梨CCT蛋白复合体调控RNP韧皮部运输机理研究

Grafting is the main way of asexual propagation in fruit trees, the interaction between rootstock and scion after grafting will affect the growth of the tree, resistance, flowering, fruit yield and quality. With the development of molecular biology technology in recent years, it was found that plant RNA especially endogenous mRNA can move through companion cell into the phloem sieve tube for long-distance transport and then unload to the sink to control the character between stock and scion. The long-distance transport mRNAs need to combine with some protein to form ribonucleoprotein complexes (RNP) for movement. In our previous study, we found that PbPTB3 can assist the long-distance transport of PbWoxT1 in phloem. However, it is not clear that how the RNP macromolecular complex formed by PbWoxT1-PbPTB3 assemble and enter into the phloem sieve tube through the companion cell. We used PbPTB3 as a bait to screen a yeast two-hybrid library and obtained a protein, PbCCT5, interacted with PbPTB3 directly. Domain analysis verified that this protein was a chaperonin complex containing eight subunits. This study intends to further analyze the function of chaperone protein CCT in RNP complex long-distance transportation by means of interaction analysis, transgenesis and grafting, and finally clarify the mechanism of chaperone protein assisting long-distance trafficking of mRNA. Our study will provide a new idea for the breeding of new transgenic rootstock of fruit trees.

嫁接是果树最主要的无性繁殖方式，嫁接后砧木与接穗之间相互作用会影响树体生长发育、抗性、开花、果实产量和品质等。近年来分子生物学等技术不断发展，人们发现植物内源mRNA可以通过伴胞装载进入韧皮部筛管长距离运输至库端，卸载后调控砧穗之间性状。同时发现mRNA并不能单独运输，需要与一些蛋白结合以RNP复合体形式传递。前期研究发现PbPTB3可辅助PbWoxT1韧皮部长距离运输，但PbWoxT1-PbPTB3形成的RNP复合体如何组装并通过伴胞进入韧皮部筛管的机制尚不清楚。本项目以PbPTB3为诱饵筛选获得一个与PbPTB3直接互作的蛋白PbCCT5，经结构域分析证实其为伴侣蛋白CCT复合体的8个亚基之一，进一步通过互作分析、转基因、嫁接等方法解析伴侣蛋白CCT在RNP复合体长距离运输中的功能，最终明确伴侣蛋白协助mRNA长距离运输的作用机理。本项目的研究将为果树新型转基因砧木育种提供全新的思路。

植物内源mRNA分子在韧皮部中长距离运输进而影响植物生长发育。嫁接植物mRNA分子在砧木与接穗间运输需要与RNA结合蛋白结合成核糖核蛋白(RNP)复合体，保护mRNA分子在韧皮部中长距离运输，然而该RNP复合体如何组装并促进mRNA转运的机制尚不清楚。PbWoxT1 (WUSCHEL-RELATED HOMEOBOX transport 1)是前期发现的砧穗间运输性mRNA分子，能够与PbPTB3结合形成RNP复合体在韧皮部中长距离运输。本项目通过酵母双杂交筛库分析发现了一个杜梨中新的蛋白质PbCCT5，它是真核生物中伴侣蛋白CCT (Chaperonin Containing T-complex Polypeptide-1)复合体的一个亚基，与PbPTB3相互作用并促进其与PbWoxT1 mRNA在杜梨韧皮部长距离运输。通过酵母双杂交和双分子荧光互补证实PbCCT的8个亚基通过1，4，2，5，7，8，6，3的顺时针排列方式形成一个八聚圆环。蛋白酶K消化和RNA免疫共沉淀证实了PbCCT复合体能够将PbPTB3和PbWoxT1组装成RNP复合体。通过在杜梨叶片中过表达GFP-PbWoxT1和GFP-PbPTB3，同时分别沉默PbCCT复合体各亚基，发现任何PbCCTs亚基的局部沉默都会减少PbWoxT1-PbPTB3复合体从叶片到叶柄的系统性运输。同样，转基因嫁接实验表明，转基因砧木中NtCCTs的沉默抑制了PbPTB3和PbWoxT1 mRNA向野生型接穗的长距离运输。综上所述，PbCCT复合体在韧皮部中将PbPTB3和PbWoxT1组装成RNP复合体，进而促进PbWoxT1在杜梨韧皮部中长距离运输。