Identification And Function Exploration Of SPR1 Interaction Proteins In Poplar

Microtubules are polymerized by α-tubulin and β-tubulin in the form of heterodimer,which form cytoskeleton with microfilaments and intermediate fibers.Microtubules play an important role in plant cell morphology,plant growth and development,wood formation,stress tolerance,and so on.The dynamic changes and functions of microtubule arrays are regulated by tubulin and microtubule associated proteins.SPR1 is one of microtubule associated proteins,which is specific in plants.Various studies have shown that SPR1 is involved in the regulation of the arrangement of cortical arrays,thus affecting the growth direction of plant cells.And it can promote the depolymerization of microtubules under salt stress and improve the tolerance of plants to salt stress.At present,there are few reports on the function of tubulin in forest trees,especially on the molecular mechanism of microtubule regulating the morphology and wood properties of forest trees,and the function of SPR1 protein in forest trees and its regulatory mechanism on microtubules are not clear.To obtain living material for observing the morphology of microtubules,in this study,primers were designed based on the Pt TUA5 and Pt TUB16 genes of Populus trichocarpa,which is used to clone 84KTUA5 and 84KTUB16 genes of 84 K poplar by homologous cloning,four plant overexpression vectors of 84KTUA5 and 84KTUB16 fused with red fluorescent protein m Cherry were constructed respectively,and then,transformed into 84 K poplars after transient expression verification in tobacco;Using the SPR1 sequence of Populus trichocarpa as a template to design primers,which is used to clone 84KSPR1 genes of 84 K poplar by homologous cloning,screening the candidate interaction proteins of 84KSPR1 by yeast two-hybrid technique,The Bi FC technology was used to verify the interaction between 84KSPR1 and its candidate interaction proteins,and to explore the interaction between 84KSPR1 interaction proteins.The main results are as follows:1.12 transgenic lines of 84KTUA5 and 24 transgenic lines of 84KTUB16 with stable inheritance were obtained,which provided background materials for further research on the function of poplar microtubules.2.through yeast two-hybrid technology,11 candidate proteins that may interact with 84KSPR1 protein were screened out.And then,it was found that 10 of them could interact with 84KSPR1 by Bi FC,and the interaction site of IQD14 and 84KSPR1 is on the microtubule array.In addition,the interaction between 84KSPR1 interacting proteins was also verified by Bi FC,and no interaction was found between EF1A、HIR1 and DRM4,however,IQD14 could interacts with EF1A、HIR1 and DRM4 respectively.Based on the previous studies,It is speculated that IQD14、Ca M and 84KSPR1 may form a polyprotein complex to regulate each other’s activity.And IQD14 may mediate the interaction between 84KSPR1 and other interacting proteins(such as EF1 A,HIR1,DRM4),thereby regulating the dynamic changes of the microtubule array.This study provides new clues for exploring the function and the regulatory mechanism of microtubules of SPR1 protein in forest trees.