Transcriptional Level Analysis Of Four Water Deficit Responsive Genes In Various Genotypic Maize Seedlings

To further understand how maize responds to drought stress,we constructed the cDNA library that maize seedling specially responds to water stress utilizing one drought-tolerant inbred "81565",and have identified 5 EST sequence.After the blast analysis,we identified the characters of three of them.One is the tryspin-like serine proteases gene(DegP),others are the cofactor-independent phosphoglycerate mutase (PGAM-i) and the gene encoding the G subunit of NADH dehydrogenase complex. Other two genes were designated as E4 and F4,without clear function.Tryspin-like serine proteases(DegP) widely involved in heat stress response of eukaryote and prokaryote,also take part in repair action against photoinhibition of higher plants.The AtDegP1,AtDegP5,AtDegP8 and AtDegP2 enzymes in Arabidopsis degrade the photodamaged D1 protein to rescue the PSâ…¡photosynthesis.One facultative halophyte,its name is ice plant(Mesembryanthemu crystallinum).The PGAM-i gene of this plant,which transcriptional level is induced by several abiotic stress.The encoding protein of E4 gene is a membrane integration protein with DUF6 and TPT domain.To disclose how the 4 maize genes,DegP,PGAM-i,E4 and F4 response water stress,we employed real time quantitative PCR technology and analyzed 4 genes expression profile of 6 maize inbred lines under water stress(20%PEG treatment).The 6 maize inbred lines "81565","87-1","R09","200B","Dan340" and "ES40" have different drought-tolerant ability in field surrounding.DegP gene has been identified that takes part in heat shock response and repair action against PSâ…¡photoinhibition of higher plants.For finding out the function of DegP gene in maize drought-tolerance,we cloned the maize DegP gene.Based on the DegP gene sequence,we analyzed the primary character of DegP gene using biological information method.Our results are mainly as follows:1.Utilizing the drought-tolerant maize inbreed "81565",the techniques of Suppression subtractive hybridization(SSH) and reverse Northern hybridization,we isolated and identified 5 up-regulated EST sequence from the water stressed maize seedlings.Three of them are the genes encoding DegP,PGAM-i,and the G subunit protein of NADH dehydrogenase complex,respectively.The functions of others are not identified.2.The expression of DegP,PGAM-i,E4 and F4 genes respond to water deficit in drought-tolerant inbred "81565".DegP,PGAM-i,E4 and F4 genes express certain level under normal condition.After 6 h of PEG treatment,water stress repressed the expression of 4 genes.Their expressional level all decreased compared with the level of control treatment.But the expressional level up-regulated with the treatment time elongation.PGAM-i gene expressional level was 1.4 times compared with control level after stress treatment 12 h.DegP gene expression increased to 0.6 times after stress treatment 24 h.The expressional level of E4 and F4 genes were up-regulated and were 1.2 times and 1.6 times compared with control level after 24 hours, respectively.3.The 4 genes expression profiles of 6 maize inbred lines under water stress have similarity and difference.The 4 genes expression profiles of drought-tolerant lines "87-1" and "R09" were similar with "81565" line.The expression of DegP gene of drought-tolerant line "200B" was repressed by water stress,but after stress 24 h, the expressional level of PGAM-i was induced and was 6 times compared with the control level,the expression level of E4 and F4 genes were induced to 2.6 and 2 times compared with the control level,respectively.The DegP and E4 genes expression of Drought-sensitive "Dan340" were down-regulated.After 24 h treatment,the PGAM-i gene expression level recovered,F4 gene expression was induced to high level.The 4 genes expression in another drought-sensitive inbred line "ES40" were all repressed, the expression level decreased along the treatment time.The sign also indicated the drought-sensitive character of"ES40" line in molecular level.4.This research based on the EST sequence of DegP,cloned the DegP gene from the inbred 87-1.We have identified the deletion mutation occurred in the 87-1 DegP gene,and analyzed the sequence diversity at the mutation site.Alignment analysis identified the DegP gene is the homolog of Arabidopsis AtDegP8.the identity of amino acid sequence between them is 66%.Other member of DegP gene family was also found in maize genome,which amino acid sequence was more similar with Arabidopsis AtDegP1 gene,the N terminal amino acid sequences of them were aligned,the identity is 53%.The research demonstrated that DegP,PGAM-i,E4 and F4 gene play a role in maize drought response,and their expression level contribute to the plant tolerance to water stress.The comparative analysis between 6 inbred lines revealed the different regulation model for the 4 genes.The 4 genes up-regulation in 3 drought-tolerant lines "81565","R09","87-1" indicates the 3 inbred lines have rapid regulation ability.The 4 genes up-regulation in line "ES40" were repressed along the treatment time,this is accordant with the drought-sensitive trait of"ES40".The DegP family genes in higher plants contribute to the acquirement to adapting abiotic stress.The high identity of amino acid sequence between maize DegP and AtDegP8 suggest that the maize DegP is the ortholog of AtDegP8.The AtDegP1, AtDegP5,AtDegP8 proteins locate in the lumen of thylakoid,the AtDegP2 protein locates in stroma.The four proteins and the FtsH protein together eliminate the damage by photoinhibition,and recover to the normal photosynthesis activity.These experiments showed that DegP gene family play important role in maize drought tolerance,and in maize production under water stress.The maize DegP gene have different genotype,one is the deletion mutation allele in inbred line "87-1",which presumed that it could not synthesis the intact structural and functional DegP protein.The relationship between this genotype and the ability of drought tolerance in different lines is still deciphered.