【作者】 杨洋；

【导师】 王小佳；

【作者基本信息】 西南大学 ， 园艺学， 2009， 博士

【摘要】 自交不亲和（self-incompatibility,SI）是一个广泛存在于被子植物中古老的遗传性状。植物体通过漫长的进化形成了有利于异花授粉的SI遗传机制,使显花植物避免近亲繁殖和自交衰退,有利于保持物种的多样性和独立性,为物种的生存和发展提供了有力的保障。在甘蓝中,柱头对花粉的识别决定于S位点的两个紧密连锁同时呈现高度多态性的基因:①S位点富含半胱氨酸基因（S locus cysteine-rich protein,SCR）,又称为S位点蛋白质11基因（S-locus protein11,SP11）,该基因编码一个位于花粉外壳的小分子极性亲水性蛋白。通过突变体分析、序列比对、转基因和授粉鉴定等一系列的证据表明它就是雄性决定因子。②S位点受体激酶基冈（S locus receptor kinase,SRK）编码一个跨膜的丝氨酸/苏氨酸受体激酶,该激酶位于柱头乳突细胞膜上,富含糖基化位点的N端S区域突出于细胞膜外,C端丝氨酸/苏氨酸激酶区整合于细胞膜内。它作为自交不亲和信号传导的雌性决定因子,决定柱头特异的S基因型并传递自交不亲和信号。同一等位S基因型的SRK与SCR相互作用是诱发自交不亲和信号传导途径的第一步,启动了整个自交不亲和信号传导途径。具有自交不亲和性的甘蓝在自花授粉后,当花粉黏附到柱头上,花粉携带的可扩散SCR信号分子就被转运到柱头表面,通过进一步的扩散与位于乳突细胞原生质膜上的SRK信号受体相互作用。SRK接收信号后被激活,引起细胞内信号的级联反应,并最终抑制花粉的萌发。在复杂的SI信号传递网络中,目前已经确定的参与自交不亲和信号传导的因子只有SRK,SCR,THL1,THL2,ARC₁和MLPK,还有很多信号因子没有被发现。2003年,吴能表通过³²P标记放射自显影从自交不亲和植物中获得一特异的未知磷酸化蛋白,推测为授粉引发的自交不亲和相关因子,并有可能作为自交不亲和信号传导的下游因子,为自交不亲和性的分子机理研究提供新的研究内容。本研究以高度自交不亲和性结球甘蓝ZQ为材料,克隆了自交不亲和信号传导中的雌雄决定因子,并进行了体外表达分析,建立了适用于SRK与SCR相互作用的体外检测体系,为进一步的化学调控试剂的筛选,实现SRK-SCR复合体聚合与解离的人为调控提供理论和技术基础。同时为编码基因的定点突变以及使用大规模蛋白质相互作用的研究方法如酵母双杂交、质谱鉴定、蛋白芯片以及基于生物信息学的分析方法,深入研究SRK与SCR相互作用的机理提供了一个相互作用体系的技术平台。另外选用结球甘蓝高度自交不亲和性材料E₁设计兼并引物结合3’-RACE对自交不亲和相关基因进行筛选和分析,以期为自交不亲和未知的下游信号的分离和鉴定提供新的信息与线索。主要工作及结果总结如下:1.S位点富含半胱氨酸蛋白/S位点蛋白11（SCR/SP11）编码区的克隆及表达采用巢式RT-PCR,以甘蓝ZQ的花药cDNA为模板,扩增了ZQ的SCR序列。SCR_fZQ序列分析结果表明ZQ材料的S基因型是属于甘蓝S₂₈单倍型。按已知S单倍型的分类分析,ZQ属于第Ⅰ类S单倍型,具有高度的自交不亲和性,这与田间检测的自交亲和指数为0.004结论一致。进一步的编码区克隆子SCR_ZQ序列分析表明:SCR_ZQ编码SCR_ZQ蛋白信号肽C端的三个氨基酸VEA和成熟肽,预测编码蛋白大小为7.8kD。编码的64个氨基酸中包含有SCR蛋白家族中保守的8个半胱氨酸和1个甘氨酸特征型氨基酸位点,另外还含有2个不保守的半胱氨酸。对这10个半胱氨酸形成的二硫键的分析表明,除保守的Cys57不能形成蛋白内二硫键,其余保守半胱氨酸均形成了蛋白内二硫键以稳定SCR_ZQ的蛋白构型。三维结构的分析表明SCR_ZQ蛋白呈皱缩螺旋状,包含一个α-螺旋和三个反向平行的β-片层。SCR_ZQ经转化酶切及连接后构建出融合表达载体pET43.1a-SCR_ZQ,并诱导获得了大小约为74kD的Nus·A-SCR融合表达蛋白。通过正交设计,定量分析各表达蛋白所得的校正相对光强值,SCR_ZQ在各诱导条件下均有高表达量,其中各表达因素对融合蛋白的表达量影响均未达到显著水平。SCR_ZQ表达菌液经蛋白纯化试剂盒处理后,能得到明显无杂带的纯化蛋白。2.S位点受体激酶胞外域编码区（mSRK）的克隆及表达2.1 S位点受体激酶胞外域编码区（mSRK）的克隆及序列分析在甘蓝ZQ柱头中成功克隆出mSRK长度为1319bp的cDNA序列,核苷酸序列的比对分析与SCR_fZQ序列比对分析结果一致:ZQ材料的S基因型与甘蓝的S₂₈基因型序列完全一致,其次与油菜的S₅₄亲缘关系很近。mSRK编码的氨基酸序列分析,推导编码氨基酸439个,编码蛋白大小49.3 kD。前16个疏水性氨基酸为信号肽序列,最后10个氨基酸为跨膜域序列,中间含有作为S多基因家族标志的12个保守的半胱氨酸。另外还含有6个N-糖基化位点及多个活性位点。mSRK编码蛋白高级结构分析结果表明:mSRK包含了B-Lectin结构域,SLG结构域以及PAN_APPLE结构域。其中PAN_APPLE结构域中三个极性氨基酸位点P（350）,T（352）,R（369）推测为蛋白或糖的结合位点。对mSRK蛋白分区建模,获得两个mSRK 3D模型。模型1对应mSRK蛋白第14位至264位氨基酸,包含了B-Lectin结构域和部分SLG结构域,形成了如海马状构型,含有两个12股β-棱状Ⅱ折叠和海马腹部一小段连接区域。模型2对应mSRK蛋白第285至391位氨基酸,覆盖了部分SLG结构域及PAN APPLE结构域。主要由1个α-螺旋和5个β-片层构成,含有12个保守的半胱氨酸和推测的3个蛋白或糖的结合位点。进一步的功能位点预测,保守性较高的氨基酸功能活性位点集中在mSRK蛋白中的半胱氨酸位点上,由此推测mSRK蛋白中半胱氨酸有重要的生物学功能。12个半胱氨酸中保守的有10个半胱氨酸被预测到可以形成五对蛋白内的二硫键,而另外两个半胱氨酸Cys366和Cys370或是保持自由状态,或者是与其它蛋白中的半胱氨酸残基配对形成二硫键。2.2 S位点受体激酶胞外域编码区（mSRK）的原核表达本研究构建了两种形式的mSRK的原核表达载体。pET43.1a-Nus·A-mSRK表达载体中含有Nus·A融合肽,用于增加mSRK在大肠杆菌中的可溶性表达量和表达活性,有利于进一步分析它与SCR蛋白的相互作用。而去除了Nus·A融合肽的pET43.1a-mSRK表达载体的构建,主要是排除Nus·A融合肽中含有的His标签在相互作用检测系统中可能造成的假阳性影响,作为Nus·A-mSRK蛋白与SCR_ZQ蛋白相互作用验证的重要补充。将构建的pET43.1a-Nus·A-mSRK与pET43.1a-mSRK表达载体在大肠杆菌BL21中成功表达,分别表达出大小约为112.0kD和50.0kD的目的蛋白。在Nus·A-mSRK原核表达中表达温度对蛋白表达量的影响极显著,最佳诱导表达条件为:温度25℃;IPTG浓度0.1 mmol·L^-1;时间1h。mSRK受表达条件影响不显著。通过超声破碎裂解表达菌液,加入Ni⁺磁珠回收目的蛋白,获得了纯化的Nus·A-mSRK蛋白。3.SCR_ZQ与mSRK蛋白相互作用的体外检测利用SCR_ZQ融合表达蛋白上的组氨酸标签能与Ni⁺磁珠螯合,将SCR_ZQ融合表达蛋白分别与构建的两套原核表达系统pET43.1a-mSRK/pET43.1a-Nus·A-mSRK的诱导表达蛋白及Ni⁺磁珠在适宜于mSRK与SCR_ZQ相互作用的蛋白提取液里孵育2h,洗涤纯化后,对纯化复合物进行SDS-PAGE检测,我们验证了mSRK蛋白与SCR_ZQ蛋白在体外是能够相互结合,形成了稳定的结合复合物。同时也说明mSRK蛋白与SCR_ZQ蛋白在体外的相互作用并不是产生于瞬间的结合。4.授粉引发的甘蓝SI相关基因的探讨根据吴能表发现的磷酸化蛋白的N端氨基酸序列设计兼并引物进行3’-race扩增,然后对所得核酸序列进行编码氨基酸的分析,我们筛选候选基因并进行EST序列的电子拼接。分析扩增得到的六条序列,均不符合编码这个磷酸化蛋白N端序列。但是同时,我们发现了一条新序列并命名为NG,目前在其它物种中未找到与之同源的序列,只是在甘蓝的EST数据库中找到可以拼接的序列表达标签。NG按照分析出的可读框,编码的氨基酸序列与拟南芥热激蛋白相关因子有较高的同源性。初步的RT-PCR检测表明NG基因在柱头有高量表达,在叶片中微量表达,花药,花茎和根中均无表达。然后我们将NG序列按照推测的编码框设计引物,扩增编码区后连接到pET43.1a表达载体上,构建了pET43.1a-NG表达载体。在大肠杆菌BL21中成功诱导表达并纯化出约91.2kD的目的融合蛋白,以利于新序列NG推测编码蛋白的功能研究。更多还原

【Abstract】 Self-incompatibility（SI） is widely distributed genetic mechanism of angiosperm.Plant evolved SI mechanism for cross pollination that allows plants with perfect flowers to avoid inbreeding and keep the diversity and independent,so survive and development could be guaranteed.In the self-incompatibility（SI） response of Brassica oleracea L.var.capitata.L.,the recognition of self-related pollen by the stigma epidermis is affected by two tightly linked and highly polymorphic genes which located in the S locus:①The S locus cysteine-rich protein gene SCR[also designated SP-11]encodes a small hydrophilic and positively charged peptide that is localized to the pollen coat.A series of studies such as mutation analysis,sequence alignment,transgenic pollen acquisition and pollination bioassay finally proved that SCR is the male determinant of SI.②The S locus receptor kinase gene SRK encodes a single-pass transmembrane serine/threonine kinase,which displayed with its glycosylated N-terminal S domain external to the cell and its C-terminal kinase domain within the plasma membrane of the stigma epidermis.As a female determinant of SI,it shows the specificity recognition of stigma and transfer SI signal to downstream element.An allele-specific interaction between the SRK and SCR is the first and determined step to start SI signal transduction.When SI Brassica oleracea L.pollinated with“self’pollen,the SCR signal molecules within the pollen coat were carried onto the surface of stigma epidermis,a further diffusion through the cell wall makes it interact with the SRK receptor on the plasma membrane and activates the SRK kinase. In turn,this activation presumably triggers a signaling cascade that inhibits pollen hydration and germination.Many signal factors involved in sophisticated SI signal transduction,but only few factors which are SRK,SCR,THE1,THL2,ARC₁ and MLPK were found.Isolation of an unknown phosphoprotein from Brassica oleracea L.,which is assumed as a SI related protein,by D.Wu in 2003 might gives a clue to further studies.In this paper,the highly self-incompatible cabbage（Brassica oleracea L.var.capitata.L）‘ZQ’ was taken as plant materials.Female and male determinates of SI were cloned and expressed in vitro respectively and an interaction detection system between the two determinates was established.By the research we went to provide the theoretical and technical way for further study on mechanism and reagent control of the interaction between SRK and SCR.On the other hand,RT-PCR and 3’-RACE were used to clone the SI related gene in cabbage‘E₁’ for new information and clues.The main results were as below:1.Cloning and expressing of the coding sequence of S locus cysteine-rich protein/S locus protein 11（SCR/SP-11）The cDNA of SCR was cloned from pollen total RNA of the cabbage.‘ZQ’ by nested RT-PCR. Sequence analysis shows that ZQ belongs to the class-ⅠS haplotypes S₂₈ of Brassica oleracea L., This S class haplotypes exhibit strong SI phonotype gives another proof besides the low ISC（index of self-compatibility）.Analysis of the coding sequence of SCR indicated that it encodes a 7.8kD protein covered the predicted three amino acid residue VEA in the C terminate of signal peptide and the whole mature peptide of SCR_fZQ amino acid sequence.Besides the eight conserved cysteine residues and one conserved glycin residue,two nonconservative cysteine residues were found in the SCR_ZQ encoding amino acid sequence.The predicted disulfide bonds in SCR_ZQ showed that:Except for Cys57 could not form the disulfide bonds with any other cysteine residues in the protein,the other conserved cysteine residues formed the disulfide bonds to stabilize SCR protein.The 3D model indicated that SCR_ZQ formed a crimple coil,including anα-helix and three-stranded antiparallelβ-sheets.After SCR_ZQ was transformed into DH5αpET43.1a and the pMD18-T-SCR_ZQ were double digestion and recovering,later ligation by T4 DNA ligase constructed expression plasmid pET43.1a-SCR_ZQ.The recombinant strain BL21/Pet43.1a-SCR_ZQ were induced by IPTG to express Nus·A-SCR_ZQ fusion protein.The expression products were analyzed by SDS-PAGE,and result showed that SCR_ZQ fusion protein was expressed at expected molecular weight 74kD. Orthogonal design was used for analyzing the expression adjusted volume.The result showed that inducing conditions have little influence on expression of SCR_ZQ fusion protein.When expression process was finished,SCR_ZQ was purified by MagneHis^TM Protein Purification System and analyzed by SDS-PAGE.2.Cloning and expressing of the extracellular domain of S locus receptor kinase （mSRK）2.1 Cloning and sequence analysis of the extracellular domain of S locus receptor kinaseThe 1319 bp cDNA of mSRK was amplified from the total RNA of stigma.of‘ZQ’.Nucleotide sequence analysis of mSRK reached the same conclusion of SCR_ZQ sequence alignment:‘ZQ’ shared the same sequence with Brassica oleracea L.S₂₈,and was closely related to Brassica rapa S₅₄. Analysis of the encoding amino acid sequence of mSRK showed that,the encoding protein contains 439 deduced amino acids with the molecule weight 49.3 kD.The first 16 amino acids encoded hydrophobic signal peptide and the final 10 amino acids were deduced transmembrane domain sequence,12 conserved cysteine residues as the sign of the S-family protein and a number of active sites including six N-glycosylation sites were scattered in the amino acid sequence of mSRK.mSRK protein structural analysis results showed that:mSRK contains B-Lectin domain,SLG-domain and PAN_APPLE domain.The three polar amino acid sites P（350）,T（352）,R（369） in PAN_APPLE domain were putative protein or sugar-binding sites.Two 3D models of mSRK were acquired from Bio-information websites.Model 1 corresponds to 14-264 amino acid residues in mSRK protein, which contains B-Lectin domain and part of SLG domain,forming a hippocampus-shaped configuration It consists of two 12β-stranded -prismⅡfold and a linker located in the abdomen of the hippocampus.Model 2 covers the 285-391 amino acid residues of mSRK protein,which encodes partial SLG domain and PAN_APPLE domain,and shows one a-helix and fiveβ-sheets,containing the twelve conserved cysteine residues and three putative protein or sugar-binding sites.Further analysis of functional sites prediction shows that functional amino acids sites are highly conserved on cysteine residues in mSRK protein,so these conserved cysteine residues might play an important role in mSRK biological functions.Predictions also indicated that 10 of the 12 conserved cysteine residues would form five pairs of disulfide bonds within the protein,while the remaining two cysteine residues Cys366 and Cys370 might remain free,or forms disulfide bonds with other proteins. 2.2 Prokaryotic expression of the extracellular domain of S locus receptor kinase（mSRK）mSRK was constructed into the prokaryotic expression vector in two forms,pET43.1a-Nus·A-mSRK expression vector is conducive to further analysis of its interaction with the SCR protein. Because the Nus·A fusion peptide could significantly increase the mSRK solubility and activity when expressed in E.coli.While mSRK expression vector without Nus·A fusion peptide was constructed to exclude the possibility of that the His tag in Nus·A fusion peptide might get false positive result in the later interaction detection,as an important supplement to verify the Nus·A -mSRK and SCR interactions.Both Nus·A-mSRK and mSRK were expressed in E.coli BL21 with their expected molecule weight about 112.0kD and 50.0kD respectively.Analysis of the expressing of mSRK protein under different induction conditions,the result shows that temperature was significantly effect the expression of Nus·A-mSRK,so best-induced conditions were suggested as follows:temperature:25℃;concentration of IPTG:0.1 mmol·L-1;time:1 hour.While there were insignificant differences on expression of mSRK in different inducing conditions.The BL21 Bacterial cells which expressed Nus·A-mSRK protein were first sonicated and then resuspend in the cell lysis reagent.Aftet incubated with Ni-Particles and recovered by Magnetic stand,purified Nus·A-mSRK fusion protein was finally obtainded.3 In vitro assay of the interaction between SCR and SRKmSRK and Nus·A-mSRK prokaryotic expression proteins were incubated with SCR_ZQ fusion protein respectively for 2 hours in suitable interaction reagents.Because SCR_ZQ fusion protein contained a 6×His Tag,which could chelate with Ni⁺,the complex of SCR_ZQ and SRK interaction product could purified by the affinity between Ni⁺ and Magnetic stand,all products were analyzed by SDS-PAGE.The result showed that SCR_ZQ could combine with mSRK in vitro and possibly form a stable complex.4 Probe into SI related gene induced by pollination of Brassica oleracea L.According to the N-terminal amino acid sequence of phosphoprotein found by Dr.Wu, degenerate primers were designed for 3’-race,and then analysis the encoding amino acid of the obtained nucleotide sequences,we screened out a candidate gene and e-splicing it with EST sequences,expect to find out the full-length sequence encoding phosphoprotein.However,by analyzing the seven amplified sequences,we finally failed to get a sequence encoding the same amino acid with the N-terminal phosphoprotein.But we found out a new sequence,and designed as NG,no sequences could be found homologous to it in other species,only a few ESTs could align in the Brassica EST database.The deduced amino acid sequence of NG had the highest homology with the heat shock protein-like factor of Arabidopsis thaliana.RT-PCR analysis showed that the expression of NG gene is higher in stigma,lower in leaf but have no expressions in other tissues. The putative opening reading frame of NG were cloned and ligated to pET43.1a vector to express the NG in vitro,finally a molecular weight of 91.2kD of NG fusion protein was expressed and purified for further study.更多还原