Study On Function Of Mitochondria Protein TIM22-2 In Arabidopsis

In flowering plants,the developments of embryo and endosperm are important for the seed formation,which have laid a good beginning for the new generation.Because Arabidopsis has a small genome,clear genetic background,short growth cycle,closed flower pollination,abundant seeds,small plant type,and its growth environment is easy to control,so it is often used to study the regulation mechanism of genes related to plant development,and these results are expected to used in rice or other food crops to make great contribution in increasing yield,quality,and resistance.Mitochondrion is an organelle that not only provides energy,but also participates in the regulation of cell division,growth and differentiation,as well as formation and development of organs such as leaves and embryos.There are many proteins inside the mitochondria,and a large number of them have been found to be involved in the development of plant embryos.In this article,several mutants of the mitochondrial protein TIM22-2 in Arabidopsis were used as experimental materials.Using phenotypic observation,genetic transformation,living embryo isolation,q RT-PCR,immunoprecipitation,mass spectrometry,and other molecular biology,cell biology,and genetic experimental techniques to perform functional studies on Arabidopsis gene TIM22-2,we found that it played an important role in embryonic development and mitochondrial protein transport.The main results obtained are as follows:1.Two mutants,tim22-2-1 and tim22-2-2,of the T-DNA insertion of the TIM22-2 gene were obtained from the Arabidopsis mutant library ABRC.The insertion site of T-DNA in the mutant tim22-2-1 was on the second exon.The embryo phenotype of this mutant was that most embryos stagnated in the globular stage and could not continue to develop.The T-DNA insertion site in the mutant tim22-2-2 was on the first exon,its embryos stagnated as early as the globular stage,and could develop into cotyledon stage,or at the latest with that the shapes were abnormal to some extent.In order to explore whether the differences in the mutation sites would affect the phenotype of the corresponding mutants,CRISPR-Cas9 gene editing approach was performed and the transgenic mutants tim22-2-crs of the target gene were obtained.One of the mutants was designed with an editing site in the second exon and was named tim22-2-cr3.Two lines of the mutant tim22-2-cr3 had albino ovules in all siliques,meaning that they produced an ovule abortion phenotype.Sequencing was used to check the sequence editing status,it was found that there was no edited in one line,but in some leaves of the other line,a base A was inserted before the 566th base,and the T base of the 567th was changed into the G base.Since both lines of this mutant were common chimera plants,they were not observed further.After screening the mutant tim22-2-cr1 edited on the first exon,the plants with stable inheritance at the editing site were obtained,and the editing result of this mutant was that the 169th base T was mutated to base C,and the 191th base C was deleted.In addition,the embryonic phenotype of the mutant tim22-2-cr1 was very similar to that of tim22-2-1.Another important research object of seed development was endosperm,therefore,the endosperm of the mutants was also observed by fluorescence.Compared with the wild type,the endosperm nuclei of tim22-2-cr1 and tim22-2-1 mutants at 5 days after pollination were not normally cellularizated.In addition,the number of endosperm nuclei in mutant tim22-2-1 decreased and the volume was expanded.The endosperm of the mutant tim22-2-2developed normally in one part and retarded in others with abnormal cellularization.Combining the phenotypic characteristics of embryo and endosperm,it was speculated that TIM22-2 gene played an important role in Arabidopsis seed development.2.The vector that TIM22-2 gene promoter fused with GUS was constructed and genetically transformed,and the transgenic plants was stained and observed.It was found that GUS signal was significantly expressed in vascular tissue of leaves,apical meristems,styles,filaments and inflorescence,indicating that TIM22-2 gene was widely expressed in vegetative and reproductive organs.After introducing the GFP tag after the promoter of TIM22-2 and obtaining the transgenic plants,we observed that embryos at different stages all had fluorescent signals,which implied that the TIM22-2 gene might be involved in embryo development.Then the subcellular localization experiment of the TIM22-2 protein was performed,and the two-week-old transgenic seedlings of p TIM22-2-CDS-GFP were used as experimental materials to extract protoplasts.The Mito Tracker staining agent was used to fluorescently locate mitochondria,and GFP fluorescence was used to localize TIM22-2protein.The study found that both fluorescent signals could co-localize on the mitochondria,making it clear that the protein was a mitochondrial protein.In order to prove that the GFP tag on the subcellular localization vector did not affect the function of the target protein,this vector was also used to restore the mutant tim22-2-2 and it was found that there were no abortive seeds in the siliques of the homozygous mutant.3.Using the promoter of the embryo-specific gene ABI3 to drive the CDS of the TIM22-2gene,the mutant’s abortive phenotype was partially restored,and partially recovered homozygous mutant tim22-2-2^{p ABI3::TIM22-2CDS}plants were obtained.The seeds of this homozygous mutant could mature,germinate into seedlings,and bloom smoothly.However,after the siliques were produced in the plants,they were shorter and thinner,and the ovules were not formed,so they could not produce the next generation.Even so,the partially restored homozygous mutant plants still were valuable genetic materials for further studying this gene.In addition,Arabidopsis p35S::TIM22-2-HA seedlings that grew after three to four weeks were used to induce callus,mitochondrial proteins were extracted,and the purpose was to use Immunoprecipitation（IP）technology to search for proteins that might interact with TIM22-2.After mass spectrometry detection of IP samples,bioinformatics analysis was performed on the obtained samples,and a total of 3444 proteins were obtained.Among them,533 were mitochondrial proteins,including 30 outer membrane proteins,53 inner membrane proteins,31 matrix proteins,3 interstitial proteins,and 416 mitochondrial proteins with unknown localization.Furthermore,the analysis of these 117 mitochondrial proteins with known localization was mainly carried out.Among them,B14.7,TIM17-2,TIM23-2 and TIM22-2 were members of the PRAT（Preprotein and amino acid transporter）family,TIM44-2 and TIM50 were both TIM23 protein complex subunits,AT5G27395 was a highly co-expressed protein with TIM22-2,and TIM9 was a small molecular chaperone subunit in the intermenbrane space.The discovery of these Arabidopsis mitochondrial proteins provided important information for further studying plant mitochondrial proteins and their transport pathways.4.After constructing the p35S::TIM23-2-HA vector and obtaining homozygous transgenic plants of Arabidopsis,RNA was extracted from the two-week-old seedlings,and the expression level of the target gene of TIM23-2 was significantly increased through the test.It was proved that HA tag did not affect the function of TIM23-2 protein.The gene editing vectors of AT5G27395,TIM17-1,and TIM23-2 were constructed using CRISPR technique,and the genes were continuously transformed to study the biological functions of the genes.In addition,gene editing vectors of AT5G27395,TIM17-1 and TIM23-2 were constructed using CRISPR technique to study the biological functions of target genes.