的蛋白编码序列由1986bp构成,即后者在序列上比甘蓝A4RC1少编码2个氨基酸序列；②比对发现3种ARC1编码序列存在93.8%的一致性,在144处的核苷酸存在差异,其对应的蛋白质之基存在99.4%的序列相似性和92.2%序列一致性,即仅有51处的氨基酸存在差异；两两序列比对发现BnARC1与BrARC1基因在序列上的更为接近；③推导的3种ARC1蛋白都包含相同的结构域,其共有的组成为：UND(N-terminal domain)、中间J的U-box (U-box domain)和C端的ARM (armadillo repeat),并且3种ARC1蛋白在二级结构和三维结构上都极为相似。3.运用PCR技术从甘蓝克隆出BoSRK基因的CDS,并对所克隆的序列连同下载的31条SRK序列进行生物信息学分析；①SRK基因由7个外显子和6个内含子构成,编码816-860个氨基酸,第一个外显子编码SRK的信号肽和胞外结构域的氨基酸；②尽管不同类型SRK的编码区核甘酸序列与对应的蛋白质序列均有较高的相似度,但胞外结构域之间存在着的3个高变区造成了SRK蛋白呈现多态性,高变区氨基酸可能与同单元型的SCR/SP11相互识别发挥重要作用；③SRK的胞外域与S基因座另一个基因的编码产物SLG有着极为相似的序列和结构,可能是源于进化过程中S域的基因倍增。4.以甘蓝幼根为材料,制备小量游离的细胞核,然后通过移动界面法制备出染色质纤维,接着以重复基因序列.5S rRNA为探针验证染色质纤维的制备效果；①从幼嫩根获取的游离细胞核能够有效的避免叶绿体色素的干扰,用来制备的染色质纤维有利于后面的原位杂交信号的检测。②中期染色体致密程度较高,以2～4kb的单拷贝或低拷贝核酸序列为探针,在中期染色体杂交信号检出率较低,仅能达到5%；将杂交前的染色体用胃蛋白酶酶解的时间适当延长,可以使染色体变的更加松散,能够适当提高杂交信号检出率；以有丝分裂前中期染色体和粗线期染色体为载体,杂交信号的检出率可以有较大的提高；以染色质纤维为载体,杂交信号检出率可以达到较为理想的状态。5.以SRK基因在中期染色体和粗线期染色体上的杂交,绿色的杂交信号仅存在于一对同源染色体上,暗示SRK在甘蓝的染色体上可能只有一个拷贝,但难以确定SRK基因在染色体上的具体位置；搜索甘蓝和白菜的染色体和基因数据库得知：SRK基因位于甘蓝7号染色体上8732037-8675105bp的核酸区段,对应白菜的7号染色体(17204000-17300000bp)的染色体区段。6.ARC1基因在甘蓝和白菜基因组中仅有一个拷贝,位于甘蓝4号染色体的正链的(35662808-35664799bp)核酸区段,对应于Brassica rapa的4号染色体(15107313-15109298bp)区段的正链。7.EXO70A1基因在中期染色体及粗线期染色体定位的结果并不一致。以EXO70A1基因的DNA序列为探针,搜索甘蓝和白菜的染色体和基因数据库得知：EXO70A1基因在甘蓝基因组中仅有一个拷贝,位于甘蓝9号染色体(39669464-39673197bp)区段的负链,与中期染色体原位杂交结果不同的原因可能是由于甘蓝基因组中存与EX070A1基因序列相近的同源基因造成；对应于Brassica rapa的10号染色体(17076191-17079967)区段的负链。8S位点受体激酶(SRK)和臂重复蛋白(ARC1)是芸薹属植物自交不亲和反应的2种重要的信号元件,SRK-ARC1的互作可能决定着自交不亲和反应下游信号的级联传导构建的重组表达载体在酵母细胞中无毒性和自激活作用产生。本研究从结球甘蓝与羽衣甘蓝柱头中克隆出的SRKJ、ARC1与EXO70A1基因的全长和局部编码区,分别连接至载体pGADT7和PGBKT7,然后应用酵母双杂交系统检测和分析这些蛋白质两两之间的相互作用。羽衣甘蓝ARC1存在5个连续的臂重复区,与结球甘蓝ARC1的氨基酸序列一致性达到98%；3个实验组Y2HGold[pGBKT7-ARC1]×Y187[pGADT7-SRKJ]、 Y2HGold[pGBKT7-ARC1T3]×Y187[pGADT7-SRKJ]和Y2HGold[pGBKT7-ARClT4] x Y187[pGADT7-SRKJ]能够在缺陷型培养基QDA/X/AbA上生长,证明三组融合酵母都激活了报告基因的表达；2个实验组Y2HGold[pGBKT7-ARClT1]×Y187[pGADT7-SRKJ]、Y2HGold[pGBKT7-ARC1T2]×Y187[pGADT7-SRKJ]不能在四缺培养基上生长,这两个实验组中的报告基因并未激活,说明ARC1T1-SRKJ和ARC1T2-SRKJ均未发生互作。表明互作的区域位于连续的臂重复区,两种甘蓝ARC1的氨基酸差异不足以引起互作区所需的正确构象的改变。9.EXO70A1可能是ARC1下游的另一种信号元件,它和ARC1的互作可能对SI反应产生重要的作用。本研究从甘蓝型油菜与羽衣甘蓝柱头中克隆出ARC1与EXO70A1基因的全长和局部编码区,分别连接至载体pGADT7和PGBKT7,然后应用酵母双杂交系统检测和分析这些蛋白质两两之间的相互作用。结果发现：ARC1蛋白在羽衣甘蓝与甘蓝型油菜中存在45个氨基酸的差异,其序列相似度与一致性分别达到95.9%和93.9%；推导的EX070A1在羽衣甘蓝和甘蓝型油菜中仅有6个氨基酸的差异,其序列相似度与一致性分别达到99.4%和98.9%；EX070A1蛋白在种内和物种间都保持着高度的保守性,其保守的程度高于ARC1。在二倍体酵母细胞中,全长的ARC1与EXO70A1之间存在较强的相互作用,能激活4个报告基因(ADE2、HIS3、AUR1-C和MEL1)的表达；去除C-端(包括臂重复区)316个氨基酸残基后的ARC1N与EXO70A1之间表现出较弱的相互作用,只能激活3个报告基因(ADE2、AUR1-C和MEL1)的表达,表明ARC1的臂重复区可能并不位于ARC1与EXO70A1的互作界面的核心区段,ARC1的N-端结构域对ARC1-EXO70A1互作起关键作用；同时发现不同ARC1-EXO70A1组合的互作强度相当,其可能原因是ARC1和EXO70A1在甘蓝与甘蓝型油菜中存在的这些序列差异并未影响ARC1-EXO70A1互作界面的构象。由此可以得出如下结论：1.ARC1和EXO70A1在芸薹属植物中整体高度保守；EXO70A1在酵母转化株和甘蓝各器官中的组成型表达有所差异,暗示该蛋白可能在植物细胞中具有多种重要的功能。SRK基因的保守性低于RC1和EXO70A1,其编码产物SRK的多态性源于S域的3个高变区,同时也是造成芸薹属植物自交不亲和不断进化的主因。2.利用幼嫩根为材料能够制备较好效果的无壁细胞核。荧光原位杂交技术和运用BLAST搜索基因组用于DNA序列的染色体定位得到相同或相近的结果,但是针对基因组中出现与目的序列相似度较高的DNA序列时,在染色体上原位杂交的结果可能会出现一定的误差,表明原位杂交存在一定的局限性。3.SRKJ和ARC1发生相互作用,和SRKj发生互作的区域位于ARC1的C端结构域。ARC1能够与EXO70A1发生较强的互作,其与EXO70A1发生互作的区域位于ARC1的N端包括U-box的构成的结构。这同时表明ARC1蛋白是一种多功能的蛋白,在SI反应中发挥极为重要的作用。更多还原

【Abstract】 Brassica have sporophytic self-incompatible (SSI) characteristics which prevent self-fertilization by rejection of’self pollen’by the stigma. Initial SSI response results from interactional binding between SRK and SCR encoded by two genes at S locus. Then the response of recognition between stigma and self pollen is transmitted from outer cells to internal cells. At the same time, ARC1is activated by the kinase domain of SRK. That activated of ARC1can interact with EXO70A1possibly triggers subsequent cascade reactions which results in self-incompatibility. After acquiring the genes of SRK, ARC1and EXO70A1, bioinformatics analysis was done in the thesis, and orientation of three genes at the chromosomes were confirmed by means of fluorescence in situ hybridization and Blast. At the same tine, the interaction of corresponding three proteins was analyzed by use of yeast two-hybrid system.1In this study, CDS and the corresponding gDNA of EXO70A1were cloned by PCR from Brassica oleracea, Brassica rapa and Brassica napus. Sequence analysis was carried out by means of bioinformatics. RT-PCR was used to analyze expression characteristics of EXO70A1in Brassica oleracea. And BoEXO70A1was transformed into yeast Y187in order to its expression in yeast. The results showed:The gene lengths of BnEXO70A1, BrEX070A1and BoEXO70A1were3797bp,3752bp and3770bp respectively, and all consisted of12exons and11introns; their identity positions reached91%. Sequence conservativation was higher in exons than that in introns (excluding4th,5th,6th and8th intron). CDS of three EXO70A1genes consisted of1917base pairs and sequence similarity was97.1%. Three proteins of EXO70A1conduced all consisted of638amino acids; consensus positions and identity positions of three EXO70A1were99.8%and98.1%respectively. BnEXO70A1, BrEXO70A1and BoEXO70A1had similar secondary structures and three-dimensional structures. All introns start from the sequence GU and end with the sequence AG (in the5’to3’ direction). They are referred to as the splice donor and splice acceptor site, respectively. Another important sequence "CU(A/G)A(C/U)" in all introns of EXO70A1genes was located20～50bases upstream of the acceptor site. The corresponding sequence lengths of all12exons of EXO70A1were identical among three Brassica species and Arabidopsis thaliana; and similarity of4coding sequences was90.1%. Consensus positions and identity positions of four EXO70A1proteins reached99.8%and93.7% respectively. EXO70A1was a subunit of EXO70family which showed high conservative in Magnoliophyta. It showed low expression of BoEXO70A1in Y187. EXO70A1was a constutive gene which could be expressed in stamens, young stems, petals, pistils, young roots and leaves. However, its expression quantity is different in different tissues, with highest expression quantity in pistils and lowest one in stamens.2. CDS and the corresponding gDNA of ARCl were cloned by PCR from Brassica oleracea, Brassica rapa and Brassica napus. Sequence analysis was carried out by means of bioinformatics. The results showed:There existed no introns in ARC1from three kinds of plants. CDS of BoARC1gene consisted of1992base pairs (bp). However, the counterparts of BnARC1and BrARCl both consisted of1986bp. Identity positions of three ARC1’s, CDS reached93.8%and there existed differences of144base pairs among them. The consensus positions and identity positions of three ARC1proteins were99.4%and92.2%respectively, there were differences of51amino acids among them. Three ARCl had identical domains and motifs, namely N-terminal UND (N-terminal domain), U-box (U-box domain) and C-terminal ARM (armadillo repeat). In addition, three ARCl had highly similar secondary structures and three-dimensional structures.3. CDS of BoSRK6was cloned, and31sequences of SRK were obtained from Genbank. Sequence analysis was carried out by means of bioinformatics. The results showed:SRK consisted of seven exons and six introns. It encoded proteins consisting of816to860amino acids. Signal peptides and S domain of SRK were encode by the first exon. Though different SRK had similar protein sequences, SRK showed high polymorphism due to three highly variable regions in its extracellular domain which played an important role in the interaction between SRK and SCR/SP11. The extracellular domain of SRK was highly similar to SLG in structure and sequence. That possibly originated from the gene duplication in the S locus.4. Young roots from Brassica oleracea were used to prepare for cell-wall free nuclei. The method of receding interface was used for subsequent preparation of chromatin fibers, and fluorescence in situ hybridization (FISH) with5S rRNA-targeted was selected to test the quality of chromatin fibers. The results showed that:Cell-wall free nuclei from young roots could be used to prepare for chromatin fibers free of pigments from chloroplast, which was favorable for subsequent FISH. The compactness of plant metaphase chromosomes and the structure of the plant cell wall and cytoplasm provide a great obstacle to FISH for single-copy or low-copy DNA sequences whose lengths were2-4kb or so. Only about5%slides were detected with FISH signal. If prolonging the time of chromosomes treated with pepsin, more FISH signals could be detected because of chromosome deaggregation. More signals could be detected when FISH on prometaphase chromosomes and pachytene chromosomes. FISH onto the chromatin fibers could get the optimal results.5. Localization of SRK:FISH onto metaphase chromosomes, pachytene chromosomes and chromatin fibers for gene localization on chromosomes with SRK gene as probes. At the same time, the location of SRK was verified by searching with BLAST at BRAD Brassica genome database (http://brassicadb.org/brad/blastPage.php). The result of FISH onto mitotic metaphase chromosomes is identical to that onto pachytene counterparts. Only one pair of green signal was detected on a pair of homologous chromosomes, which indicated that only one-copy SRK gene possibly existed in Brassica oleracea’s genome. However, it was hard to define correctly the localization of SRK in the genome of Brassica oleracea because of the compactness of Brassica oleracea’s chromosomes. SRK gene lay in the region of8732037-8675105bp of the B. oleracea’ chromosome7by searching BRAD Brassica genome database. The corresponding region in Brassica rapa’s genome was the region of8732037-8675105bp in the chromosome7.6. Localization of ARC1:The searching result by BLAST showed that:ARC1was also a single-copy gene in both Brassica oleracea’genome and Brassica rapa’ genome. It lay in the region of35662808-35664799bp of B. oleracea’s chromosome4. It located at the region of17204000-17300000bp in Brassica rapa’s chromosome4.7. Localization of EXO70A1:The result of FISHing onto metaphase chromosomes and pachytene chromosomes for EXO70A1localization on chromosomes with EXO70A1gene as probe showed that:Only one hybridization signal was detected on mitotic metaphase chromosomes of Brassica oleracea, but sometimes two or three hybridization signals were detected on Brassica oleracea’s pachytene chromosomes. After searching searching at BRAD Brassica genome database with BLAST, only one copy EXO70A1gene was detected and it lay in the region (39669464-39673197bp) of Brassica oleracea’s chromosome9. In contrast, it lay in the region (17076191-17079967) of Brassica rapa’s chromosome10. The result was different from that of FISH on pachytene chromosomes. That was possibly because there were one or two highly homologous genes of EXO70A1lying in Brassica oleracea’s genome. 8. S locus receptor kinase (SRK) and ARM repeat containing (ARC1) are two key SI elements in Brassica. Interaction of SRK-ARC1possibly plays essential role in downstream SI signal transmission. In this study, in order to identify the interaction of SRKJ-ARC1during the course of SI, the segmental and full-length CDS of SRKJ and ARC1were amplified from B. oleracea var apitata and Brassica oleracea L var. acephala and then ligated to the plasmids of pGADT7and PGBKT7respectively. Then two recombinant plasmids were transformed to yeasts Y187and Y2HGold. At last the interaction of SRKJ-ARC1was tested by yeast two-hybrid system. The results showed that:The bait was confirmed not toxic to yeast and not to activate the expression of reporter genes by the test for autoactivation and toxicity. There existed five ARM repeats in ARC1, which had98%similarity at amino acid level between B. oleracea var acephala and B. oleracea var apitata. That three experimental groups Y2HGold[pGBKT7-ARC1]×Y187[pGADT7-SRKJ]、Y2HGold[pGBKT7-ARC1T3]×Y187[pGADT7-SRKJ] and Y2HGold[pGBKT7-ARC1T4]×Y187[pGADT7-SRKj] could grow on QDO/x/A nutritional media with transcription activation of the reporter genes AUR1-C, MEL1, HIS3and ADE2indicated that there existed interaction between ARC1from B. oleracea var acephala and SRK from B. oleracea var capitata and the interaction domain was located at the domain of ARM repeats, and the difference at amino acid level with the ARC1of B. oleracea var capitata was not enough to change the conformation in the interaction region. All above mentioned provides some insights into the molecular mechanism of self-incompatibility in Brassica.9. EXO70A1is possibly an important SI element in Brassica. EXO70A1-ARC1possibly plays essential role in downstream SI signal transmission. In order to identify the interaction of EXO70A1-ARC1during the course of SI, the segmental and full-length CDS of ARC1and EXO70A1were amplified from B. oleracea and Brassica napus and ligated to the plasmids of pGADT7and PGBKT7respectively. Then two recombinant plasmids were transformed to yeasts Y187and Y2HGold. At last the interaction of EXO70A1-ARC1was tested by yeast two-hybrid system. The results showed that:Sequence analysis showed that ARC1consisted of663amino acids in Brassica oleracea and661amino acids in Brassica napus, and there existed45amino acids difference between them. Sequence alignment showed that similarity positions and identity positions reached95.9%and93.9%between BoARC1and BnARCl, whereas there existed only six-amino-acid difference between BoEXO70A1and BnEXO70A1. The Similarity positions and identity positions reached99.4%and98.9%between BoEXO70A1and BnEXO70A1respectively. The homology of EXO70A1was higher than that of ARC1. Yeast two-hybrid results indicated that the strong interaction existed between ARC1and EXO70A1, and it could activate the expression of four reporter genes (ADE2, HIS3,AUR1-C, and MEL1) in diploid yeasts. However, low interaction existed between EXO70A1and ARC1N with316amino acids deleted from C-terminal, and it only activated the expression of three reporter genes (ADE2, AUR1-C, and MEL1). This provides an insight that the interface of interaction between ARC1and EXO70A1may not consist of the domains of arm repeats in ARCl. N-terminal domains of ARC1play an essential role in the interaction of ARC1-EXO70A1. The influences of the differences in amino-acid composition between B1ARCl and BnARCl on the interaction of ARC1-EXO70A1couldn’t be detected by means of yeast two-hybrid system, which probably resulted from that the binding interface between ARC1and EXO70A1was not altered by sequence difference of two proteins in two Brassica species.Thus conclusions can be drawn that:1. ARC1and EXO70A1, in Brassica species, are both high conservative; EXO70A1perhaps plays very important roles in plant cells because of its different expression characteristics in different plant tissues. The conservation of SRK was relatively slightly lower than that of ARC1and EXO70A1in Brassica. High polymorphism of resulted from the three high variable regions in SRK, and it was an important reason of molecular evolution of S locus genes leading to self-incompatibility of Brassica.2. Young roots can be prepared for cell-wall free nuclei. Gene localization using FISH and searching genome databases can obtain the same or similar results. However, FISH seems not to be suitable for the orientation of DNA sequences with which there are one or more highly similar sequences. It indicated that FISH has some limitations.3. ARC1can interact with SRK and EXO70A1. The interface of interaction between ARC1and kinase domains of SRK consists of the domains of arm repeats in ARC1. N-terminal domains of ARC1, containing U-box, interact directly with EXO70A1.更多还原