Functional Analysis Of The Transcription Factor ZYGOTE1/2 Involved In Zygote Activation In Arabidopsis Thaliana

Fertilization of higher plants is a turning point in the generational transition.During fertilization two parallel events occur: sperm and egg cells fuse to form a diploid embryo,and another sperm fuses with the two polar nuclei of the central cell to form a triploid endosperm.The egg cell develops into a zygote after fertilization.The zygote develops into an embryo that further develops into a mature plant.The transition from egg cells to zygote development is a key point of genetic control during early embryogenesis.It represents that the development of zygote no longer depends on maternal factors provided by egg cell,but is controlled by zygote.Genome-wide gene activation in the zygote,termed zygotic gene activation,is one of the conditions for transition from egg cells to zygote.However,the gene activated by zygote plays a very important role in embryo development.However,due to technical limitations,the molecular mechanism of these genes has not been further studied.Based on the results obtained in previous work about transcriptome of egg cells and zygotes,the zygote-activated transcription factors ZYG1 and ZYG2 were identified.The Arabidopsis plants with mutant on these two genes were used as research object to investigate the functions of ZYG1 and ZYG2 genes in the early development process of embryos.By studying the expression pattern of ZYG1 gene,phenotype of mutant plants after hybridization,recovery experiments,and promoter methylation verification,etc.,an attempt was made to explore the role of ZYG1 gene in the early embryonic development of Arabidopsis thaliana.The experimental results were summarized as follows:1.Identification and screening of zygote-activated genes: 11 genes were selected through our laboratory’s transcriptome sequencing results of egg cells and zygotes obtained in the previous period.These genes were not expressed in egg cells,but were highly expressed in zygotes.The expression of these genes during embryonic development was further analyzed by transgene,and the de novo zygote transcription factor ZYG1 and its homologous gene ZYG2 were screened.2.ZYG1 and ZYG2 fusion proteins expressed in embryo after fertilization: By observing the expression patterns of proZYG1::ZYG1-GFP and proZYG2::ZYG2-GFP in embryos and mature tissues,it was found that the GFP signal was not expressed before fertilization,and expressed all phases after fertilization,like zygote,two-celled embryo,four-celled embryo,globular-stage embryo,heart-stage embryo,torpedo-stage embryo,early cotyledon,and mainly expressed in the root tip at the later stage of cotyledon.3.ZYG1 and ZYG2 are involved in early embryo development: mutants on ZYG1 and ZYG2 were screened out,and it was observed that single mutant could not cause seeds abortion.zyg1-1 zyg2-1/+ and zyg1-2/+ zyg2-2 were obtained through the cross in a reciprocal manner.It was found that the numbers of seeds abortion obeys law of segregation and affects early embryo development4.ZYG1 and ZYG2 are not imprinted genes: In order to verify whether ZYG1 and ZYG2 have the imprinting effect,the transgenic plants of the fusion protein model vector of the two genes are crossed with wild-type plants,and the embryos within 1 to2 days after pollination are observed.The expression of the fluorescent signal,it was found that the transgenic plants did not affect the expression of the fluorescent signal in the embryo,either as a maternal or a paternal parent.5.ZYG1 promoter has no obvious methylation modification :To verify whether the specific expression pattern of ZYG1 is controlled by methylation due to its promoter activity,we isolated the root tip tissue that express ZYG1 and the leaves that did not express ZYG1.The bisulfite method was used to analyze the methylation of the ZYG1 promoter region of two samples.The results showed that the ZYG1 promoter had no obvious methylation modification.6.Preliminary exploration of downstream signaling of ZYG1: construct p ZYG1:LEC1-GFP,transgene it into zyg1-2/+ zyg2-2 mutant plants,and observe the expression of fluorescence signals in the embryo.Observing the expression of fluorescence signals in embryos,we selected mutant plants with LEC1 expression.