Cloning Of ZmCPB1 And Association Analysis Of It's Polymorphisms And Kernel Weight-Related Traits In Maize(Zea Mays L.)

Grain type and grain weight are important components of maize yield.Therefore,it is important to clone the genes related to grain type and grain weight in order to elucidate the complex molecular genetic mechanism of yield traits and improve maize yield.Because of the large genome and multiple repeat sequences of maize,it is difficult to study the genes related to corn kernel weight by using the map-based cloning technique.According to the collinearity of gramineous crops,homologous cloning of known genes related to grain weight of other gramineous crops in maize has become the main method to study the genes related to grain weight of maize.In this study,a direct homologous gene ZmCPB1 of rice grain weight related gene OsCPB1 was cloned from maize,and its spatiotemporal expression pattern and subcellular localization of its encoded protein were analyzed.Fifteen small populations composed of backbone inbred lines from four major maize germplasm in China were used to analyze the correlation between polymorphism of ZmCPB1 gene and grain size and grain weight of maize.The main results are as follows:(1)Based on the protein sequence information of rice OsCPB1 gene,we identified four genes with high homology in maize.The similarity between one gene encoding protein and OsCPB1 gene coding protein was 88.87%,which contained the conserved region of CPB1/D11 responsible for panicle morphogenesis and grain size,and the sequence of conserved region was identical to that of OsCPB1,so we named it ZmCPB1.The full-length 4448bp(GenBank accession number is GRMZM2G1071999),have 9 exon.The total length of CDS is 1466 bp and encode 481 amino acids(GenBank accession number is DAA37378.1).The ZmCPB1 protein belongs to cytochrome P450 family and contains the conserved D,E,I,J,K,L6 alpha-helix of the cytochrome P450 family,the heme binding region Fxx GxRxCx G is highly conserved between the helix I and the helix L at the C-terminal.According to the B73 reference genome sequence,the DNA sequence with long 1500 bp upstream of ZmCPB1 gene coding region was cloned.Sequence analysis software predicted that the promoter contained specific expression elements in endosperm tissues,Brassinolide response elements and other expression regulatory elements.Transmembrane domain prediction showed that ZmCPB1 gene encoded protein had a transmembrane domain,and subcellular localization prediction revealed that it might be located in endoplasmic reticulum.(2)The fusion expression vector of 35S::Zm CPB1-RFP and 35S::HDEL-GFP was constructed.The fusion expression vector was transiently expressed in tobacco epidermis by transient infection.The fluorescence distribution was observed under confocal laser microscope.The expression indicated that ZmCPB1 was located in the endoplasmic reticulum.(3)QRT-PCR technique was used to analyze the tissue specificity of ZmCPB1 expression.The results showed that ZmCPB1 was mainly expressed in panicle,and the highest level of expression was found in the endosperm 10 days after pollination..At the same time as starch synthesis in maize endosperm cells,ZmCPB1 may play an important role in the positive regulation of grain weight and ear yield formation in maize.(4)Fifteen small populations composed of backbone inbred lines from four major maize germplasm in China were used to analyze the correlation between polymorphism of ZmCPB1 gene and grain size and grain weight of maize.The results showed that the expression of ZmCPB1 was positively correlated with grain length and ear length,respectively,with a correlation of 0.049(P<0.05)and 0.033(P<0.05).However,there was no significant correlation between the polymorphism of ZmCPB1 gene cDNA sequence and its encoded protein sequence in the population and the grain size and grain weight related traits of maize.(5)Using Napin as promoter,the seed specific plant expression vector of ZmCPB1 gene was constructed,which can be used to identify the biological function of ZmCPB1 gene.In conclusion,ZmCPB1,a direct homologous gene of rice grain weight related gene OsCPB1,was cloned from maize.The results of bioinformatics analysis indicated that ZmCPB1 may play an important role in regulating ear formation and grain development,spatio-temporal expression pattern analysis showed that it was the highest expression in endosperm 10 days after pollination.Correlation analysis with corn grain weight showed that there was a significant positive correlation between its expression and corn kernel length and ear length.These results suggested that ZmCPB1 may play an important role in the regulation of grain endosperm development,which laid a foundation for further study on its biological function and molecular regulation mechanism in maize grain development.