Functional Analysis For ZmES32 Of Maize NAC Gene Family That Is Specifically Expressed In Endosperm

Corn is an important cereal,fuel and fodder crop in the world,and China is one of the world’s largest countries in corn producing and consuming.Corn occupies an important position in the national economy,not only is the energy source of human and animal,but also the raw materials in medical attendance,brewing and chemical and many other industries.Starch is the major composition of maize endosperm,which,accounting for about 80% of corn grain,is the key factor to maize yield and quality and the important parts of storage of nutrients.It is of great significance to the genetic improvement of crop quality and cultivating high yield and perfect quality of corn,through carrying out the research of corn starch biosynthesis pathways and digging starch synthesis-related genes.Based on the result of screening the endosperm-specific expression transcription factors by genome-wide association analysis,we cloned the maize endosperm-specific candidate gene ZmES32,and detected its function in starch synthesis metabolic pathways by molecular biology techniques.The major results are as follows:(1)Bioinformatics analysis and forecast for the endosperm-specific candidate gene ZmES32 indicates that it belongs to the NAC transcription factor family,with the cDNA sequence length of 1176 bp,and encodes 391 amino acids,isoelectric point 5.81 and molecular weight of 42.13 KDa.ZmES32 contains the nuclear export sequence,while has no a signal peptide and obvious transmembrane structure.Transcriptome sequencing data shows that ZmES32 specificity expression in the endosperm after pollination.(2)The tissue-expression pattern analysis of ZmES32 was performed by qRT-PCR,which showed that ZmES32 was highly expressed in the 10 d,15d and 20 d endosperm after pollination,and nearly had no expression in other tissues.It was preliminary determined that ZmES32 was expressed in the endosperm specificity.(3)Subcellular localization analysis indicated that ZmES32 was a nuclear localization protein.Yeast one-hybrid experiment showed that ZmES32 had transcriptional activation activity,and the active region was located from 335 to 392 amino acids.DNA binding analysis showed that ZmES32 could specificity bind to the cis-element of CATGTG,which revealed ZmES32 was a classic transcription factors gene.(4)Statistical analysis for grain of transgenic rice showed that compared to the control zhonghua 11,the thousand seed weight,grain length and width,total starch content,soluble sugar content,amylopectin blue value and maximum absorption peaks were all decreasing,while the amylose content was increasing with a largest average difference of 2.81%,as well as the corresponding blue value of amylose and maximum absorption peak were higher than the control.In addition,scanning electron microscope analysis of rice grain revealed that the grain internal structure had changed and showed loose arrangement between starch grains and protein body,also with chalkiness phenomenon.These results indicated that ZmES32 made an impact on the size and weight of the transgenic rice,meanwhile,increased the amylose content.(5)The expression analysis for 17 starch synthesis-relative endogenous genes in the transgenic rice grain showed that these genes had changed in their expression.For example,the OsGBSSI,relating to amylose synthesis,showed higher expression lever in transgenic rice than in wild rice zhonghua 11.On the contrary,The expression level OsGBSSII,amylopectin synthesis-related gene,was decreased.These results revealed the ZmES32 was involved in the regulation of starch synthesis pathways in rice.