Identification And Activity Analysis Of Specific Expression Regulatory Elements In Maize Endosperm

Maize seed development begins with a double fertilization event producing a diploid embryo and triploid endosperm,respectively.Endosperm is a storage tissue that accounts for80% to 85% of the weight of mature grains and provides nutrients for embryo development.The developmental state of endosperm directly affects the yield and quality of maize.In-depth study of maize endosperm development will help to increase maize yield and improve its quality traits.Duplication genes generated by the duplication of DNA fragments often show expression differentiation in functional differentiation,and different tissue expression specificities between gene copies are presented.Studies have found that a large number of genes related to endosperm development,starch and protein storage synthesis and accumulation are expressed in maize endosperm.Most of these genes are produced by replication and are specifically expressed in endosperm after long-term evolution.The expression properties of genes are inseparable from the cis-acting elements of their regulatory regions.Studies have found that there are cis-acting elements in the promoters of endosperm-specific expression genes that bind to transcription factors to regulate their specific expression.More than ten endosperm-specific expression elements have been identified in grain storage protein genes of cereal crops.In order to further reveal the formation and regulation mechanism of endosperm tissue expression specificity,this study analyzed the endosperm-specific expression elements based on the differences in regulatory sequences between endosperm-specific and non-endosperm-specific expression replica gene pairs that undergo tissue expression differentiation.The four sets of maize transcriptome data from different stages and parts of endosperm were combined to screen and identify endosperm-specific genes.Phylogenetic analysis was used to obtain endosperm-specific and non-endosperm-specific expression replica gene pairs,and the differences in regulatory sequences were compared to identify endosperm-specific expression candidate regulatory elements.At the same time,the common elements of endosperm-specific highly expressed genes were analyzed,and they were also used as candidate elements.Then the transient expression vector of the candidate element was constructed,and the expression activity and specificity of the candidate element in the endosperm were analyzed by gene gun transformation of maize endosperm for transient expression and GUS histochemical staining.The main findings are as follows:1.Based on the transcriptome data of maize endosperm development,1137endosperm-specific expression genes were identified,of which 531 were 6-8 DAP(Days after pollination,DAP),482 were 9-13 DAP,and 236 were 14-34 DAP.106 genes were highly expressed in both 6-8 DAP and 9-13 DAP periods,and 6 genes were highly expressed in both9-13 DAP and 14-34 DAP.2.The functional enrichment analysis of maize endosperm-specific genes found that the maize endosperm-specific genes of 6-8 DAP were mainly involved in the proliferation and differentiation of early embryo and endosperm cells;the genes of 9-13 DAP were mainly involved in protein synthesis and modification;14-34 DAP The genes are mainly involved in the metabolism of starch and carbohydrates.3.Combined with phylogenetic analysis,we identified replicative gene pairs(ie,one replica was specifically expressed in the endosperm,and the other was expressed in other tissues of the endosperm),and a total of 196 pairs were identified.The differences in regulatory sequences were analyzed,and 7 elements were identified as candidate regulatory elements for endosperm-specific expression,including 6 new elements and 1 reported element.The genes with extremely high expression levels and specific expression in the endosperm were regarded as endosperm-specific highly expressed genes,and a total of 200 genes were identified.By analyzing the common elements of their regulatory sequences,10 candidate regulatory elements for endosperm-specific expression were identified,and 16 candidate regulatory elements had different functions.The function related to the specific expression of endosperm has not been reported.4.A transient expression vector containing candidate regulatory elements was constructed,and the maize endosperm was transiently transformed by gene gun and GUS histochemical staining was used to analyze the expression activity and tissue specificity of endosperm-specific expression candidate regulatory elements.It was found that conserved fragments containing MYB-like sequence elements could The driver GUS gene is only expressed in the endosperm,and has endosperm-specific expression characteristics in the seed coat,embryo and endosperm.The conserved fragment containing the combination of ACGTATERD1,GTGANTG10,WRKY71 OS,and ABRELATERD1 elements can also drive the high expression of GUS gene only in the endosperm,indicating that the combination of elements is responsible for the specific high expression of the gene in the endosperm.