【作者】 顾洪雁；

【导师】 亓宝秀； 常维山；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 G蛋白是普遍存在于真核生物细胞中的一个与GTP结合蛋白家族,与膜受体偶联(Heterotrimeric GTP binding proteins),由α,β,γ三个亚基组成异三聚体复合物,分子量100kDa左右。哺乳动物目前发现23种左右的Gα,6种Gβ,14种Gγ蛋白,每个亚基都由多基因编码,从而构成多种多样的可能组合方式。目前已在豌豆、玉米、菠菜、拟南芥、燕麦、大豆、大麦、水稻等多种植物细胞中检测到了G蛋白的存在。植物中G蛋白数目很少,任一G蛋白亚基在各种植物中只有1到2个成员。如拟南芥中只有1个Gα,1个Gβ和2个Gγ。相互作用的只有少数几个蛋白,包括与Gα互作的AtPirinl (PRN1)、prephenatedehydratase1(PD1)、THYLAKOID FORMATION1(THF1)和PLDa1,以及与Gβ互作的N-MYCDOWN-REGULATED1(NDL1)。植物G蛋白的空间结构目前了解极少,主要是参考动物的G蛋白进行推测。Ga亚基有两个独立区域组成,一个区域(GTPase区)含有GTP结合域,这个区域参与GTPase结合。另一个区域即完整的α螺旋区域,它是由一个长的中心螺旋和5个短的螺旋组成,该区域是识别效应物的区段。目前对拟南芥G蛋白的了解很有限,需要进一步研究G蛋白的结构与功能,信号转导机制,及其相互作用因子。因此我们发展了含有连接肽的串联亲和层析技术(命名为TAPa-LP),用以研究拟南芥G蛋白的互作因子,蛋白结构及其生理作用。串联亲和纯化(tandem技术通过靶蛋白质一端嵌入一个特殊的蛋白质标签(TAP tag),不破坏靶蛋白质序列,经过两步连续的亲和纯化获得接近自然条件的特定蛋白质复合体,然后用质谱技术或Edman降解法进行蛋白质鉴定。融合蛋白的功能可能改变或丧失(有的不影响),这也是TAP方法固有的一个缺点,解决的办法是增加一个柔性连接肽,使两边的蛋白质空间结构各自独立,减少干扰。本实验设计的连接肽为GGGGSGGGGGS,核酸序列为GGAGGAGGAGGATCAGGA GGAGGAGGAGGATCA,并构建了5套载体：p2300EC-GPT, p2300EC-GT, p2300EC-QT, p2300EC-QPT和p2300EC-FPT,这5种类型中有3种有连接肽,另2种不含连接肽。TAPa-LP系统揭示了G蛋白在植物的发育过程起着极其复杂的作用。我们发现在叶、茎、花和果实的发育过程中GTP-Ga是一种正调控因子,而GTP-Ga在种子花青素和根的发育过程中为负调控因子。Gaβγ在主根、茎和果实的发育过程是正调控因子,而在叶片的表皮细胞横向增值的过程中起着负调控作用。也就是说在拟南芥发育过程中,GTP-Ga在不同的组织分别发挥4种正调控和2种负调控作用。三聚体Gap丫发挥着3种正调控和1种负调控作用。Gaβγ和GTP-Ga在根和叶的发育中起着相反的调控作用。我们推测·Gaβγ和GTP-Ga的比值影响着或强烈影响植物的生长。在gpal和fpt中GPA1的缺失导致Gapy和GTP-Ga两者均缺失,植物的生长并没有受到明显的影响,这可能是因为G蛋白参与的正负调控的双双缺失造成的。这也说明G蛋白在植物的发育过程中并不是主要的调控元件,可能存在着另外的不为G蛋白参与的调控模式。gpt在T2代的类型中长的高大而粗壮,可能是35S启动子较强的功能提高了Gaβγ和GTP-Ga的含量。qpt在T1代生长健壮但在T2代生长的极其柔弱,主根几乎不再生长,最终是全体死亡。其原因极可能是T1代的激素水平不同于T2种子的水平。看来植物生长的调控不仅依赖于Gaβγ和GTP-Ga的含量,而且也依赖于激素水平,不同的植物激素进行着复杂的植物的生长调控。通过TAPa或linker-TAPa对Ga功能的干涉情况,来判断Ga亚基在空间结构上调控区域所在的空间位置。从实验情况来看,TAPa或linker-TAPa标签有时会影响Ga的功能。Ga的三维结构呈现出两个独立的区域,根据SWISS-MODEL预测的融合蛋白的结构,呈现出三个独立的区域,也就是比Hamm报道的多了TAPa区域。当G、GPT、 QT和QPT中的一种或2种或3种能明显引起拟南芥某种表型发生变化时,调控区域可能定位在C端区域,因为TAPa干扰了G蛋白的功能,靠近C端最有可能的调控区是A stem, I primary root, A primary root, A silique size, I Seed color和A leaf。Gβγ二聚体结合在Ga的N端区域,功能区多在C端。在筛选拟南芥G蛋白α亚基GPA1蛋白互作因子过程中,成功获取了Gγ1和Gβ两个互作因子,并首次获取了完整的G蛋白,从而进一步明确了各亚基之间的相互作用的关系。用改良的含有连接肽或不含连接肽的TAPa纯化系统对融合蛋白空间结构的影响上,可以获取一定的空间结构信息,结合蛋白质空间结构的预测模型和理论,可以探索蛋白质的结构与功能的关系。用改良的含有连接肽或不含连接肽的TAPa纯化系统建立的转基因体系中,可以根据各基因型相同与不同的变化来观察表型的对应变化,从而推知基因可能发挥的某项生理功能。总之,含有连接肽的蛋白纯化系统主要有三个方面的作用：一是其主要的功能就是进行互作因子的鉴定；其次是推知诱饵基因可能的生理功能；三是探知诱饵蛋白的结构信息,这一作用仍是摸索性的,是否可行需要进一步探讨。由于蛋白质空间结构的极端复杂性,科学技术研究手段的局限性(较为常用的方法有x射线晶体衍射方法、NMR等),以及蛋白质预测理论的停止不前,到目前为止,仍然没有大的进展。我们发现了蛋白质在磁场作用后吸光度的变化,根据此变化规律发展了物理学中的光学定律,并试图通过建立数学模型来预测蛋白质的空间结构。单色光通过磁化的蛋白质溶液时,在不同方向所测的吸光度竟然有不同的值,这种现象不符合朗伯-比尔定律。通过实验我们推知蛋白质在磁场中发生了偏转并进行了定向。推导出吸光度A不仅与蛋白质浓度与比色皿内径成正比,而且与蛋白质分子的受光面积成正比,这就大大发展了朗伯-比尔定律(A=K·C·b·SS)。对任何一种蛋白溶液,我们根据新的朗伯-比尔定律,依据MATLAB7.0.1可方便的绘制出蛋白质的三维构象。溶菌酶与牛血清白蛋白的分子形状我们已绘制出来。蛋白质空间结构的研究仍然是当今生命科学的热点和难点。我们发展出一种新的探知蛋白质空间结构的方法,同时也说明磁场对蛋白质确实具有明显的作用,但与当前磁场对细胞或生物体的影响的理论解释不同,这为电磁学对生物的作用机理的探讨提供了一种新的思路。我们相信新的朗伯-比尔定律的运用,在光学、电磁学与分子生物学领域将会具有重要意义。总之,我们发展了TAP-LP纯化系统,对拟南芥G蛋白的复合体进行了成功的纯化,该方法优势在于极大的克服了(仍不能完全克服)以往TAP干扰诱饵蛋白的弊端,提高了成功的可能性。并且发展了物理学中的光学定律(朗伯-比尔定律),通过建立数学模型已成功的预测出BSA和溶菌酶的三维结构。更多还原

【Abstract】 The G-proteins act as critical molecular switches in diverse signal transduction pathways in eukaryotes. Similarly, in plant, the G-proteins play regulatory roles in multiple developmental processes ranging from seed germination and early seedling development to root development and organ shape determination. The repertoire of G-protein signaling complex is much simpler than in metazoans. Specifically, Arabidopsis, has only one canonical G alpha, one G beta, and two G gamma subunits, only one Regulator of G-protein Signaling (RGS) protein and several proteins, including AtPIRIN1, PLD1, PD1, and THF1. Ga subunits contain two domains, a domain involved in binding and hydrolyzing GTP and a unique helical domain that buries the GTP in the core of the protein. We only have known about a preliminary of the spatial structure of G-proteins in metazoans, but know little about spatial structure of G-proteins in plant. So future studies are expected to identify the mechanisms by which G-proteins regulate phenotypic and developmental plasticity and to clarify of structure-function relationships in G-proteins or Ga. People urgently need some new methods of Arabidopsis protein complex isolation.Therefore, we describe the application of tandem affinity purification-linker peptide (TAP-LP) strategy to the study of Ga subunit in Arabidopsis.Rigaut described the tandem affinity purification (TAP) system in yeast, a number of studies have arisen in recent years demonstrating the applicability of such a system in many other different organisms, such as mammalian cells, insect cells, tobacco leaves et al., TAP tags sometimes not only affect bait protein function, but also can react to or bind with other proteins. It is a serious problem if the protein that you want to produce is TAPa fusion proteins. But we have obtained important information that regulatory domains are located in G-protein N-terminal region or C-terminal region because tag protein is due to interference with the function of N-terminal region or C-terminal region of bait protein.In order to take full advantage of this situation, we suggest TAP system construct two types:one is a linker sequence between a bait protein and TAPa, the other is no linker sequence between a bait protein and TAP tags. The design of linker or no linker may play an important part in the fusion protein. Linker peptide can independently fold protein domains in a fusion construct.We had to try a few candidate sequences (3-20aa long) with varying degrees of flexibility/rigidity by computer. Sequences made up of Gly and Ser. By varying the number of Gly-Ser pairs we can modulate the flexibility of the linker. Ultimately, we have designed a linker peptide, GGGGSGGGGGS, its nucleic acid sequence is GGAGGAGGAGGATCA GGAGGAGGAGGAGGATCA, then we have constructed5vectors composed of linker or no linker. Our results demonstrate that the TAPa-linker peptide (TAPa-LP) system sometimes can enhance the activity of TAP fusion proteins in Arabidopsis.The TAPa-LP system provides us with a new research method of Ga subunit in Arabidopsis. Subsequently, we forecasted three-dimensional structure of fusion proteins by’ SWISS-MODEL. TAPa-LP system revealed that G-proteins play a complex regulation role in the different stage in plant development.We found that GTP-Ga is a positive regulator in leaf, stem, flower and silique development whereas GTP-Ga is a negative regulator in primary root and anthocyanins synthesis in seed, while heterotrimeric complex is a positive regulator in primary root, stem, and silique development, whereas heterotrimeric complex is a negative regulator for cell proliferation in leaf blade’s width direction. That is to say, GTP-Gα act as4positive regulators and2negative regulators, and heterotrimeric complex act as3positive regulators and1negative regulators. GTP-Gα and heterotrimeric complex play reversal roles in the root and leaf development. We speculate that the ratio of GTP-Gα and Gaβγ impact on or strongly impact on the growth of plants. Lack of GTP-Gα and Gaβγ in gpa1-4and fpt do not obviously showed a significant difference in plant growth, suggesting that lack of positive and negative regulation of G protein does not affect the growth of plants, and G-proteins is not the primary regulation of plant growth in the major regulation components. gpt is the highest among all plant types in the T2plants because gpt with35S promoter-GPA1promote the ratio of GTP-Gα and Gaβγ.The size of qpt plant morphology appeared to show tall and strong in T1stage but qpt have mostly died and a few grew very weak at an early age in the T2stage, while the size of gpt plant morphology grew normal size in T1stage but gpt is the strongest and highest among all plant types in the T2plants in the early age, hormone level of T1seed is different from level of T2seed. So regulations of plant growth depend not only on the ratio of GTP-Ga and Gaβγ but also on the hormone level. Plant growth is regulated by the plant hormones gibberellins (GA), brassinosteroids (BR), abscisic acid (ABA) and auxin (Ueguchi-Tanaka et al.,2000; Ullah et al.,2002), but gpt and qpt appeared to show the greatest differences between T1and T2stage. We call this phenomenon as the elongation effects of hormone, because hormones in seed of transgenic plant come from parental generation. We speculated that a model of G-protein’s role mechanism perhaps is dependent on the hormone signal transduction pathway or/and affect hormone synthesis. However, in Arabidopsis, we expected to reveal more components of the heterotrimeric G-protein signal transduction pathways, and to identify the mechanisms by which G-proteins regulate phenotypic and developmental plasticity.Our studies showed that TAPa or linker-TAPa sometimes impaired bait protein function in G-protein-TAPa or G-protein-linker-TAPa fusion proteins, previous studies also reported this problem, such as FHY1and HFR1. It is a serious problem if the protein that you want to produce is TAPa fusion proteins. But we have obtained important information that regulatory domains are located in G-protein region because tag protein is due to interference with the function of bait protein. It is now known that Ga display two independent regions in the three dimensional structure. In terms of logic, regulatory domain is likely located in the C-terminal region of Ga when one, two or three of GT, GPT, QT and QPT play a role in terms of G-protein phenotypic, Such as A stem, I primary root, A primary root, A silique size, I seed color and A leaf.It is well known that N-terminal region of G-protein can bind the Gβγ dimmer, so we speculated that regulatory domains are liable to be in the C-terminal region of G-protein rather than in the N-terminal region. Of course, this model needs a lot of experiments to be further validated.Several known interactions involving Ga were confirmed, that is to say, TAPa-LP system revealed that several interactions of Arabidopsis G-proteins have one Gp and one Gγ1. Point out with great regret, we have no found many novel potential interactions.We conclude that TAPa-LP, in combination with MS, can be used as an effective method for the studies of the Arabidopsis G-proteins.When monochromatic light passes through a homogeneous absorbing medium, the absorbance is proportional to the growth of concentration and thickness of the medium, which is Lambert-Beer law. The shade selection of protein solution magnetized for certain time from different angles makes different absorbance, which does not meet the Lambert-Beer law. Accordingly, we derive that the absorbance A is not only proportional to the concentration and thickness of the medium, but also proportional to the light area Ss of certain direction. For the same protein solution, we can obtain the absorbance A of six directions, and thus get six SS, the relative ratio of which will inevitably reveal plentiful information of the protein shape. The conformation of protein can be easily drawn out by software (MATLAB7.0.1) developed by computer. We have drawn out the molecular shape of lysozyme and bovine serum albumin. In brief, we have developed Lambert-Beer law (A=K-C-b-Ss) and a new method of exploring protein spatial structure.To sum up, we have developed TAPa-LP purification strategy applied to Arabidopsis protein complex isolation. We have developed Lambert-Beer law (A=K·C·b·Ss) and a new method of exploring protein spatial structure.更多还原