Functional Analysis Of Stress-responsive Gene OsUF3 In Rice

With the rapid development of genomics,the sequencing data of many unknown functional genes has attracted wide attention.With the successful decipherment of many biological genes,a breakthrough has been made in the study of genomics.Therefore,these unknown functional genes of rice are considered.It is an integral part of its genetic blueprint.At present,approximately 20% to 40% of the genes encoding proteins in all eukaryotic genomic sequences belong to unknown functional genes.To understand the mechanism of adversity response in rice and discover new rice stress response genes,we used the Affymetrix Rice Gene Microarray(containing 51279 transcripts)to analyze gene expression profiles of rice Pei'ai 64 S under drought,low temperature,and high temperature stress.A candidate gene of interest was screened among stress-response genes.Because it has no obvious structural features and no relevant reports,it was named the unknown functional gene OsUF3(Oryza sativa unknown function).Analysis of GEO expression data showed that OsUF3 was mainly induced by drought and low temperature.To further verify the role of OsUF3 gene in stress,over-expression vector(D-163+1300: OsUF3),antisense-suppressed expression vector(D-163+1300: OsUF3),Green Fluorescent protein vector(D-163+1300: OsUF3-GFP)and promoter vector(p CAMBIA3301:OsUF3-P)were constructed.The binary expression vector was used to transfer these chimeric genes into rice Nipponbare by Agrobacterium-mediated transformation.The results of the research were as follows:1)According to the bioinformatics analysis,OsUF3 has no intron;the open reading frame is 558 bp and encodes 186 amino acid residues;the molecular weight of the protein is 43.25 KD,and the isoelectric point is about 4.95.2)Analysis of promoter regions upstream of OsUF3 revealed multiple cis-acting elements associated with stress responses,such as TGACG-mofit,GC-motif,HSE,et al.3)Real-time fluorescent quantitative analysis showed that the expression of OsUF3 was lowest in ABA treatment,highest in drought treatment,and more regular in Na Cl treatment;OsUF3 gene expression increased under high temperature treatment.4)Agrobacterium-mediated D-163+1300: OsUF3-GFP vector was transiently expressed in tobacco leaves.The results showed that OsUF3 was located in the nucleus.5)Stress experiment results showed that OsUF3 over-expression transgenic line Ox-2 was sensitive to salt and low temperature stress but insensitive to ABA.6)The GUS activity of OsUF3 promoter transgenic plants showed that under normal growth conditions,the expression of OsUF3 was not significantly different from that of control Nipponbare;the root color of OsUF3 promoter transgenic plants after GUS staining became darker after 6 days of low temperature treatment(blue Color),while the control group,Nipponbare,showed no significant change,indicating that OsUF3 expression was up-regulated in roots at low temperature.7)Field statistics showed that the plant height of mature plants of Nipponbare and OsUF3 overexpressing lines Ox-2 and Ox-4 were 91.5,75.5,and 77 cm,respectively.The panicle length of the three were 18.15,17.6 and 14.83 cm,there were no significant difference between Ox-2 and Nipponbare,but Ox-4 was significantly shorter than Nipponbare;the lengths of panicle neck were 6.3,2.05,and 1.53 cm,respectively,and Nipponbare had a longer than overexpressed lines.In addition,over-expressed lines mature was late,and the kernel wasn't full-filled.In this study,overexpressing strains showed sensitivity to low temperature and Na Cl treatments compared to Nipponbare,and the center of gravity for later work will be placed on transcription factors associated with low temperature and salt stress to explore the mechanism and signaling pathways of OsUF3 in response to stress.Subcellular localization could not be successfully localized in rice protoplasts.In the later work,it strived to localize in rice cells to verify whether the gene was also located in the nucleus in rice,supporting the results of tobacco transient expression.At present,observations of agronomic traits have been performed on field data and it has been found that there are significant differences between them and the control.However,this is only a single set of data for the T2 generation,which may be caused by the overexpression of the target gene,or may be due to external experimental conditions,such as tissue culture.Next,we plan to observe the statistics of T3 and T4 generations and analyze whether the phenotype is stable to the offspring.In addition,we also plan to hybridize the T3 generation with the relevant phenotype to the control Nipponbare to determine whether the trait is caused by overexpression or mutation.