A Preliminary Study Of The Cloning And Function Of Dark Response Genes In Maize

Zea Mays L.is an annual gramineous plant with the highest yield and planting area among the three major grain crops in China.It is also an important grain crop in the world.It has high edible and industrial value.Maize native to tropical and subtropical regions has strong photoperiod sensitivity and is suitable for growing under short sunshine conditions.However,the main producing areas of maize in China are mainly in northeast,North and northwest China under long sunshine conditions.To meet the needs of an increasingly diversified maize industry,we need more varieties of maize adapted to the northern regions and with different advantages and benefits.The introduction of tropical and subtropical maize germplasm resources can solve the demand for excellent maize characters,but the photoperiodic sensitivity of tropical and subtropical maize is one of the key factors restricting the large-scale cultivation of excellent maize varieties in the north.Therefore,it is of great significance to study the photoperiod of maize.To study the photoperiodic reaction of maize is the key to solve the problem of photoperiodic sensitivity of maize,and it is also one of the effective ways to enlarge the varieties of fine maize in north China.At present,the research on the Photoperiod of maize is based on the light reaction of photoperiod.Although some achievements have been made,there is still no breakthrough.This study innovatively started from the dark reaction of maize photoperiod reaction,in order to study maize photoperiod reaction from different perspectives,and thus to provide new research ideas for solving maize photoperiod sensitivity problem.Based on the dark response protein sequences obtained from previous maize proteomics analysis,the preliminary study of the cloning and function of the maize dark response gene was carried out.The main research contents and results are as follows:(1)According to the obtained maize dark response protein sequences,NCBI and other gene databases were used to obtain the corresponding maize dark response gene sequences.Four maize dark response genes(Zm DRG12,Zm DRG15,Zm DRG16 and Zm DRG19)were obtained from maize B73 using PCR.Four maize dark response gene clone vectors PMD18-T-Zm DRG12,PMD18-T-Zm DRG15,PMD18-T-Zm DRG16 and PMD18-T-Zm DRG19 were obtained using PMD18-T as the skeleton.(2)Bioinformatics analysis of four maize dark response genes revealed that Zm DRG12 gene encodes a protein with 105 amino acid residues,which has three possible phosphorylation sites and is a stable protein whose function is related to protein transport.It is speculated that Zm DRG12 protein may be located in thechloroplast and bind to Sec Y so that the protein transport can proceed smoothly.The Zm DRG15 gene encodes a protein with 585 amino acid residues,which has 61 possible phosphorylation sites and is a stable protein,which is predicted to be located in the chloroplast and its function is related to the GTP binding protein.The Zm DRG16 gene encodes a protein with 654 amino acid residues,which has 67 possible phosphorylation sites and is an unstable protein.It is predicted that Zm DRG16 protein is located in chloroplast and may be involved in photoperiodic reactions regulated by blue light and ULTRAVIOLET A.The Zm DRG19 gene encodes a protein with 411 amino acid residues,which has 34 possible phosphorylation sites and is a stable protein.It predicts that the Zm DRG19 protein is located in the mitochondria,and its function is related to glutamate dehydrogenase.It was found that the proteins encoded by the four maize dark response genes were non secreted hydrophilic proteins without transmembrane movement,and the four maize dark response genes all had many cis acting elements related to light response.It was speculated that the four genes might participate in the photoperiod response of maize and affect the growth and development of Maize;(3)Using the technology constructed by the carrier,the fusion expression vector PA7-GFP-Zm DRG12,PA7-GFP-Zm DRG15,PA7-GFP-Zm DRG16 and PA7-GFP-Zm DRGD2 were obtained with p A7-GFP as the skeleton.Using rice protoplasts for subcellular localization,it was found that four maize dark response gene proteins were all located in chloroplasts.(4)Three heterologous expression vectors PCAMBIA-1301-Zm DRG12,PCAMBIA-1301-Zm DRG16 and PCAMBIA-1301-Zm DRG19 were obtained with PCAMBIA-1301 as the skeleton.The dark response genes of maize were transferred to Arabidopsis thaliana by agrobacteria-mediated inflorescent impregnation.The transgenic Arabidopsis lines L1,L2,L3,L5 and L7 were obtained by resistance screening and PCR verification.It was found that Zm DRG12 transgenic Arabidopsis plants had the phenotype of advancing bolting.Southern blot and RT-q PCR were used to detect Zm DRG12 transgenic Arabidopsis line L2.It was found that Zm DRG12 was highly expressed in Arabidopsis thaliana.It is believed that the high expression of Zm DRG12 in Arabidopsis is the reason for bolting advancing in transgenic Arabidopsis.(5)The expression pattern of Zm DRG12 gene in maize B73 was obtained by RT-q PCR.It was found that the expression of Zm DRG12 has a circadian rhythm and is highly expressed in the dark,confirming that Zm DRG12 is a dark response gene.