The Molecular Mechanism Of APPR-1-P Processing Enzyme Regulates Embryonic Development And Stress Responses

The embryonic development of higher plants,especially angiosperms,is subject to precise genetic regulation,which has been a research hotspot in plant developmental biology.Embryonic development includes a series of important events such as pollination,fertilization,cell division and differentiation,and organ morphogenesis.Embryonic development is the basis of plant formation and plays an important role in plant growth and development and generational alternation.Studying the embryonic development process is of great significance for understanding the whole cell fate of plants and its molecular mechanisms.The APPR-1-P processing enzyme family has two members,AT1G69340 and AT2G40600.In this paper,one member of the appr-1-p processing enzyme family,AT1G69340 gene,was selected as the research object.Lab preliminary results showed that the homozygous mutant of this gene can cause embryonic lethality,indicating that the gene may regulate embryonic development.To further investigate the function of this gene,we conducted the following studies.Research content 1: The action mechanism of AT1G69340 gene regulating embryo development.The main experimental results were as follows:1.The T-DNA insertion mutants(cs808255 and cs839860)of the AT1G69340 gene were ordered from the TAIR website,and the position of the T-DNA insertion into AT1G69340 gene was identified by PCR and sequenced.The T-DNAs of the cs808255 and cs839860 mutants were inserted into the eighth and first exons of the AT1G69340 gene,respectively.Further genotypic identification also revealed that there were no homozygotes in the two mutant progeny.2.Phenotypic analysis was performed on the seeds of AT1G69340 T-DNA insertion mutants at different developmental stages,some abortive seeds were generated in heterozygous mutants.Further observation of the embryos of the hybrid mutant seeds showed that about one quarter of the seed embryos developed slowly and eventually stagnated,showing defects in embryonic development and unable to become normal mature embryos.3.It was found that the loss of AT1G69340 function caused the mutant embryo to develop more slowly than the wild type and eventually stagnated.The results of GUS transgenic lines showed that the gene was mainly expressed in the cotyledons,roots,and leaves,stems,anthers and other tissues of mature plants,and was also widely expressed in embryos and endosperm.These results suggested that AT1G69340 was expressed in the embryo and endosperm and regulated the process of embryo development.4.By constructing p CAMBIA3301-GFP-AT1G69340 fusion expression vector transforming the resultant construct into onion epidermal cells via gene gun bombardment or into Arabidopsis thaliana through Agrobacterium-mediated flower dipping,the localization of AT1G69340 protein in cells was determined and it was found that AT1G69340 expressed in both nucleus and cytoplasm.5.The p CAMBIA3301NH-AT1G69340 overexpression vector was constructed and transformed into heterozygous lines of at1g69340 mutant by Agrobacterium tumefaciens.The functional complementary transgenic lines were screened,and the complementary transgenic homozygous lines could generate fertile offspring,indicating that AT1G69340 gene can complement homozygous embryonic lethal phenotype of the T-DNA insertion mutant.Research content 2: The molecular mechanism of AT1G69340 gene involved in salt stress response.The main experimental results are as follows:1.Salt stress treatment was carried out on AT1G69340 T-DNA insertion mutant,complementary transgenic lines and wild type Arabidopsis thaliana,the result showed that the complementary transgenic lines were less salt-tolerant than wild-type and heterozygous mutant,combined with the detection results of AT1G69340 gene expression level,after salt treatment the expression level of AT1G69340 gene in the heterozygous mutant was not significantly different from the wild type,while the expression level of gene in complementary transgenic lines significantly increased.2.In order to analyze the mechanism of AT1G69340 gene participating in salt stress responses,the expression of AT1G69340 under salt stress and the expression of stress response marker gene in AT1G69340 mutant and its complementary transgenic lines were detected.In complementary transgenic lines,the expression levels of SOD1,SOD2 and other genes were lower than those of wild type and mutant,while the expression levels of SOS1,CAT1 and other genes did not significantly change.It remains to be explored whether AT1G69340 regulates the down-regulation of SOD1,SOD2 and other genes.