Arabidopsis Thylakoid Lumen Protein Knockout Mutants Creation And Preliminary Analysis

Chloroplast is the main organelle of photosynthesis in higher plants.It consists of chloroplast envelope,thylakoid and stroma.Stroma is the place where dark reaction takes place,while thylakoid is the place where light reaction takes place.Thylakoid is composed of thylakoid membrane and thylakoid lumen.There are more than 80 lumenal proteins that can be identified in Arabidopsis thaliana.At present,the important proteins in lumenal include Deg1 protein who involved in the degradation of PSII protein,carotenoids synthesis proteins,CYP38 protein,plastocyanin（PC）,carboxy terminal processing protease of D1 protein,and peripheral proteins Psb O、Psb P and Psb Q of photosystem II.In addition,they also include Pro-Immune factors,molecular chaperones,proteins related to hydrolysis.Although luminal proteins are not directly involved in photoelectron transport,they also play important roles in photosynthesis、the structure and genesis of thylakoids.With the in-depth study of chloroplast proteomics,the functions of important proteins located in different regions of chloroplast have been gradually clarified,which lays a foundation for a comprehensive understanding of the molecular members and their respective functions of plant chloroplast proteome.However,there are still many gaps in the protein research of lumenal.At present,the main function of Arabidopsis lumenal proteome is to help protein folding、catalyze the hydrolysis and antioxidation of thylakoid protein,it is still to be explored whether the protein which has been proved to be located in the lumen of thylakoid but whose function is unknown also has been above.Whether their existence is redundant or essential.Through PPDB（the plant protein database）and TAIR（https://www.arabidopsis.org/）,13 proteins with unknown functions in lumenal were screened out in this experiment.At present,there is no detailed annotation on TAIR website.This study aims to explore the function of these proteins by reverse genetics.The homozygous T-DNA mutants of AT1g14590、AT1g51350、AT1g62130、AT2g26340、AT3g09490、AT3g29240、AT3g44020、AT3G53560、AT5g46560 were obtained by NASC seed bank.The gene knock-out mutants of AT1g33780,AT1g51400,AT2g37400 and AT5g02590 were obtained by CRISPR-Cas9 gene editing technology.And identify these mutants are knock-out at the DNA level and RNA level.In order to explore whether these proteins play a role in photosynthesis,the obtained T-DNA homozygous mutants were stressed by high-light,low-light and drought,and AT5g46560mutant with early flowering phenotype was found under low light and normal light conditions.The reverse mutation verification test and Blue Native-PAGE test were carried out for this mutant.The analysis showed that the deletion of AT5g46560 protein resulted in the accumulation of PS II and Cytb₆f complexes.Therefore,it was speculated that AT5g46560might affect the photosynthesis of Athaliana by influencing the assembly or stability of photosynthesis macromolecular complexes,leading to the emergence of early flowering phenotypes in plants.In this experiment,13 gene knock-out mutants with unknown function were obtained,and their bioinformatics analysis and preliminary phenotype analysis were carried out.The gene knockout mutants provide the basis for the further study of the function of unknown proteins in lumen.