The Function And Expression Regulation Of Terpene Synthase Gene TPS6 In Zea Mays

As a staple crop and industrial raw materials,maize has an important role in national economy.Disease,herbivore and drought are the key factors to maize yield loss and quanlity.The results have showed that terpene synthases play an important role in stress responses in maize.There are a lot of researches focus on the maize terpene synthase genes during the past three decades,however,we have known little about the function and expression regulation of terpene synthase genes.The TPS6 gene and its promoter were the main research object in the study.The purpose was to elucidate the function and expression regulation mechanism of TPS6 gene.Quantitative real-time PCR was used to systematically detect the expression ofTPS6 gene in maize inbred line B73 seedlings.The expression of TPS6 gene was extremely low in the leaf of maize seedlings under normal conditions.After the jasmonates,salicylic acid,abscisic acid,ethylene and JA/ET treatment,the expression of TPS6 was no significant raise in the leaf of maize seedlings.There was also no significant increase in TPS6 expression in leaf after herbivore(Armyworm)treatment.However,TPS6 gene in leaf was significantly induced after Ustilago maydis treatment,and its expression increased significantly with the time extension,reaching the peak on the 6th day.Under normal conditions there was a certain expression of TPS6 gene in maize seedlings root.After treated with different hormones,the expression of TPS6 was intensely induced only by ABA.We detected the TPS6 expression was increase in root after armyworm or U.maydis treatment leaf It showed there was a signal transmission process from leaf to root induced the expression of TPS6.A high-throughput screening of an Arabidopsis transcription factor library using the region from-400 to-1 of the TPS6 promoter as the bait identified DREB1A transcription factor.Through maize transcriptome sequencing analysis,we got 34 from 2332 transcription factors which had the same expression pattern with TPS6.The 34 transcription factors mainly concentrated in the AP2/ERF(9),bHLH(7),MYB(4)and WRKY(4)subfamily.The validation of transcription factors was currently under way.The TPS6 promoter sequencing results of 126 maize inbred lines showed that the region from-100 to-1 of TPS6 promoter was conservative among the 126 inbred lines.Compared with B73,the region from-100 to-1 of TPS6 promoter in 73 inbred lines was consistent with B73,while 53 inbred lines had a 8 bp(CATGCATC)deletion in the region.The 8 bp deletion contained a RY-repeat cis-element.The proportions of 8 bp deletion in different breeding subpopulations and adaptable inbred lines were great difference which might be due to the selection results during the process of maize domestication.Analysis of TPS6 expression in 126 maize inbred lines showed that TPS6 gene was not expressed in inbred lines which had 8 bp deletion in TPS6 promoter under normal conditions or ABA treatment It indicated that the 8 bp sequence for TPS6 expression was very important.Plant expression vectors of TPS6 overexpression and CRISPR-Cas9 were constructed to obtain transgenic maize.GC-MS analysis showed that ?-macrocarpene and its derivative zealexin-Al could be detect in the TPS6-OX transgenic plants,while the related products were not detected in the TPS6-CRISPR transgenic plants.Compared with B73,the drought and disease resistance of TPS6-OX transgenic maize had significantly increased,but the insect resistance had no obvious difference.This result had a certain relevance with the qPCR result.In the next step,we would analyse the mechanism of PS6 gene in different tissues(aboveground and underground)participating in diifferent anti-adversity processes(biotic and abiotic).All ofthe results in this study demonstrated that the expression of TPS6 ne in maize was mainly controlled by ABA signal pathway.The Arabidopsis thaliana DREB1A transcription factor could bind to the TPS6 promoter.The 8 bp sequence(CATGCATC)in TPS6 promoter was polymorphic in maize inbred lines,which was very important for the expression of TPS6 gene.TPS6 overexpression could improve the drought tolerance,disease resistance in maize,but no obvious effect to insect resistance.When knocked out TPS6 in maize,it showed more susceptibility to disease.It was great significance to analyze the expression regulation mechanism of terpene synthase gene TPS6 and to cultivate anti-adversity maize varieties.