Cloning And Functional Analysis Of Histone H3K36 Methyltransferase In Wheat

Wheat is one of the most commonly used crops in the world.Flowering is essential for its developmental transition from vegetative to reproductive stages.The flowering time regulaton is important for wheat,which directly affects grain yield.Studies had shown that histone methylation modification is closely related to the regulation of flowering time.Histone methyltransferase(SET Domain Group,SDG)is the key factor of histone methylation modification.Here,we isolated the Ta SDG7,Ta SDG8,and their homoeologous(A,B,D).Ta SDG7 and Ta SDG8 both could participate in the methylation modification of H3K36.The temporal and spatial expression pattern of identified genes were analyzed.The gene silencing based on barley stripe mosaic virus(BSMV)and fuctional recovery experiment using Arabidopsis mutants sdg7/8(+ /-)were operated,which preliminarily validated the biological function of SDG7/8 in developmental regulation.The main findings are as follows:1.Combining bioinformatics and RT-PCR technology,homoeologous(A,B,D)of Ta SDG7/8 were identified and their c DNA sequence characteristics were analyzed.Six homoeologous of Ta SDG7 were localized on the long arm of chromosome 5 and 7,respectively.According to the location,named as Ta SDG7-5AL,Ta SDG7-5BL,Ta SDG7-5DL and Ta SDG7-7AL,Ta SDG7-7BL,Ta SDG7-7DL,respectively.Genomics analysis indicated that Ta SDG7 were composed of 11 exons and 10 introns.Ta SDG7-5AL/BL/DL encoded 261,362 and 447 amino acids,respectively,with the molecular weight of 30.39 k Da,41.35 k Da and 50.86 k Da.Ta SDG7-7AL/BL/DL encoded 338,340 and 338 amino acids,respectively,with the molecular weight of39.08 k Da,39.38 k Da and 39.04 k Da.Structural analysis of Ta SDG7 protein indicated each of the six homeologues had two identical SET and AWS conserved domains,which play important biological functions in the process of histon methylation.Three-dimensional structure predictions revealed that Ta SDG7-5DL was composed of three α-helices and ten β-sheets.The Ta SDG7-5AL/BL has 186 and 86 amino acids deletions as compared to Ta SDG7-5DL,respectively,the deletions of one α-helix and two β-sheets at the N-terminal were found.Ta SDG7-7AL /BL/DL were all composed of four α-helices,thirteen β-sheets and irregular curl.Three homoeologous of Ta SDG7-5L may play different functions during the processes of grain development.Homoeologous of Ta SDG8 were localized on the short arm of chromosome 2,named as Ta SDG8-2AS,Ta SDG8-2BS and Ta SDG8-2DS,respectively.Genomics analysis indicated that Ta SDG8-2AS/BS were composed of 17 exons and 16 introns,and Ta SDG8-2DS was composed of 11 exons and 10 introns.Ta SDG8-2AS/BS/DS encoded 1088,1070 and 707 amino acids,with the molecular weight of 120.68 k Da,119.46 k Da and 78.00 k Da,respectively.Structural analysis of Ta SDG8 protein indicated that homoeologous of Ta SDG8 contain three conserved domains,namely SET,AWS and zf-CW.As compared to Ta SDG7,the Ta SDG8 have two identical conserved domains(SET and AWS,which implied that both of these genes have similar functions.Ta SDG8 contains the zf-cw conserved domain,which could specially binds to DNA and promotes the interaction between proteins.The sturcture differce between them might indicated that the transferase activity of Ta SDG8 with zf-cw domain were increased.The mechanism will be verified in the further experiment.Three-dimensional structure predictions revealed that the three homoeologous of Ta SDG8-2S were all composed of two α-helices and thirteenβ-sheets.Evolutionary tree cluster analysis showed that Ta SDG7-5AL/BL/DL,Ta SDG7-7AL/BL/DL and Ta SDG8-2AS/BS/DS all belong to the fifth subfamily of the SDG family,namely the Suv subfamily.2.In order to verify the activity of Ta SDG7 and Ta SDG8 in vitro,a prokaryotic expression vector was constructed.SDS-PAGE results revealed that the fusion protein GST-Ta SDG7-5L was expressed successfully,with the molecular weight of 66.0 k Da.But the expression level of Ta SDG8-2S was very low.The expression conditions will be further optimized and the activity expression in vitro will be manipulated.3.To investigate temporal and spatial expression patterns,the c DNAs of roots,stems,leaves,embryos,endosperm and dried seeds islated from "Jing 841" and "Chinese Spring" were used as templates.q RT-PCR results indicated that the highest expression level of the homologous of Ta SDG7-5AL/BL/DL and Ta SDG8-2AS/BS/DS were detectedin leaves,the lowest expression level were found in endosperm.further analysis showed that the expression level of Ta SDG7/8-B was higher than that of Ta SDG7/8-A and Ta SDG7/8-D,which might indicated that the Ta SDG7 and Ta SDG8 derived from the B genome might play an important role in wheat.The expression level of Ta SDG7-7AL/BL/DL on chromosome seventh were very low,which implied that Ta SDG7-7AL/BL/DL might not be involved in the regulation of wheat development.4.Vernalization is an important pathways to control the flowering time in wheat.The winter-type “Jing 841”,the semi-winter “Zheng Yumai 9987” and the spring“Chinese Spring” were used as materials to explore the response of Ta SDG7/8 to vernalization treatment.q RT-PCR results showed that the expression of Ta SDG7-5L was up-regulated with shorter time in “Jing 841”than that of in “Zheng Yumai 9987”and “Chinese Spring”,which might be implied that Ta SDG7-5L was closely invloved in the vernalization pathway.Further analysis found that expression of Ta SDG7-5AL were the highest has and expression of Ta SDG7-5DLwere the lowest during the process of vernalization treatment.The expression of Ta SDG8-B was higher than that of Ta SDG8-A/D,indicating that the B genome of Ta SDG8 plays an important role in Vernalization pathway.5.Seed is not only an important organ for storage but also an essential seeding material.Therefore,the regulation of seed development and germination process has important influence on the growth and development of wheat.“Chinese spring” and“Zheng Yumai 9987” were used as material,the expression characteristics of Ta SDG7/8 in wheat grain development showed that Ta SDG7-5L was up-regulated in“Zheng Yumai 9987”,and the expression level reached to the highest at 20 d,which is the key period for grain filling and is related to the size of grain formation.The time of high expression of Ta SDG8-2S in “Chinese spring” was earlier than in “Zheng Yumai 9987” at 15 DAP,indicating that the different expression pattern of Ta SDG8-2S may be closely related with the regulation in grain size.During seed germination period,q RT-PCR results demonstrated that the highest expression level of Ta SDG7-5L was observed at 24 h after imbibition.The expression level of Ta SDG7-5L in germinating 12 h embryos was the highest in "Chinese Spring".For three homologous genes,the expression level of Ta SDG7-5 AL is higher than that of Ta SDG7-5BL and Ta SDG7-5DL.The expression level Ta SDG8-2BS >Ta SDG8-2A/DS were found,which indicated that the roles of A,B and D homologous genes are different during seed germination.The fuction of themduring the developmental will be further explorated.6.Ta SDG7/8 was overexpressed in Arabidopsis mutants sdg7/8(+ /-),and four strains of Ta SDG8 transgenic Arabidopsis have been screened.In the next step,the progeny will be screened to obtain pure transgenic plants.Meanwhile,gene silencing techniques mediated by barley stripe mosaic virus(BSMV)was worked in wheat.q RT-PCR analysis showed that the expression of Ta SDG8 was significantly down regulated after inoculation with BSMV-Ta SDG8,indicating that the target gene was degraded,but no phenotypic was founded compared with the control.On the base,the the number of tillers increased obviously and obvious dwarf phenotype was observed when the expression level of Ta SDG7 and Ta SDG8 were suppressed simultaneously by using BSMV-Ta SDG7 and BSMV-Ta SDG8 inoculation.This results indicated that Ta SDG7 and Ta SDG8 might have complementary effect,but the mechanism needs to be found in further study.RNA interference vector of Ta SDG8-2S was constructed to obtain a permanent transformant.Now,a number of RNAi transgenic materials of T0 generation have been obtained and the molecular identification of T1 generation are still undergoing.Next,RNA interference vector of Ta SDG7 will be constructed,and transgenic materials with double mutant of Ta SDG7 and Ta SDG8 will be screened,which will be used to verify the biological function of Ta SDG7 and Ta SDG8 in developmental regulation in wheat.