Research On ZmTFCB Regulation Of Microtubule Homeostasis And Multi-omics Mining Of Maize Kernel Genes

Kernel is the main organ for the formation of maize yield and quality.Mining the key genes that affect maize kernel development and analyzing its mechanism can provide theoretical basis and excellent genetic resources for the genetic improvement of maize yield and quality.The purpose of this study is to clone the gene ZmTFCB that affects kernel development by using the maize kernel mutant ifcb\ verify the function of ZmTFCB through complementation tests,subcellular localization,expression analysis;through yeast double hybrid(Y2H),dual fluorescein enzyme complementation(LCI),immunofluorescence and other experiments analyzed the molecular mechanism of ZmTFCB regulating the homeostasis of microtubules in vivo and affecting kernel development;at the same time,using proteomic genome-wide association analysis(pGWAS)and multi-omics integration analysis methods to explore the candidate genes that affect kernel development,and verify their functions through CRISPR-Cas9 technology to construct a regulatory network for kernel development.The main findings are as follows:1.Compared with the wild type,the(fcb mutant has slower kernel development,impaired embryo and endosperm development,and the germination rate is only about 19% of that of the wild type;the seedlings of the tfcb are extremely weak,with few and underdeveloped roots.The two-leaf stage began to wither and die.2.By map-based cloning,the candidate gene controlling the tfcb mutant was located in the 348 kb physical interval on the second chromosome of maize.This interval contains 8protein-coding genes on the B73 reference genome;expression and sequencing analysis shows that Gene](ZwTIFCS)is the primary candidate gene;the results of the maize complementation test show that the ZmTFCB expression line can restore the phenotype of the kernel mutation and is the gene that causes the(fcb mutation.3.There are 2 copies of ZmTFCB in the tfcb mutant,one copy is consistent with the inbred line B73,and the other copy has 3 bases missing in the 7th exon,resulting in a protein ZmTFCB-h that lacks the DEI motif;ZmTFCB is localized in the cytoplasm,the deletion of DEI does not affect its subcellular localization;ZmTFCB is a constitutively expressed gene with the highest expression level in female ears.4.ZmTFCB encodes a microtubule folding cofactor B,which is related to a-tubulin folding and microtubule formation.BN-PAGE and molecular sieves were used to separate and determine the tubulin in the endosperm of wild-type and mutant kernels.It was found that the total amount of a-tubulin in the endosperm of mutant kernels was significantly reduced and mainly in monomeric form,while that in the endosperm of wild-type kernels the a-tubulin mainly exists in the form of heterodimers.The changes of microtubules were observed by immunofluorescence,and it was found that the cortical microtubules per unit area in the wild type were dense and airanged horizontally,while the cortical microtubules in the mutant had half the density of the wild type and were close to the oblique and longitudinal an*angement.The sensitivity of the tube inhibitor oiyzalin is reduced.5.The changes in endosperm cell nucleus replication are a common phenomenon in Dek-like kernel mutants.Flow cytometric analysis of wild-type and mutant kernel endosperm1 1 and 15 days after pollination found that the proportion of polyploid cells in the mutant kernel endosperm was significantly lower than that of the wild type.The results of western blot showed that the accumulation of cell cycle-related proteins in the mutant was reduced,indicating that the endoreduplication of the mutant kwenel endosperm cells was blocked.It can be seen that ZmTFCB also affects the endoreduplication of endosperm cells.6.TFCB is an important cofactor for a-tubulin folding,and ZmTFCB directly interact with a-tubulin.In order to analyze the mechanism of ZmTFCB.the relationship between the potential interacting proteins screened from the kernel yeast librarx' and the possible interacting proteins reported in humans and yeasts and ZmTFCB was verif ied by Y2 H and LCI.The results show that ZmTFCB directly interacts with the cytoplasmic localized ZmCCT5(TCP-1(CCT)s subunit)chaperone protein and ZmTFCE to mediate the folding of a-tubulin;ZmTFCB regulates the dynamics of microtubules by interacting with the microtubule additive end protein END-BINDING 1(ZmEBI);ZmTFCB-h cannot interact with ZmTFCE due to the deletion of the DEI motif,and cannot participate in the correct folding of a-tubulin,but can participate in the dissociation of a/p-microtubule dimers through a ZmTFCE-independent pathway.7.Evolutionary analysis shows that TFCB is conserved in huinans.animals and plants.In order to clarify the functional conservation of ZmTFCB.a knockout line ot AtTFCB gene was constructed using CRISPR-Cas9 technology.The knockout line of heterozygous genotype produced 1/4 abortion seeds,and its abortion phenotype could be restored by overexpression of ZmTFCB.indicating that TFCB has a relatively conserved function during the seed development of monocotyledonous and dicotyledonous plants.8.In order to dig out genes that affect kernel development from the proteomics level of the population,qualitative and quantitative analysis was performed on the proteome of the kernels of 210 mazie inbred lines 20 days after pollination,and 468 protein spots were identified.GO annotation and KEGG analysis showed that 54% of the identified proteins have annotation information,while 46% of the proteins have unknown functions? which means that there are still many genes related to their development to be discovered in the kernel.9.Using the 1.25 M SNP data and the quantitative value of the identified protein spots to perform a genome-wide association analysis based on a mixed linear model,46 quantitative trait loci(pQTL)affecting 40 protein spots were identified,including 13 cis pQTL and 33 trans pQTL.The unreported trans pQTL candidate gene RH38 of P2704 was transformed into maize using CRISPR-Cas9 technology.The results showed that individuals with heterozygous genotype produced 1/4 of the mutant kernels,indicating that RH38 is a key gene affecting kernel size.The above results indicate that pQTL analysis is an effective way to dig out candidate genes for kernel size.10.The expression QTLs(eQTLs),metabolite QTLs(mQTLs),quality traits QTLs and kernel size QTLs identified from the same related population and multiple RIL populations were integrated with pQTLs to construct three main pathways affecting maize kernels developmental regulatory network.