甘蓝自交不亲和信号传导元件ARC1、SRK的克隆与体外表达及其相互作用研究
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摘要
植物的自交不亲和性(self-incompatibility,SI)在植物界中普遍存在,是植物为了防止近亲繁殖、促进物种分化、保持遗传多样性,而在长期进化过程中形成的一种复杂而完善的重要机制。它实质上是一种雌蕊识别自体花粉和异体花粉,阻止自体花粉萌发的信号传导过程,涉及从授粉到受精过程中发生的花粉和雌蕊之间的相互作用,几乎一半以上的显花植物具有自交不亲和性,涉及到70多个科、250多个属。
甘蓝是典型的具有孢子体型自交不亲和性的芸薹属植物,这类SI在遗传上受一个带有复等位基因的多态性S基因座所控制。最近几年,对芸薹属自交不亲和性的决定性因子研究方面,取得了长足的进步。迄今为止,已分离到3类与S位点连锁的基因,分别编码SLG(S-locusglycoprotein,S位点糖蛋白)、S位点富半胱氨酸蛋白SCR(S-locus cystein-rich protein)/SPll(S-locus protein 11,S位点蛋白11)和SRK(S-locus receptor kinase,S位点受体激酶)。三个基因(SLG、SRK、SCR)的相继分离与鉴定,意味着由SRK介导的自交不亲和信号传导的上游事件越来越清晰了。来自花粉中的SCR/SP11在SLG的协助下与柱头上的跨膜蛋白SRK的胞外域相互识别,当两者具有相同单倍型时,SRK发生自我磷酸化,引起细胞内信号的级联反应,最后抑制自花花粉的萌发、水合或花粉管伸长。但是,对SRK介导的细胞信号传导下游磷酸化蛋白研究相对于上游受体来说要缓慢得多。在整个信号传导途径中,ARC1是下游事件中最为感兴趣的蛋白之一,它能够与SRK相互作用,并将信号传递给下游因子。因此在自交不亲和信号传导过程,SRK与ARC1的结合与否以及复合体的存在和变化是证实这个分子过程和调控自交不亲和反应的关键。为了探索这些问题,必须首先鉴定ARC1与SRK之间的相互作用,调控二者的解离与聚合,深入分析二者的作用机理,建立相互作用化控试剂的筛选平台,从而为下一步筛选ARC1的下游因子创造条件。
本研究以高度自交不亲和性甘蓝E1为材料,克隆了自交不亲和信号传导元件ARC1和SRK基因,并进行了表达分析。体外证实了ARC1与SRK的相互作用,建立了适用于SRK和ARC1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-ARC1复合体聚合与解离的人为调控提供理论和技术基础。主要工作及结果总结如下:
1甘蓝ARC1臂重复蛋白编码基因的克隆及其在大肠杆菌中的表达
1.1 ARC1基因的克隆与序列分析
采用PCR和RT-PCR技术,以甘蓝E1基因组DNA和柱头总RNA为模板,扩增了ARC1的DNA和cDNA序列,经测序和序列分析表明,DNA序列无内含子存在,扩增ARC1基因的编码序列为1 992 bp,编码663个氨基酸残基,分子量为73.5 kD。序列同源性分析表明,甘蓝E1的ARC1基因(B.oleracea E1 ARC1)与羽衣甘蓝ARC1基因(B.oleracea kale ARC1,NCBI登录号:EU344909)、油菜ARC1基因(B.napus ARC1,NCBI登录号:AF024625)、甘蓝20010197 ARC1基因(B.oleracea ARC1 20010197~([188]))、甘蓝ARC1基因(B.oleracea ARC1,NCBI登录号:AJ417335)、拟南芥中泛素蛋白连接酶(A.thaliana PUB17,NCBI登录号:NM102674)、水芹ARC1基因(A.thaliana cress ARC1,NCBI登录号:AY150512)的同源性分别为97%、94%、93%、95%、70%和70%。作为芸薹属5个基因,B.oleracea E1 ARC1、B.oleracea kale ARC1、B.oleracea ARC1 20010197、B.oleracea ARC1、B.napus ARC1之间的碱基差异很小,而作为十字花科中不同属的两个基因B.oleracea E1 ARC1和A.thaliana ARC1,在碱基上却存在相对较大的差异,编码的碱基差异大多数集中在ARC1的N端,说明ARC1基因存在较强的保守性,而且C端区域的保守性强于N端。在甘蓝与油菜的序列中均存在碱基的缺失,但这种缺失往往是以密码子形式的缺失,并没有引起移码突变。同源氨基酸序列进行比对显示,B.oleracea E1 ARC1基因编码的氨基酸序列与B.oleracea kale ARC1、B.oleraceaARC1 20010197、B.napus ARC1、A.thaliana ARC1所编码的氨基酸序列相似性分别为97.9%、97.6%、93.2%和53.2%。建立进化树后分析发现,B.oleracea E1 ARC1与B.oleracea kale ARC1、B.oleracea ARC1 20010197、B.oleracea ARC1的亲缘关系较近,而与B.napus ARC1、A.thalianaPUB17、A.thaliana cress ARC1的亲缘关系较远,更充分说明了ARC1基因的较强保守性,而且种间的保守性高于属间的保守性。在甘蓝E1的ARC1蛋白质序列中有一个U-box结构,5个臂重复序列。U-box结构位于第282个氨基酸和第347个氨基酸之间,臂重复序列编码42个氨基酸残基,油菜ARC1的U-box结构位于第283个氨基酸和第347个氨基酸之间。而在拟南芥中臂重复序列只有1个,这种较大的差异可能是导致ARC1作为与自交不亲和信号传导有关的基因在两个不同属之间的功能有所不同的原因。对甘蓝E1的ARC1蛋白的活性位点分析发现,ARC1蛋白中具有犰狳的ARM活性位点,该位点在芸薹属中的保守性很差,同时还发现其它磷酸化活性位点,包括10个蛋白激酶C磷酸化位点、1个亮氨酸拉链位点、2个基于cAMP-和cGMP蛋白激酶林磷酸化位点、11个酪蛋白激酶Ⅱ磷酸化位点、5个N-糖基化位点、8个N-肉豆蔻酰化位点、1个氨基化位点、1个酪氨酸激酶磷酸化位点,这些位点的存在与ARC1蛋白参与信号传导过程中依赖的磷酸化作用是相辅相成的。另外,拉链结构的存在,可能与ARC1的二聚化有关,或者是下一级信号传导元件的结合位点。ARC1蛋白的三维图形覆盖了400个氨基酸(第264位到第663位氨基酸),该段蛋白的结构整体上是右手螺旋,形成了类似海马状构型,腹部中空,形成大沟,背部形成小沟,小沟区域正是ARM结构域,可能为它与其他蛋白的结合提供了相应位点。
1.2 ARC1在大肠杆菌中的表达
提取pMD18-T-ARC1和pET43.1a质粒,经EcoRⅠ和KpnⅠ双酶切,纯化回收ARC1和pET43.1a片段,用T4 DNA Ligase连接以构建表达质粒pET43.1a-ARC1,经PCR以及EcoRⅠ和KpnⅠ双酶切鉴定。提取pET43.1a-ARC1质粒,转化表达宿主菌BL21,IPTG诱导表达ARC1融合蛋白,表达产物的SDS-PAGE结果显示,在约135 kD处有重组蛋白表达特异带出现,其蛋白质相对分子质量与预计值相符。在诱导的过程中,IPTG浓度影响不大,但诱导温度(℃)对ARC1蛋白的表达影响很大,最佳诱导条件为:温度32℃;IPTG 1.0 mmol·L;时间4 h。诱导表达的融合蛋白ARC1主要是以包涵体的形式存在,但通过细胞破碎及蛋白纯化试剂盒纯化后,纯化产物经SDS-PAGE检测,泳道基本没有杂蛋白条带,纯化结果比较理想。
2甘蓝SRK激酶结构域编码序列的克隆与其在大肠杆菌中的表达
2.1 SRK_(K1)基因的克隆与序列分析
以甘蓝E1为材料,特异扩增SRK激酶结构域编码区序列(SRK_(E1))。测序结果显示扩增的DNA序列和cDNA序列分别为1 711 bp和1 241 bp,DNA序列包含6个外显子、5个内含子,内含子大小分别为89 bp、107 bp、89bp、102 bp和78 bp。5个内含子的5'和3'端完全符合经典的GT-AG法则。同源氨基酸序列比对表明,SRK_(E1)与sRK_6、SRK_(910)、SRK_(18)、SRK_(29)氨基酸序列同源性分别为88.2%、85.7%、87.2%和85.7%。SRK_(E1)的氨基酸序列包含了跨膜结构域的后16个氨基酸和整个激酶结构域,跨膜结构域的Cys和激酶活性位点Lys并没有发生突变。用Blastp程序对SRK_(E1)的氨基酸序列进行保守结构域分析发现,该序列中存在蛋白激酶结构域和酪氨酸激酶结构域。蛋白激酶结构域位于第78位氨基酸和第361位氨基酸之间,编码284个氨基酸。在系统进化分析中发现,不同单元型SRK依据序列同源性高低分为两类(ClassⅠ和ClassⅡ),ClassⅠ表现为高度自交不亲和,ClassⅡ(SRK_(15)、SRK_(60)、SRK_2、SRK_(29)和SRK_5)的自交不亲和性较弱,SRK_(E1)与SRK_(45)、SRK_3聚类在一起。因此,依据不同单元型SRK序列的多态性从分子层面上证明了E1材料的高度自交不亲和性。
2.2 SRK_(E1)在大肠杆菌中的表达
提取pMD18-T-SRK_(E1)和pET43.1a质粒,经BamHⅠ和HindⅢ双酶切,纯化回收SRK_(E1)和pET43.1a片段,用T4 DNA Ligase连接以构建表达质粒pET43.1a-SRK_(E1),经PCR以及BamHⅠ和HindⅢ双酶切鉴定。提取pET43.1a-SRK_(E1)质粒,转化表达宿主菌BL21,IPTG诱导表达SRK_(E1)融合蛋白,表达产物的SDS-PAGE结果显示,在约107 kD处有重组蛋白表达特异带出现,IPTG的浓度和诱导温度对该蛋白表达量的影响不大,且表达的SRK_(E1)融合蛋白主要包涵体形式存在。SRK_(E1)融合蛋白最佳的诱导表达条件为:温度37℃;IPTG 0.5 mmol·L~(-1);时间4h。
3 ARC1与SRK_(E1)体外相互作用的方法建立
免疫共沉淀法鉴定蛋白质相互作用原理表明,蛋白质X与蛋白质Y相互作用,如蛋白质X与其抗体免疫沉淀,则蛋白Y会与蛋白质X及其抗体一起沉淀下来。依据该原理利用pET43.1a作为表达载体表达ARC1、SRK_(E1),巧妙利用了pET43.1a-ARC1、pET43.1a-SRK_(E1)融合蛋白序列中的6×His标签与Ni~+相结合的特点,探索性地建立了体外检测蛋白质间相互作用的新方法。将pET43.1a-SRK_(E1)融合蛋白与Ni~+结合,结合后的复合物作为“诱饵蛋白”,把体外表达的ARC1融合蛋白溶于适于相互作用的缓冲液中作为“靶蛋白”溶液,两者4℃条件下孵育后利用pET43.1a-SRK_(E1)上结合的Ni~+与磁力架吸附性将“诱饵蛋白”与靶蛋白的复合体纯化出来。本研究对ARC1与SRK_(E1)相互作用的检测,条带清楚,明确证实了两者相互作用,得到了理想的结果,说明我们建立的体外检测蛋白相互作用方法是可行的。本方法与免疫共沉淀法鉴定蛋白质相互作用相比,不需要制备抗体,简化了实验操作,避免了因抗体特异性不强可能带来的假阳性结果。另外本文建立的检测体系可作为ARC1与SRK相互作用的化控试剂筛选平台,从而为深入分析两者相互作用的机理以及分离和鉴定自交不亲和未知的下游信号传导元件提供新的信息与线索。
4 ARC1与SRK_(E1)相互作用的体外检测
将在大肠杆菌BL21中诱导表达的pET43.1a-SRK_(E1)融合蛋白Ni~+结合,结合后的复合物作为“诱饵蛋白”。将在大肠杆菌BL21中诱导表达的pET43.1a-ARC1融合蛋白作为“靶蛋白”,并溶解在适于相互作用的缓冲液中,“诱饵蛋白”和“靶蛋白”溶液在4℃条件下孵育2 h,每隔15 min轻轻翻转混匀一次,然后利用pET43.1a-SRK_(E1)融合蛋白结合的Ni~+与磁力架吸附性将诱饵蛋白和蛋白的复合体纯化出来,产物经SDS-PAGE检测,结果表明ARC1蛋白与SRK_(E1)蛋白在体外能够相互结合,可能形成稳定的复合体,同时也说明二者的相互作用并非产生于瞬间的结合。
Many bisexual flowering plants possess a reproductive strategy called self-incompatibility(SI) that enables the female tissue(the pistil) to reject self but accept non-self pollen for fertilization.SI response represents cell signal transduction process that allows the pistil to distinguish between self and non-self pollens and prevented self pollens germination.Pollination and fertilization in flowering plants involve a series of complex events with tightly regulated cell-cell interactions and signaling between the pollen and the pistil.More than half flowering plants possess a SI and involve 70 family,250 genus.
Brassica oleracea L.is a classical Brassicaceae plant with the sporophytic self-incompatibility that was controlled by a single multi-allelic S locus genetically.In recent years,much progress has been made in determining the male and female S determinant in Brassica species.To date,in the signal transduction pathway of self-incompatible Brassica,three highly polymorphic genes with linked S locus have been identified.One is the S locus glycoprotein(SLG) gene that encoded a secreted glycoprotein abundantly present in the papillar cell of the stigma surface,one is S-locus receptor kinase(SRK)gene,the other is S-locus cystein-rich(SCR) gene.One after the other,three gene(SLG、SRK、SCR) isolated and characterized implies that upstream events of the SRK-mediated signal transduction pathway are more perspicuous.In the signal transduction pathway,ARC1 is a most interesting target that can interact with SRK specifically and transfer self-incompatible signal. Therefore,ARC1 band to SRK or not and stability of the formed compound is the key to regulate SI responses.In order to answer the former questions,we test the interaction between ARC1 and SRK, further analyze interaction mechanism,regulate separation ang aggregation of ARC1-SRK,and establish a screening system for chemical regulation of the interaction.Sequentially,these studies provide condition for further screening downstream targets of ARC1.
In this paper,the highly self-incompatible Brassica oleracea E1 was taken as plant material. ARC1 and SRK were cloned,expressed respectively and in vitro confirmed interaction between interaction ARC1 and SRK.A detection system between the two determinates was established.By the research we provide the theoretical and technical foundation for further selecting controlled reagent and artificially regulating the aggregation and dissociation of SRK-ARC1 complex.The main results were as below:
1 Cloning of coding sequence of ARC1 from B.oleracea E1 and expression in E.coli BL21
1.1 Cloning and sequence analysis of ARC1 gene
The DNA and cDNA fragments of ARC1 were amplified from genomic DNA and stigma total RNA of Brassica oleracea E1 by PCR and RT-PCR methods.Sequencing assay demonstrated that no intron existed in DNA sequences,and the cDNA of ARCl was 1,992 bp in length,encoding 663 amino acids,molecular weight was 73.5 kD.The nucleotide sequence of B.oleracea E1 ARC1 respectively showed 97%,94%,93%,95%,70%and 70%sequence identity to B.oleracea kale ARC1(Accession number in NCBI is EU344909),B.napus ARC1(Accession number in NCBI is AF024625),B.oleracea 20010197ARC1,A.thaliana PUB17(Accession number in NCBI is NM102674),and A.thaliana ARC1(Accession number in NCBI is AY150512).Little difference was showed in the bases of five ARC1 alleles(B.oleracea E1 ARC1,B.oleracea kale ARC1,B. oleracea 20010197ARC1,B.oleracea ARC1 and B.napus ARC1),but more difference in N-terminal of B.oleracea E1 ARC1 and A.thaliana ARC1.This explained that the C-terminal of ARC1 had more conservative than N-terminal.In addition to there were miss bases in the nucleotide sequence of B.oleracea and B.napus,and this miss bases with 'codon' didn't bring code mutation.The alignment amino acid sequence of B.oleracea E1 ARC1 respectively showed 97.9%,97.6%,93.2% and 53.2%sequence identity to B.oleracea kale ARC1,B.oleracea ARC1 20010197,B.napus ARC1 and A.thaliana ARC1.Phylogenetic tree of ARC1 proteins showed that B.oleracea E1 ARC1 was the closest to B.oleracea kale ARC1,B.oleracea ARC1 and B.oleracea ARC1 20010197,but the furthest to A.thaliana PUB17、A.thaliana cress ARC1.Database searches with both the DNA and amino acid sequences revealed that there were five arm repeat domains of C-terminal encoded amino acid 42 and a U-box domain that was located between amino acid 282~(th) and 347~(th) in B. oleracea E1 ARC1.In contrast to B.oleracea E1 ARC1,five arm repeat domains and a U-box domain that was located between amino acid 283~(th) and 347~(th) existed in B.napus ARC1,only an arm repeat in A.thaliana cress ARC1 and A.thaliana PUB17.The significant difference potentially determined the function discrepancy of ARC1in B.oleracea and A.thaliana.Prediction of active site of ARC1 showed that B.oleracea E1 ARC1contained active site of armadillo and the active site motif were poorly conservative in B.oleracea.In addition to ARC1 also contained Protein kinase C phosphorylation site,cAMP-and cGMP-dependent protein kinase phosphorylation site,Casein kinaseⅡphosphorylation site,Leucine zipper site,N-glycosylation site,N-myristoylation site, Amidation site,Tyrosine kinase phosphorylation site.They probably took part in the signal transduction of SI.In ARC1 protein,the three dimension models covered the 264-663 amino acid residues which wereα-helix and formed a hippocampus-shaped configuration and a wide groove and a narrow groove,which may be the binding sites of other proteins.
1.2 Expression of ARC1 in E.coli BL21
The plasmid pMD18-T-ARC1 and pET43.1a were extracted,and then they were digested by restriction enzymes EcoRⅠand KpnⅠfor purification and recovering of ARC1 cDNA and pET43.1a fragments.Two fragments were ligated by T4 DNA ligase to construct expression plasmid pET43.1a-ARC1,identified by PCR and two restriction enzymes digestion by EcoRⅠ/ KpnⅠ,then pET43.1a-ARC1 was transformed into E.coli BL21.The recombinant strain BL21/ pET43.1a-ARC1 were induced by IPTG to express ARC1 fusion protein.The expression product were analyzed by SDS-PAGE,result showed that ARC1 fusion protein was expressed with molecular mass as expected at 135 kD.The primary analysis of inducing condition showed that inducing temperature has insignificant influence on expression of ARC1 fusion protein,but,IPTG concentration is little.So best-induced conditions were suggested as follows:temperature,32℃; concentration of IPTG,1.0mmol·L~(-1);time:4 hour.When expression process was finish,ARC1 was purified by MagneHis~(TM) Protein Purification System and analyzed by SDS-PAGE.The result showed the only ARC1 band in the gels,although ARClwas inclusion.
2 Cloning of coding sequence of SRK kinase domain from B.oleracea E1 and expression in E.coil BL21
2.1 Cloning and sequence analysis of SRK_(E1) gene
The coding sequence of SRK kinase domain(termed SRK_(E1)) was amplified from Brassica oleracea E1 in this paper.Sequence analysis showed that the amplified fragment of DNA and cDNA were 1 711bp and 1 241bp respectively,SRK_(E1) gene contained 6 extrons and 5 introns.The two ends of 5 introns complied with GT-AG rule.Alignment of the amino acid sequence of SRK_(E1) with SRK_6, SRK_(910),SRK_(18),SRK_(29) showed that the identity was 88.2%、85.7%、87.2%and 85.7%respectively. The amino acid sequence of SRK_(E1) began from 4~(th) amid acid of the transmembrane domain, including 16 amid acids of latter transmembrane domain and the whole kinase domain,Cysteine of transmembrane domain and Lysine of active-site have no mutation,showing the amplified cDNA fragment of SRK_(E1) could be used to express the kinase domain of SRK.Analyzing conserved domains of SRK_(E1) by Blastp showed that the coding region of SRK contained tyrosine kinase domain and protein kinase domain,and protein kinase domain localized between amino acid 78 and 361.The phylogenetic analysis showed that different SRK alleles were classified into two groups, and classⅡ,on the basis of their nucleotide sequences.ClassⅠS haplotypes represent highly self-incompatibility,classⅡS haplotypes represent poorly self-incompatibility,and SRK_(E1) clustered with SRK_(45)、SRK_3.So,Based on sequence diversity,the high self-incompatibility of E1 was demonstrated on molecular level.
2.2 Expression of SRK_(E1) in E.cali BL21
The plasmid pMD18-T-SRK_(E1) and pET43.1a were firstly extracted from each own strains,and then they were digested by restriction enzymes BamHⅠ/HindⅢ.After the SRK_(E1) cDNA and pET43.1a fragments were purified and recovered,they were ligated by T4 DNA ligase to construct expression plasmid pET43.1a-SRK_(E1),identified by PCR and two restriction enzymes digestion by BamHⅠ/HindⅢ,then pET43.1a-SRK_(E1) was transformed into E.coli BL21.The recombinant strain BL21/ pET43.1a-SRK_(E1) were induced by IPTG to express SRK_(E1) fusion protein.The expression product were analyzed by SDS-PAGE,result showed that SRK_(E1) fusion protein was expressed with molecular mass as expected at 74 kD,The primary analysis of inducing condition showed that IPTG concentration and inducing temperature only have a little influence on expression of SRK_(E1) fusion protein.SRK_(E1) fusion protein expressed in E.coli BL21 was mainly insoluble.
3 Establishment way of interaction in vitro between ARC1 and SRK_(E1)
Based on the principle of co-immunoprecipitation method,we put forward a new method in this study.The fusion protein of pET43.1a-SRK_(E1) contains a 6×His target,which can combine with Ni~+. The product of pET43.1a-SRK_(E1) combined with Ni~+ was used as 'bait',ARC1 solution as 'target'.
The mixture of 'bait' and 'target' is,then,incubated at 4℃.The protein complex was purified from mixture through the absorbability of Ni~+ with magnetic stand.In this study,via method,we have got perfect result.This suggests that this method is valuable to validate interaction between proteins. Compared with coimmunoprecipitation method,the new method shows advantages of antibody free, simple operation,and less fault result because of its weak specificity of antibody.In addition, constructed detection system is taken as a screening flat of chemical regulation reagents of the interaction between ARC1and SRK,which provides new information and clues for further analyzing mechanism of interaction and isolating and identifying unknown downstream targets in signal transduction pathway of self-incompatibility.
4 In vitro assay of the interaction between ARC1 and SRK_(E1)
The pET43.1a-SRK_(E1) fusion protein expressed in E.coli BL21 contained a 6×His-Tag,which can combine with Ni~+.SRK_(E1) fusion protein having combined with Ni~+ was used as 'bait'.The pET43.1a-ARC1 fusion protein expressed in E.coli was solved in buffer suited for interaction between ARC1 and SRK_(E1).The SRK_(E1) solution as a 'target','bait' and 'target' were incubated at 4℃for 2 h,mixing gently for every 15 min,then the complex of ARC1 and SRK_(E1) was purified by the affinity between Ni~+ and Magnetic stand,all products were analysis by SDS-PAGE.The result showed that ARC1 and SRK_(E1) could act with each other to combine and possibly form a stable complex,and the interaction was not bond momentarily.
甘蓝是典型的具有孢子体型自交不亲和性的芸薹属植物,这类SI在遗传上受一个带有复等位基因的多态性S基因座所控制。最近几年,对芸薹属自交不亲和性的决定性因子研究方面,取得了长足的进步。迄今为止,已分离到3类与S位点连锁的基因,分别编码SLG(S-locusglycoprotein,S位点糖蛋白)、S位点富半胱氨酸蛋白SCR(S-locus cystein-rich protein)/SPll(S-locus protein 11,S位点蛋白11)和SRK(S-locus receptor kinase,S位点受体激酶)。三个基因(SLG、SRK、SCR)的相继分离与鉴定,意味着由SRK介导的自交不亲和信号传导的上游事件越来越清晰了。来自花粉中的SCR/SP11在SLG的协助下与柱头上的跨膜蛋白SRK的胞外域相互识别,当两者具有相同单倍型时,SRK发生自我磷酸化,引起细胞内信号的级联反应,最后抑制自花花粉的萌发、水合或花粉管伸长。但是,对SRK介导的细胞信号传导下游磷酸化蛋白研究相对于上游受体来说要缓慢得多。在整个信号传导途径中,ARC1是下游事件中最为感兴趣的蛋白之一,它能够与SRK相互作用,并将信号传递给下游因子。因此在自交不亲和信号传导过程,SRK与ARC1的结合与否以及复合体的存在和变化是证实这个分子过程和调控自交不亲和反应的关键。为了探索这些问题,必须首先鉴定ARC1与SRK之间的相互作用,调控二者的解离与聚合,深入分析二者的作用机理,建立相互作用化控试剂的筛选平台,从而为下一步筛选ARC1的下游因子创造条件。
本研究以高度自交不亲和性甘蓝E1为材料,克隆了自交不亲和信号传导元件ARC1和SRK基因,并进行了表达分析。体外证实了ARC1与SRK的相互作用,建立了适用于SRK和ARC1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-ARC1复合体聚合与解离的人为调控提供理论和技术基础。主要工作及结果总结如下:
1甘蓝ARC1臂重复蛋白编码基因的克隆及其在大肠杆菌中的表达
1.1 ARC1基因的克隆与序列分析
采用PCR和RT-PCR技术,以甘蓝E1基因组DNA和柱头总RNA为模板,扩增了ARC1的DNA和cDNA序列,经测序和序列分析表明,DNA序列无内含子存在,扩增ARC1基因的编码序列为1 992 bp,编码663个氨基酸残基,分子量为73.5 kD。序列同源性分析表明,甘蓝E1的ARC1基因(B.oleracea E1 ARC1)与羽衣甘蓝ARC1基因(B.oleracea kale ARC1,NCBI登录号:EU344909)、油菜ARC1基因(B.napus ARC1,NCBI登录号:AF024625)、甘蓝20010197 ARC1基因(B.oleracea ARC1 20010197~([188]))、甘蓝ARC1基因(B.oleracea ARC1,NCBI登录号:AJ417335)、拟南芥中泛素蛋白连接酶(A.thaliana PUB17,NCBI登录号:NM102674)、水芹ARC1基因(A.thaliana cress ARC1,NCBI登录号:AY150512)的同源性分别为97%、94%、93%、95%、70%和70%。作为芸薹属5个基因,B.oleracea E1 ARC1、B.oleracea kale ARC1、B.oleracea ARC1 20010197、B.oleracea ARC1、B.napus ARC1之间的碱基差异很小,而作为十字花科中不同属的两个基因B.oleracea E1 ARC1和A.thaliana ARC1,在碱基上却存在相对较大的差异,编码的碱基差异大多数集中在ARC1的N端,说明ARC1基因存在较强的保守性,而且C端区域的保守性强于N端。在甘蓝与油菜的序列中均存在碱基的缺失,但这种缺失往往是以密码子形式的缺失,并没有引起移码突变。同源氨基酸序列进行比对显示,B.oleracea E1 ARC1基因编码的氨基酸序列与B.oleracea kale ARC1、B.oleraceaARC1 20010197、B.napus ARC1、A.thaliana ARC1所编码的氨基酸序列相似性分别为97.9%、97.6%、93.2%和53.2%。建立进化树后分析发现,B.oleracea E1 ARC1与B.oleracea kale ARC1、B.oleracea ARC1 20010197、B.oleracea ARC1的亲缘关系较近,而与B.napus ARC1、A.thalianaPUB17、A.thaliana cress ARC1的亲缘关系较远,更充分说明了ARC1基因的较强保守性,而且种间的保守性高于属间的保守性。在甘蓝E1的ARC1蛋白质序列中有一个U-box结构,5个臂重复序列。U-box结构位于第282个氨基酸和第347个氨基酸之间,臂重复序列编码42个氨基酸残基,油菜ARC1的U-box结构位于第283个氨基酸和第347个氨基酸之间。而在拟南芥中臂重复序列只有1个,这种较大的差异可能是导致ARC1作为与自交不亲和信号传导有关的基因在两个不同属之间的功能有所不同的原因。对甘蓝E1的ARC1蛋白的活性位点分析发现,ARC1蛋白中具有犰狳的ARM活性位点,该位点在芸薹属中的保守性很差,同时还发现其它磷酸化活性位点,包括10个蛋白激酶C磷酸化位点、1个亮氨酸拉链位点、2个基于cAMP-和cGMP蛋白激酶林磷酸化位点、11个酪蛋白激酶Ⅱ磷酸化位点、5个N-糖基化位点、8个N-肉豆蔻酰化位点、1个氨基化位点、1个酪氨酸激酶磷酸化位点,这些位点的存在与ARC1蛋白参与信号传导过程中依赖的磷酸化作用是相辅相成的。另外,拉链结构的存在,可能与ARC1的二聚化有关,或者是下一级信号传导元件的结合位点。ARC1蛋白的三维图形覆盖了400个氨基酸(第264位到第663位氨基酸),该段蛋白的结构整体上是右手螺旋,形成了类似海马状构型,腹部中空,形成大沟,背部形成小沟,小沟区域正是ARM结构域,可能为它与其他蛋白的结合提供了相应位点。
1.2 ARC1在大肠杆菌中的表达
提取pMD18-T-ARC1和pET43.1a质粒,经EcoRⅠ和KpnⅠ双酶切,纯化回收ARC1和pET43.1a片段,用T4 DNA Ligase连接以构建表达质粒pET43.1a-ARC1,经PCR以及EcoRⅠ和KpnⅠ双酶切鉴定。提取pET43.1a-ARC1质粒,转化表达宿主菌BL21,IPTG诱导表达ARC1融合蛋白,表达产物的SDS-PAGE结果显示,在约135 kD处有重组蛋白表达特异带出现,其蛋白质相对分子质量与预计值相符。在诱导的过程中,IPTG浓度影响不大,但诱导温度(℃)对ARC1蛋白的表达影响很大,最佳诱导条件为:温度32℃;IPTG 1.0 mmol·L;时间4 h。诱导表达的融合蛋白ARC1主要是以包涵体的形式存在,但通过细胞破碎及蛋白纯化试剂盒纯化后,纯化产物经SDS-PAGE检测,泳道基本没有杂蛋白条带,纯化结果比较理想。
2甘蓝SRK激酶结构域编码序列的克隆与其在大肠杆菌中的表达
2.1 SRK_(K1)基因的克隆与序列分析
以甘蓝E1为材料,特异扩增SRK激酶结构域编码区序列(SRK_(E1))。测序结果显示扩增的DNA序列和cDNA序列分别为1 711 bp和1 241 bp,DNA序列包含6个外显子、5个内含子,内含子大小分别为89 bp、107 bp、89bp、102 bp和78 bp。5个内含子的5'和3'端完全符合经典的GT-AG法则。同源氨基酸序列比对表明,SRK_(E1)与sRK_6、SRK_(910)、SRK_(18)、SRK_(29)氨基酸序列同源性分别为88.2%、85.7%、87.2%和85.7%。SRK_(E1)的氨基酸序列包含了跨膜结构域的后16个氨基酸和整个激酶结构域,跨膜结构域的Cys和激酶活性位点Lys并没有发生突变。用Blastp程序对SRK_(E1)的氨基酸序列进行保守结构域分析发现,该序列中存在蛋白激酶结构域和酪氨酸激酶结构域。蛋白激酶结构域位于第78位氨基酸和第361位氨基酸之间,编码284个氨基酸。在系统进化分析中发现,不同单元型SRK依据序列同源性高低分为两类(ClassⅠ和ClassⅡ),ClassⅠ表现为高度自交不亲和,ClassⅡ(SRK_(15)、SRK_(60)、SRK_2、SRK_(29)和SRK_5)的自交不亲和性较弱,SRK_(E1)与SRK_(45)、SRK_3聚类在一起。因此,依据不同单元型SRK序列的多态性从分子层面上证明了E1材料的高度自交不亲和性。
2.2 SRK_(E1)在大肠杆菌中的表达
提取pMD18-T-SRK_(E1)和pET43.1a质粒,经BamHⅠ和HindⅢ双酶切,纯化回收SRK_(E1)和pET43.1a片段,用T4 DNA Ligase连接以构建表达质粒pET43.1a-SRK_(E1),经PCR以及BamHⅠ和HindⅢ双酶切鉴定。提取pET43.1a-SRK_(E1)质粒,转化表达宿主菌BL21,IPTG诱导表达SRK_(E1)融合蛋白,表达产物的SDS-PAGE结果显示,在约107 kD处有重组蛋白表达特异带出现,IPTG的浓度和诱导温度对该蛋白表达量的影响不大,且表达的SRK_(E1)融合蛋白主要包涵体形式存在。SRK_(E1)融合蛋白最佳的诱导表达条件为:温度37℃;IPTG 0.5 mmol·L~(-1);时间4h。
3 ARC1与SRK_(E1)体外相互作用的方法建立
免疫共沉淀法鉴定蛋白质相互作用原理表明,蛋白质X与蛋白质Y相互作用,如蛋白质X与其抗体免疫沉淀,则蛋白Y会与蛋白质X及其抗体一起沉淀下来。依据该原理利用pET43.1a作为表达载体表达ARC1、SRK_(E1),巧妙利用了pET43.1a-ARC1、pET43.1a-SRK_(E1)融合蛋白序列中的6×His标签与Ni~+相结合的特点,探索性地建立了体外检测蛋白质间相互作用的新方法。将pET43.1a-SRK_(E1)融合蛋白与Ni~+结合,结合后的复合物作为“诱饵蛋白”,把体外表达的ARC1融合蛋白溶于适于相互作用的缓冲液中作为“靶蛋白”溶液,两者4℃条件下孵育后利用pET43.1a-SRK_(E1)上结合的Ni~+与磁力架吸附性将“诱饵蛋白”与靶蛋白的复合体纯化出来。本研究对ARC1与SRK_(E1)相互作用的检测,条带清楚,明确证实了两者相互作用,得到了理想的结果,说明我们建立的体外检测蛋白相互作用方法是可行的。本方法与免疫共沉淀法鉴定蛋白质相互作用相比,不需要制备抗体,简化了实验操作,避免了因抗体特异性不强可能带来的假阳性结果。另外本文建立的检测体系可作为ARC1与SRK相互作用的化控试剂筛选平台,从而为深入分析两者相互作用的机理以及分离和鉴定自交不亲和未知的下游信号传导元件提供新的信息与线索。
4 ARC1与SRK_(E1)相互作用的体外检测
将在大肠杆菌BL21中诱导表达的pET43.1a-SRK_(E1)融合蛋白Ni~+结合,结合后的复合物作为“诱饵蛋白”。将在大肠杆菌BL21中诱导表达的pET43.1a-ARC1融合蛋白作为“靶蛋白”,并溶解在适于相互作用的缓冲液中,“诱饵蛋白”和“靶蛋白”溶液在4℃条件下孵育2 h,每隔15 min轻轻翻转混匀一次,然后利用pET43.1a-SRK_(E1)融合蛋白结合的Ni~+与磁力架吸附性将诱饵蛋白和蛋白的复合体纯化出来,产物经SDS-PAGE检测,结果表明ARC1蛋白与SRK_(E1)蛋白在体外能够相互结合,可能形成稳定的复合体,同时也说明二者的相互作用并非产生于瞬间的结合。
Many bisexual flowering plants possess a reproductive strategy called self-incompatibility(SI) that enables the female tissue(the pistil) to reject self but accept non-self pollen for fertilization.SI response represents cell signal transduction process that allows the pistil to distinguish between self and non-self pollens and prevented self pollens germination.Pollination and fertilization in flowering plants involve a series of complex events with tightly regulated cell-cell interactions and signaling between the pollen and the pistil.More than half flowering plants possess a SI and involve 70 family,250 genus.
Brassica oleracea L.is a classical Brassicaceae plant with the sporophytic self-incompatibility that was controlled by a single multi-allelic S locus genetically.In recent years,much progress has been made in determining the male and female S determinant in Brassica species.To date,in the signal transduction pathway of self-incompatible Brassica,three highly polymorphic genes with linked S locus have been identified.One is the S locus glycoprotein(SLG) gene that encoded a secreted glycoprotein abundantly present in the papillar cell of the stigma surface,one is S-locus receptor kinase(SRK)gene,the other is S-locus cystein-rich(SCR) gene.One after the other,three gene(SLG、SRK、SCR) isolated and characterized implies that upstream events of the SRK-mediated signal transduction pathway are more perspicuous.In the signal transduction pathway,ARC1 is a most interesting target that can interact with SRK specifically and transfer self-incompatible signal. Therefore,ARC1 band to SRK or not and stability of the formed compound is the key to regulate SI responses.In order to answer the former questions,we test the interaction between ARC1 and SRK, further analyze interaction mechanism,regulate separation ang aggregation of ARC1-SRK,and establish a screening system for chemical regulation of the interaction.Sequentially,these studies provide condition for further screening downstream targets of ARC1.
In this paper,the highly self-incompatible Brassica oleracea E1 was taken as plant material. ARC1 and SRK were cloned,expressed respectively and in vitro confirmed interaction between interaction ARC1 and SRK.A detection system between the two determinates was established.By the research we provide the theoretical and technical foundation for further selecting controlled reagent and artificially regulating the aggregation and dissociation of SRK-ARC1 complex.The main results were as below:
1 Cloning of coding sequence of ARC1 from B.oleracea E1 and expression in E.coli BL21
1.1 Cloning and sequence analysis of ARC1 gene
The DNA and cDNA fragments of ARC1 were amplified from genomic DNA and stigma total RNA of Brassica oleracea E1 by PCR and RT-PCR methods.Sequencing assay demonstrated that no intron existed in DNA sequences,and the cDNA of ARCl was 1,992 bp in length,encoding 663 amino acids,molecular weight was 73.5 kD.The nucleotide sequence of B.oleracea E1 ARC1 respectively showed 97%,94%,93%,95%,70%and 70%sequence identity to B.oleracea kale ARC1(Accession number in NCBI is EU344909),B.napus ARC1(Accession number in NCBI is AF024625),B.oleracea 20010197ARC1,A.thaliana PUB17(Accession number in NCBI is NM102674),and A.thaliana ARC1(Accession number in NCBI is AY150512).Little difference was showed in the bases of five ARC1 alleles(B.oleracea E1 ARC1,B.oleracea kale ARC1,B. oleracea 20010197ARC1,B.oleracea ARC1 and B.napus ARC1),but more difference in N-terminal of B.oleracea E1 ARC1 and A.thaliana ARC1.This explained that the C-terminal of ARC1 had more conservative than N-terminal.In addition to there were miss bases in the nucleotide sequence of B.oleracea and B.napus,and this miss bases with 'codon' didn't bring code mutation.The alignment amino acid sequence of B.oleracea E1 ARC1 respectively showed 97.9%,97.6%,93.2% and 53.2%sequence identity to B.oleracea kale ARC1,B.oleracea ARC1 20010197,B.napus ARC1 and A.thaliana ARC1.Phylogenetic tree of ARC1 proteins showed that B.oleracea E1 ARC1 was the closest to B.oleracea kale ARC1,B.oleracea ARC1 and B.oleracea ARC1 20010197,but the furthest to A.thaliana PUB17、A.thaliana cress ARC1.Database searches with both the DNA and amino acid sequences revealed that there were five arm repeat domains of C-terminal encoded amino acid 42 and a U-box domain that was located between amino acid 282~(th) and 347~(th) in B. oleracea E1 ARC1.In contrast to B.oleracea E1 ARC1,five arm repeat domains and a U-box domain that was located between amino acid 283~(th) and 347~(th) existed in B.napus ARC1,only an arm repeat in A.thaliana cress ARC1 and A.thaliana PUB17.The significant difference potentially determined the function discrepancy of ARC1in B.oleracea and A.thaliana.Prediction of active site of ARC1 showed that B.oleracea E1 ARC1contained active site of armadillo and the active site motif were poorly conservative in B.oleracea.In addition to ARC1 also contained Protein kinase C phosphorylation site,cAMP-and cGMP-dependent protein kinase phosphorylation site,Casein kinaseⅡphosphorylation site,Leucine zipper site,N-glycosylation site,N-myristoylation site, Amidation site,Tyrosine kinase phosphorylation site.They probably took part in the signal transduction of SI.In ARC1 protein,the three dimension models covered the 264-663 amino acid residues which wereα-helix and formed a hippocampus-shaped configuration and a wide groove and a narrow groove,which may be the binding sites of other proteins.
1.2 Expression of ARC1 in E.coli BL21
The plasmid pMD18-T-ARC1 and pET43.1a were extracted,and then they were digested by restriction enzymes EcoRⅠand KpnⅠfor purification and recovering of ARC1 cDNA and pET43.1a fragments.Two fragments were ligated by T4 DNA ligase to construct expression plasmid pET43.1a-ARC1,identified by PCR and two restriction enzymes digestion by EcoRⅠ/ KpnⅠ,then pET43.1a-ARC1 was transformed into E.coli BL21.The recombinant strain BL21/ pET43.1a-ARC1 were induced by IPTG to express ARC1 fusion protein.The expression product were analyzed by SDS-PAGE,result showed that ARC1 fusion protein was expressed with molecular mass as expected at 135 kD.The primary analysis of inducing condition showed that inducing temperature has insignificant influence on expression of ARC1 fusion protein,but,IPTG concentration is little.So best-induced conditions were suggested as follows:temperature,32℃; concentration of IPTG,1.0mmol·L~(-1);time:4 hour.When expression process was finish,ARC1 was purified by MagneHis~(TM) Protein Purification System and analyzed by SDS-PAGE.The result showed the only ARC1 band in the gels,although ARClwas inclusion.
2 Cloning of coding sequence of SRK kinase domain from B.oleracea E1 and expression in E.coil BL21
2.1 Cloning and sequence analysis of SRK_(E1) gene
The coding sequence of SRK kinase domain(termed SRK_(E1)) was amplified from Brassica oleracea E1 in this paper.Sequence analysis showed that the amplified fragment of DNA and cDNA were 1 711bp and 1 241bp respectively,SRK_(E1) gene contained 6 extrons and 5 introns.The two ends of 5 introns complied with GT-AG rule.Alignment of the amino acid sequence of SRK_(E1) with SRK_6, SRK_(910),SRK_(18),SRK_(29) showed that the identity was 88.2%、85.7%、87.2%and 85.7%respectively. The amino acid sequence of SRK_(E1) began from 4~(th) amid acid of the transmembrane domain, including 16 amid acids of latter transmembrane domain and the whole kinase domain,Cysteine of transmembrane domain and Lysine of active-site have no mutation,showing the amplified cDNA fragment of SRK_(E1) could be used to express the kinase domain of SRK.Analyzing conserved domains of SRK_(E1) by Blastp showed that the coding region of SRK contained tyrosine kinase domain and protein kinase domain,and protein kinase domain localized between amino acid 78 and 361.The phylogenetic analysis showed that different SRK alleles were classified into two groups, and classⅡ,on the basis of their nucleotide sequences.ClassⅠS haplotypes represent highly self-incompatibility,classⅡS haplotypes represent poorly self-incompatibility,and SRK_(E1) clustered with SRK_(45)、SRK_3.So,Based on sequence diversity,the high self-incompatibility of E1 was demonstrated on molecular level.
2.2 Expression of SRK_(E1) in E.cali BL21
The plasmid pMD18-T-SRK_(E1) and pET43.1a were firstly extracted from each own strains,and then they were digested by restriction enzymes BamHⅠ/HindⅢ.After the SRK_(E1) cDNA and pET43.1a fragments were purified and recovered,they were ligated by T4 DNA ligase to construct expression plasmid pET43.1a-SRK_(E1),identified by PCR and two restriction enzymes digestion by BamHⅠ/HindⅢ,then pET43.1a-SRK_(E1) was transformed into E.coli BL21.The recombinant strain BL21/ pET43.1a-SRK_(E1) were induced by IPTG to express SRK_(E1) fusion protein.The expression product were analyzed by SDS-PAGE,result showed that SRK_(E1) fusion protein was expressed with molecular mass as expected at 74 kD,The primary analysis of inducing condition showed that IPTG concentration and inducing temperature only have a little influence on expression of SRK_(E1) fusion protein.SRK_(E1) fusion protein expressed in E.coli BL21 was mainly insoluble.
3 Establishment way of interaction in vitro between ARC1 and SRK_(E1)
Based on the principle of co-immunoprecipitation method,we put forward a new method in this study.The fusion protein of pET43.1a-SRK_(E1) contains a 6×His target,which can combine with Ni~+. The product of pET43.1a-SRK_(E1) combined with Ni~+ was used as 'bait',ARC1 solution as 'target'.
The mixture of 'bait' and 'target' is,then,incubated at 4℃.The protein complex was purified from mixture through the absorbability of Ni~+ with magnetic stand.In this study,via method,we have got perfect result.This suggests that this method is valuable to validate interaction between proteins. Compared with coimmunoprecipitation method,the new method shows advantages of antibody free, simple operation,and less fault result because of its weak specificity of antibody.In addition, constructed detection system is taken as a screening flat of chemical regulation reagents of the interaction between ARC1and SRK,which provides new information and clues for further analyzing mechanism of interaction and isolating and identifying unknown downstream targets in signal transduction pathway of self-incompatibility.
4 In vitro assay of the interaction between ARC1 and SRK_(E1)
The pET43.1a-SRK_(E1) fusion protein expressed in E.coli BL21 contained a 6×His-Tag,which can combine with Ni~+.SRK_(E1) fusion protein having combined with Ni~+ was used as 'bait'.The pET43.1a-ARC1 fusion protein expressed in E.coli was solved in buffer suited for interaction between ARC1 and SRK_(E1).The SRK_(E1) solution as a 'target','bait' and 'target' were incubated at 4℃for 2 h,mixing gently for every 15 min,then the complex of ARC1 and SRK_(E1) was purified by the affinity between Ni~+ and Magnetic stand,all products were analysis by SDS-PAGE.The result showed that ARC1 and SRK_(E1) could act with each other to combine and possibly form a stable complex,and the interaction was not bond momentarily.
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