摘要
植物韧皮部作为植物维管束重要的组成部分,不仅充当着同化碳水化合物的运输通道,更因其内含激素、活性氧、钙离子、RNA与蛋白等信息物质而充当着协调植物系统生长与发育的信息高速公路。植物维管系统对于植物响应环境逆境变化也起着举足轻重的作用,深入了解植物韧皮部长距离信号物质及其作用可提供提高农作物生产率的手段。南瓜因其维管束分明,容易采集韧皮液而成为研究维管束系统最好的模式植物之一。目前其韧皮液内RNA转录组与蛋白质组已获得,但是我们对其韧皮液内上千RNA与蛋白的研究还只是冰山一角,仍有很多问题尚待研究。本实验室此前研究发现了RNA结合蛋白CmRBP50在南瓜韧皮液内形成了一个核糖核蛋白(ribonucleoprotein, RNP)复合体,这是目前为止在植物中获得的第一个能进行长距离运输的核糖核蛋白复合体,但是关于此复合体的形成机制还不清楚。本文以南瓜(Cucurbita maxima cv Big Max)为材料,通过生物化学、分子生物学等手段研究了南瓜韧皮液内可能的mRNA结合蛋白及基于CmRBP50核糖核蛋白复合体的形成分子机理。取得主要结果如下：
1.运用蛋白质组学手段研究了南瓜韧皮液内可能的mRNA结合蛋白。通过Poly (U)亲和柱结合液相串联质谱(LC-MS/MS)技术获得了南瓜韧皮液内29个潜在的mRNA结合蛋白。其功能包括与RNA代谢、翻译相关,与蛋白代谢、运输相关,与细胞结构、能量产生相关,与代谢相关及一些功能未知的蛋白。
2.研究了CmRBP50的磷酸化位点及其对于CmRBP50RNP形成的重要性。首先通过蛋白overlay技术明确了磷酸化修饰对基于CmRBP50RNP形成的重要性,然后通过液相串联质谱鉴定获得了四个磷酸化位点丝氨酸223、438、440和444。通过点突变及小西葫芦黄化花叶病毒(Zucchini yellow mosaic virus, ZYMV)表达系统表达并纯化获得了CmRBP50及S223A(丝氨酸223突变为丙氨酸),STripleA(丝氨酸438、440、444分别突变为丙氨酸),SQuadA(丝氨酸223、438、440、444分别突变为丙氨酸)等点突变蛋白。蛋白overlay与免疫共沉淀技术证实了以上磷酸化位点对于此RNP形成的重要性,尤其是C端Ser438、440和444突变为丙氨酸后可充分剥离CmRBP50与其他蛋白间的互作。最后,我们克隆并表达了CmRBP50的潜在互作蛋白,运用体外pull down技术证实CmRBP50与CmPP16, GTPbP及PSPL可发生体外互作,且其C端的磷酸化修饰对其互作至关重要。
3.研究了可能磷酸化CmRBP50的激酶。通过运用胶内磷酸化(In gel kinase assay)技术从维管束蛋白与韧皮液蛋白着手,鉴定了可能磷酸化CmRBP50的激酶。通过对可疑位点质谱鉴定我们得到核苷二磷酸激酶,丝／苏氨酸激酶,蛋白激酶adkl和蛋白激酶Csa000515及糖原合成酶激酶-3等可能的激酶,经论证推测最有可能的是糖原合成激酶-3。
本文通过对韧皮液内mRNA结合蛋白的初步鉴定及对首个韧皮液内发现的能进行长距离运输的基于CmRBP50的RNP形成机制研究,将为揭开CmRBP50在韧皮液中介导的生理功能打下基础,同时为研究韧皮液内其他RNP提供依据。本研究最终不仅有助于我们揭示不同器官间的通讯机制或信号转导机制,丰富植物生理学、生态学和园艺学的知识,也可为我们利用植物的通讯机制来应用到农业生产,探索出一条提高蔬菜的抗性、产量及品质的新途径,这具有十分重要的科学与现实意义。
Phloem is an important part of plant vascular system, which not only plays key roles in translocating assimilated carbohydrates, but also serves an information superhighway in coordinating with plant growth and development as it is rich in signal moleculars such as hormones, redox oxygen species, Ca2+, RNAs and proteins. Plant vascular system is also important for plants in response to environmental changes, thus understanding phloem long-distance substances and their functions would help us in improving agricultural productivity. Pumpkin(Cucurbita maxima cv Big Max) is one of the best model plants to study vascular biology as it has distinct vascular system and is easy to collect phloem sap. At present, its phloem transcriptome and proteome have been investigated, but little is known about those thousands of RNAs and proteins, still many questions wait for further study. Our previous work identified a RNA binding protein CmRBP50 which formed a ribonucleoprotein (RNP) complex in pumpkin phleom sap. This is the first RNP identified in plants which can undergo long-distanced movement, however, the molecular mechanism remained to be characterized. In this paper, pumpkin was used as material, and molecular biology, biochemistry techniques were applied to study the putative mRNA binding proteins and the molecular mechanism of CmRBP50 based RNP formation. Results were obtained as follows:
1. Proteomic analysis of putative mRNA binding proteins in pumpkin phloem sap. Poly(U) affinity column and LC-MS/MS analysis were applied and 29 putative mRNA binding proteins were identified from pumpkin phloem sap. They were RNA metabolism, translation or protein metabolism, transport or cell structure, energy production or metabolism as well as some unknown functions related.
2. We examined the phosphorylation sites on CmRBP50 and the importance of phosphorylation to the formation of CmRBP50 based RNP complex. Firstly, protein overlay assay was applied to confirm the importance of phosphorylation to CmRBP50 based RNP complex formation, and then four phoshoserines 223,438,440 and 444 were mapped as in vivo phosphorylation sites on CmRBP50. Point mutations and Zucchini yellow mosaic virus (ZYMV) viral system were used to clone and purify CmRBP50 and its mutant proteins S223A (Serine 223 to Alanine), STripleA (Serine 438,440,444 to Alanine), SQuadA (Serine 223,438,440,444 to Alanine). Protein overlay assay and co-immunoprecipitation experiments confirmed that the above phosphorylation sites were essential for CmRBP50 based RNP complex formation, especially the three serines on C terminus. The replacement of these three serines to alanine abolished the binding to its phloem interaction partners efficiently. Finally, we cloned and expressed putative interacting proteins of CmRBP50, in vitro pull-down experiments confirmed the interaction between CmRBP50 and CmPP16, GTPbP as well as PSPL, and additionally the phosphorylation on C-terminus was essential for their binding.
3. Protein kinases for CmRBP50 were analyzed and discussed. In gel kinase assay were applied to detect possible kinases for CmRBP50 from pumpkin vascular bundle proteins and phloem sap proteins. We found a nucleoside diphosphate kinase, a serine threonine kinase from vascule proteins and a protein kinase adkl, a protein kinase Csa000515 and a glycogen synthase kinase-3 from phloem sap analyzed by LC-MS/MS. Glycogen synthase kinase-3 was targeted as the candidate kinase for further study.
By studying the mRNA binding proteins in pumpkin phloem sap and the molecular mechanism of the first identified long-distance translocatable CmRBP50 based RNP complex, this paper will help us to understand CmRBP50 mediated physiological functions in phloem sap, and to study the other RNPs in phloem sap. Eventually this paper will not only help us to reveal the mechanism of communication and signal transduction pathways between distal organs, to increase the knowledge of plant physiology, ecology and horticulture, but also provide us a useful tool to improve stress defense, quantity and quality of vegetables in agricultural production, which has significant scientific and applicable meaning.
引文
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