Functional Analysis Of Microtubule Binding Protein Gene GhIQD21 In Cotton

Cotton is an important economic crop which plays an important role in Chinese national economy.It is of great economic value and strategic significance to improve fiber yield,reveal the regulation mechanism of fiber development,and cultivate high-quality cotton varieties suitable for cultivation.As an important component of cytoskeleton,microtubule skeleton plays multiple roles in cell growth and development.Microtubule binding protein(MBP)regulates the arrangement and stability of microtubules by interacting with microtubules.Plant specific IQD proteins family can not only act as calmodulin binding protein by binding with calmodulin,but also act as microtubule binding protein by binding with microtubule.IQD proteins have pleiotropic effects on plant growth and development and response to environmental changes.High expression of some IQD proteins in cotton fiber cells suggests that these IQD proteins are closely related to the development of fiber cells,but their specific functions and regulatory mechanisms are not clear.Gh IQD21 gene which has a low expression level under sphingolipid synthesis inhibitors(Fumonisin 1)treatment was specifically expressed during fiber elongation,suggesting that Gh IQD21 may be closely related to fiber length.In order to clarify the function and regulatory mechanism in fiber development,Gh IQD21 was cloned in this study.The subcellular localization and ectopic expression of Gh IQD21 were observed by constructing overexpression vector and transforming into Arabidopsis thaliana and tobacco.To analyze the relationship between Gh IQD21 and microtubules,we analyze the mechanism of Gh IQD21 regulating organ morphology.We clarified the effect of Gh IQD21 gene on fiber length by gene silencing and so on.We also verified the relationship between Gh IQD21 and calmodulin Gh Ca M7.This study laid a foundation for further research on the regulation mechanism of cotton IQD protein in the growth and development of cotton fiber cells.The main results are as follows:1.The gene,affected by Fumonisin 1 treatment,which have a predominant expression in cotton fiber elongation were selected for cloning,evolution and expression analysisAfter Fumonisin 1 treatment and the transcriptomics analysis,the gene which is predominant expression in fiber elongation period was selected.The gene sequence of10 DPA(Days post anthesis,DPA)fiber was amplified from its c DNA of Gossypium hrsutum Wild type.The results of sequence analysis showed that the gene was closely related to At IQD21,so it was named Gh IQD21.The expression characteristics of the gene were detected by fluorescence quantitative(RT-PCR)showed that the expression level reached the highest in 10 DPA fiber blast,followed by that in 6 DPA-12 DPA.2.Analyze the function of Gh IQD21 gene in transgenic lines of Arabidopsis thalianaIn order to analyze the function,we first expressed Gh IQD21 in Arabidopsis thaliana.The results showed that the expression of Gh IQD21 led to the reduction of phenotype and abnormal cell morphology.In order to explore the causes of phenotypic changes,the hypocotyls were treated with microtubule depolymerization drugs.The results showed that the sensitivity of overexpression lines was higher than that of wild type and the arrangement of MT was changed.These results indicate that the cloned Gh IQD21 is a functional sequence,and ectopic expression affects the growth and development of Arabidopsis thaliana.3.The effect of the expression level of Gh IQD21 on the growth and development of cotton fiberThrough cotton genetic transformation and virus induced gene silencing,transgenic cotton with overexpression and inhibition of Gh IQD21 was obtained.Overexpression of Gh IQD21,the fiber length became shorter than wild type.On the contrary,if the expression of Gh IQD21 was inhibited,the fiber length became significantly longer than wild type.These results indicated that either overexpression or inhibition of Gh IQD21 expression had an important effect and confirmed that Gh IQD21 may play a negative role on the development of cotton fiber.