Study On Maize Somatic Embryogenesis-related Genes ZmSERF1 And ZmYUCCA5

Maize?Zea mays L.?is one of the most important cash crops,and its yield plays an important role in the development of the country's agricultural economy.Therefore,the innovation of new maize germplasm and new maize varieties through multiple ways has become an important issue of agricultural concern.The excellent maize transformation receptor is very important for its genetic transformation technology.Because of its bipolarity,low variation of clones,high stability,high survival rate of differentiation and transplantation,somatic embryo has become a good receptor material for plant genetic transformation.But the formation of maize somatic embryo is very difficult and the research of its development process is still very few.So studying the genes related to the maize somatic embryo is very meaningful for the success of maize genetic transformation.Based on the expression pattern of maize inbred line Y423,we focused on ZmSERF1 and ZmYUCCA5 with very high gene expression.We cloned these two genes and the corresponding vectors were constructed.Then the function of ZmSERF1 gene was preliminarily studied,which made a very solid foundation for the future in-depth research.The findings were as below:1.The 840bp ZmSERF1 coding sequence was successfully cloned from somatic embryos induced by elite maize inbred line Y423.The AP2/ERF domain of ERF was found to be highly conserved by comparing the sequences of ERF protein from many plants.The sequence similarity between maize ZmSERF1 and MtSERF1 is 34.55%,and the sequence similarity with AtERF1 is 42.96%.2.The plant overexpression vector pCAMBIA3301-35s::ZmSERF1 was successfully constructed and transformed into wild-type and mutant Arabidopsis thaliana,respectively.Then we obtained the T₃ overexpression lines and complementary lines.With these lines,the ability of somatic embryo induction was measured and analyzed.The results showed that ectopic expression of ZmSERF1 gene could restore the ability of somatic embryo induction,and overexpression of ZmSERF1 gene increased the ability of somatic embryo induction.Therefore,the ZmSERF1 gene has been proved to have an important role in the induction of somatic embryogenesis.3.The plant interference expression vector p3301-UBI-SERF1?+?-intron-SERF1?-?was successfully constructed.Then through the Agrobacterium tumefaciens-mediated methods,we transferred the over-expression vector,empty vector and interfering vector into Y423-induced somatic embryos.By comparing the phenotypes of the contemporary somatic embryos,the results showed that there was a higher browning mortality of the somatic embryos,51.88%infected by the interference vector during screening and a lower rate of fresh resistant somatic embryos,17.73%.4.The yeast expression vector pGBKT7-ZmSERF1 was successfully constructed.The results showed that the ZmSERF1 protein had no toxic effect on yeast cells.And it could inhibit the self-activation of the reporter gene when the 3-AT concentration was 40mM/L.Then eight candidate interaction proteins were obtained.The subcellular localization vector pSATN1-GW-ZmSERF1 was successfully constructed and transformed into maize leaf protoplasts.The results showed that the ZmSERF1protein was localized in the nucleus.5.The 1236bp ZmYUCCA5 coding sequence was successfully cloned from somatic embryos induced by elite maize inbred line Y423.Then we proceed a bioinformatics analysis.The overexpression vector p3301-35s::ZmYUCCA5 was constructed and transformed into wild-type Arabidopsis thaliana.T₂ generation transgenic ZmYUCCA5 over-expressing Arabidopsis lines were obtained.The yeast expression vector pGBKT7-ZmYUCCA5 was constructed and it was proved that ZmYUCCA5 protein had non-toxic effect to yeast cells and had no self-activating activity for the reporter gene.Finally,one candidate interaction protein was obtained.The subcellular localization vector pSATN1-GW-ZmYUCCA5 was constructed and transferred into maize leaf protoplasts.Combining the prediction,it was indicated that the ZmYUCCA5 protein was primarily located in chloroplasts.