Studies On Evolution Of C2H2 Zinc-Finger Protein Gene Family In Brassica Campestris Subsp.chinensis And Transcriptional Regulation Of DAZ3 In Pollen Development

Pollen development acts as a central part of angiosperm reproduction and development.Pollen fertility is directly related to whether plants can be pollinated and fertilized to produce the next generation,which is very important for the yield of cruciferous vegetable crops and the quality of seeds.A series of complex regulatory processes are involved in the pollen development,in which many transcription factors play important roles.As one of the largest transcription factors families,C2H2 zinc-finger proteins can bind to DNA,RNA or protein through their zinc finger domain,and then participate in various biological processes.During flower development,C2H2 zinc finger protein act as histone methyltransferase or demethylase and participate in floral induction,and regulate the transcription of floral homeotic genes by affecting cell proliferation and hormone regulation.However,little has known about the role of C2H2 zinc-finger proteins in the transcriptional regulation of the maturity of floral organs,especially in pollen development.The study on the function and transcriptional regulation of C2H2 zinc-finger protein during pollen development will provide new knowledge for understanding the regulation mechanism of this gene family in the field,and is of great significance for exploring the reproductive development of angiosperms.In this study,Brassica campestris subsp.chinesis(syn.Brassica rapa subsp.chinensis)was used as plant material,evolution and expression analysis of C2H2 zinc-finger protein gene family provide data basis for studying the function of C2H2 zinc-finger protein during pollen development.Then,the expression of BrDAZ3 was analyzed,and the function of BrDAZ3 during pollen development was performed by RNA interference and over-expression techniques.The T-DNA insertion mutants of AtDAZ3 in Arabidopsis thaliana were identified,and the effect of the mutation of AtDAZ3 gene on pollen development was analyzed.The phenotype of atdaz3 was rescued to confirm the phenotype of mutant was caused by AtDAZ3 single gene;combining overexpression technology,the role of AtDAZ3 in pollen development was analyzed;and the transcriptional regulation of DAZ3 in pollen development was analyzed.The main results and conclusions are as follows:(1)The 243 B.rapa C2H2 zinc-finger protein gene was identified.The B.rapa C2H2 zinc-finger protein gene family was then divided into I?X subgroups by analyzing gene structure,protein domain and evolutionary tree.Q and MQ-type zinc-finger proteins are clustered in three subgroups ?,? and ?,and most of them contain EAR reprossor domains at their C-terminus;mix-type zinc-finger proteins only exist in subgroup ? and ?;the zinc finger proteins of D and T type are dispersed in other subgroups.Chromosome location,gene replication and syntenic analysis,the results showed that the expansion of B.rapa C2H2 zinc-inger protein gene family mainly originated from fragment duplication and tandem duplication.By calculating the Ka,Ks and Ka/Ks values between the orthologous and paralogous gene pairs,the results showed that the paralogous genes of B.rapa was generated by the whole genome triplication after isolation from the Arabidopsis genome,and B.rapa C2H2 zinc-finger protein had lower selection pressure and evolution rate in the evolutionary process than that of Arabidopsis C2H2 zinc-finger protein.In addition,the expression of C2H2 zinc-finger protein genes in roots,stems,leaves,flowers,siliques,sepals,petals,stamens and pistils in B.rapa was analyzed.The specific or highly expressed genes in stamens were screened.Then their expression in pollen mother cell stage,tetrad stage,uninuclear pollen stage,binucleate pollen stage and trinucleate pollen stage was analyzed,and several potential pollen function genes were selected.Analysis of co-expression network,the transcriptional regulation network of C2H2 zinc-finger protein gene during flower development was explored.(2)CDS and full-length gene sequences of BrDAZ3 were cloned,and deduced BrDAZ3 could encode a D-type C2H2 zinc-finger protein with two EAR repressor domains at the C-terminal.The spatial and temporal expression pattern of BrDAZ3 was analyzed.The expression level of BrDAZ3 in various tissues and organs was detected by RT-PCR and q-PCR.It was found that BrDAZ3 was highly expressed in stamens.The expression of BrDAZ3 in different floral bud development stages was detected,and the results showed that the expression of BrDAZ3 began to exist at uninuclear microspore stage and increased gradually in subsequent development stages until the trinuclear pollen stage.So,BrDAZ3 acts as a "late" pollen gene.Analysis of subcellular localization,the result showed that BrDAZ3 located in the cytoplasm and nucleus,which indicates that it can participate in the transcriptional regulation of nuclear proteins,as well as important biochemical metabolic activities in the cytoplasm.(3)To analysis the function of BrDAZ3,RNA interference and overexpression techniques were performed.It was found that abnormal expression of BrDAZ3 affected the development of pollen wall and pollen tube.The abnormal expression of BrDAZ3 did not affect the vegetative growth.However,the abnormal expression of BrDAZ3 resulted in abnormal cytoplasmic degradation in about half of the pollens from the uninuclear pollen stage,and gradually intensified during the subsequent development process,resulting in the collapse of pollen grains,and no intine formation.At the same time,the shape of baculum on the collapsed pollen exine were abnormally spherical.The abnormal expression of BrDAZ3 can also affect pollen tube germination in vitro.The down-regulation of BrDAZ3 expression results in about half of the pollen can not germinate,some of the germinated pollen is abnormal in shape.The abnormal outward protrusion of pollen cytoplasm causes the pollen grain to burst and divide into two petals,and the pollen tube tip curls.The up-regulation of BrDAZ3 expression results in the expansion of the pollen tube tip.These abnormal pollen germination in pistils was blocked,which eventually led to the majority of ovules could not fertilize after self-pollination,resulting in shorter siliques,and some of them had no seeds,resulting in lower seed setting rate.(4)The CDS and full length of AtDAZ3 gene were cloned,and and deduced AtDAZ3 encoded a D-type C2H2 zinc-finger protein with two EAR repressor domains at the C-terminal.The spatial and temporal expression patterns of AtDAZ3 were analyzed.The results showed that AtDAZ3 was highly expressed in pollen and began to express at floral bud stage 11(uninuclear pollen stage).With the highest expression at floral bud stage 14(mature pollen stage)and continued to express in pollen tube.That is to say,AtDAZ3 was also a "late" pollen gene.The expression of AtDAZ3 was also found in sepals and petals.The subcellular localization of AtDAZ3 was analyzed and found to be located in cytoplasm and nucleus.(5)Mutant atdaz3 has slender leaves,smaller floral organs and abnormal pollen development.The results showed that the length-width ratio of leaves increased significantly compared with wild type,and the epidermal cells of leaves became larger.The cell shape of palisade tissue near the upper epidermis was abnormal,the cell gap became larger,and the cell arrangement of spongy tissue was disordered.The width or length of sepals,petals,stamens and pistils of floral organs were significantly or extremely lower than those of wild type.The mutation of AtDAZ3 results in the degradation of pollen cytoplasm from the uninuclear pollen stage,which eventually leads to without the formation of intine,collapse of pollen grains and the shape of baculum on the collapsed pollen exine were abnormally spherical.The pollen germination of the mutant was blocked,which resulted in the failure of most ovules to fertilize and the decrease of seed setting rate after self-pollination.AtDAZ3 could completely rescue the phenotype of atdaz3.The up-regulation of AtDAZ3 expression did not affect vegetative growth and floral organ development.The cytoplasm of pollen began to degrade abnormally from uninuclear pollen stage,and finally formed collapsed microspore,but this abnormality did not affect the seed-setting rate.(6)The transcriptional regulation of BrDAZ3 and AtDAZ3 during pollen development is conservative.That is BrDUO1a,not BrDUO1b,can directly activate the transcription of BrDAZ3;AtDUO1 can directly activate the transcription of AtDAZ3,in which the MYB binding sites in the promoters of AtDAZ3 are necessary for transcriptional activation.EAR repressor domains of C-terminal of DAZ3 can interact with CTLH domains of N-terrmnal of TPL/TPR,and form heterodimers with each other.In addition,AtDAZ3 and AtTPL can interact with AtHDA6 and AtHDA19.The expression of genes involved in pollen intine,exine and pollen tube development was detected in atdaz3.The results showed that a series of genes involved in the synthesis of substances of intine and the expression of sporopollen synthesis genes of exine were abnormal.The expression of genes related to cell wall formation and modification,cell signal transduction and cell transport during pollen tube development was down-regulated or up-regulated.Cell proliferation,cell cycle and cell transport related genes were detected and their expression was also abnormal.Therefore,the model of transcriptional regulation is that DAZ3 is directly activated and regulated by DUO1.DAZ3 forms co-inhibitors with TPL,HDA6 and HDA19 to inhibit the transcription of downstream genes,then participating in the development of pollen wall and pollen tube.