Study On Function And Expression Regulation Of Terpene Synthase Gene TPS2 In Zea Mays

Maize(Zea mays L.)is an important crop as food and industrial material.But insect pests have led to a significant reduction in maize production.Maize volatile terpenes can respond to a variety of biotic and abiotic stress,and different terpene synthases can catalyze the production of different terpenes with different roles.TPS2 has been reported that catalyze the production of linalool,(E)-nerolidol,DMNT and TMTT,but its function and expression regulation mechanism are unclear.In this study,TPS2 gene and its promoter were taken as the research object,the purpose was to clarify the biological function and expression regulation mechanism of maize terpene synthase gene TPS2.Bioinformatics analysis found that there were 18 terpene synthase genes reported in maize,and some terpene synthase genes showed low sequence similarity and different function.Using the TPS2 amino acid sequence BLAST found that there were genes with very high homology in other species(sorghum,rice,brachypodium,sibiricum,Arabidopsis),indicating the evolutionary conservation of TPS2.Different plant hormones(JA,SA,ET)and armyworms treated maize inbred line B73 seedlings.The q PCR results showed that both plant hormones and insect pests induced the expression of TPS2.The TPS2 overexpression vector was constructed and transformed into maize through Agrobacterium-mediated transformation.The transgenic maize was named TPS2 OE.The results of q PCR showed that the expression of TPS2 gene in both leaves and roots of TPS2 OE was significantly higher than that of B73;P450 enzyme gene CYP92C5 has almost no expression in B73 and TPS2 OE.The expression of another P450 enzyme gene CYP92C6 in leaves is significantly higher than that in roots,and the expression level of CYP92C6 in B73 and TPS2 OE are almost the same.We detected the content of TPS2 product by gas chromatography mass spectrometry(GC-MS),and found that the content of TMTT in TPS2 OE was significantly higher than that of wild-type B73.The results of the corn borer behavior selection experiment showed that the number of eggs laid on the leaves of TPS2 OE and B73 was almost the same;compared with the control group B73,the number of larvae on TPS2 OE seedlings was less and the damage area was smaller.In order to reveal the mechanism of TPS2 expression regulation,the cis-acting elements of the promoter were analyzed by bioinformatics.TPS2 promoter contains a variety of cis-acting elements in response to stress,including hormone response regulatory elements: JA response elements,SA response elements;drought and defense response elements.In order to further explore the TPS2 promoter functional segment and its mechanism of action,the 5’-end truncated promoter was constructed to a luciferase dual-fluorescent plant expression vector.The transient expression analysis of maize protoplasts revealed that-183 to-129 were the key functional segments of the promoter,and there were negative regulatory elements in-227 to-183.Using the-500 to-1 region of the TPS2 promoter as the bait sequence,Yeast 1 hybrid(Y1H)high-throughput screening of B73 leaf and root c DNA expression libraries and two maize transcription factors were found: CCAAT-DR1-transcription factor 11(cadr11)and b ZIP transcription factor 60(b ZIP60).In summary,TPS2 gene is induced by plant hormones and herbivorous insects.Overexpression of TPS2 gene can increase the resistance of corn borer.The required functional region of TPS2 promoter is-183 to-129,and negative regulatory elements were in-227 to-183.Yeast one-hybrid screened two candidate transcription factors of TPS2promoter: cadr11 and b ZIP60.The results laid a foundation for further exploring of TPS2 gene transcriptional regulation,and provided reference for the regulation mechanism of TPS genes and the control of insect pests.