Studies On The Heterologous Expression Of The Arabidopsis Orange Gene In Tobacco Trichomes

The Orange(Or)gene was originally cloned from a natural mutant of cauliflower(Brassica oleracea var.botrytis)with an orange curd,which induces the transformation of the leucoplasts of the plant cell into a chromoplasts.It can also promotes the accumulation of a large amount of carotenoids in the plant tissue to improve nutrition values and have economical values.The surface of tobacco leaves contains many epidermal hairs,and the amount of trichome accounts for about 85%of the total epidermis.Trichome is an important synthesis and storage organ of secondary metabolites of plants.Tobacco is a model plant for the study of metabolic engineering of terpenoids due to its ability to synthesize important terpenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate(IPP,DMAPP).At present,the function of Or gene has been well-demonstrated.This protein regulates a series of important metabolic processes,but its molecular function in the trichome needs to be discovered.In this study,the trichome-specific promoter is used to express Arabidopsis thaliana Or(AtOr)in Nicotiana benthamiana trichome,which leads to the change of metabolites.This study tends to reveal the mechanism of Or,and has great theoretical value and great potential for application.The Arabidopsis thaliana OR protein contain a predicted chloroplast transit peptide sequence in amino terminal.We constructed two subcellular localization vectors of full-length AtOR and a truncated version of AtOR with the predicted chloroplast transit peptide removed.The subcellular localization results showed that the full-length AtOR was located in the chloroplast of the cell,while the truncated version of the AtOR that removed the predicted chloroplast transit peptide was attached to the chloroplast surface.A promoter of DBR2 was cloned from the genome of Artemisia annua,and AtOr and the truncated version of AtOr?cTP gene were inserted into the binary expression vector.The length of DBR2 promoter was 1797 bp.The full-length AtOr and its truncated version of the removed signal peptide(AtOr?cTP)were connected behind the promoter and then loaded into the binary expression vector.Were obtained 19 lines with overexpressing full-length AtOr and 7 lines with expressing AtOr?cTP by leaf-plate transgenic technology.The growth phenotype of transgenic tobaccos was normal,and the transferred gene did not inhibit their growth.Positive plants were detected by PCR amplification.The corresponding gene bands could not be amplified from the wild-type tobacco plants,but a size-conforming band was amplified from the transgenic tobacco,indicating that the target gene was successfully transferred to tobacco.Real-time fluorescence quantitative PCR was used to identify the positive plants.In difeerent transcription of AtOr lines,the transcription levels of AtOr were 37.21-254.45 times of NbOr.From AtOr?cTP-1 to7 lines,the transcription levels of AtOr?cTP were 34.63-143.78 times of NbOr.The trichome of T2 generation transgenic tobacco were brushed with frozen brush,and the RNA was extracted and then reversely transcribed into cDNA followed by fluorescent quantitative PCR.The results showed that the target genes transcription levels of AtOr-5-1 to AtOr-8-3 were 1.86-15.30-fold of wild-types.The target genes transcription levels of AtOr?cTP-1-1 to AtOr?cTP-2-3 were 0.91-2.62 times of wild-types in trichome.Scanning electron microscopy showed no significant differences in the morphology and morphology of glandular hair on tobacco surfaces.Real-time fluorescent quantitative PCR is used to detect the transcription levels of related genes in the carotenoid metabolic pathway and found that the transcription level of the geranylgeranyl diphosphate synthase(geranylgeranyl diphosphate synthase,GGPPS)gene was up-regulated.The GGPPS transcription levels of AtOr-5,AtOr-8,AtOr?cTP-1 and AtOr?cTP-2 were 3.72,20.17,5.56,and 7.58 times higher than those of wild type,respectively.Extraction of wild-type and transgenic tobacco pigments for analysis,the results showed that the content of chlorophyll and carotenoids in transgenic tobacco increased compared to wild type.The content of ?-carotene in AtOr-5 and AtOr-8 increased by 49.32%and 12.00%respectively compared to wild type.The content of chlorophyll a in AtOr-5 and AtOr-8 increased by 57.07%and 6.60%compared with the wild type respectively.The content of chlorophyll b in AtOr-5 and AtOr-8 increased by 82.67%and 20.01%respectively compared to wild type.The research shows that the full-length AtOR protein with signal peptide can induce the chloroplasts,which promotes the carotenoid synthesis in transgenic tobacco leaves.At the same time,our study also showed that although the artemisinin biosynthesis occurs in trichome,its role in the promoter of DBR2 gene is more pronounced in leaves.The specific mechanism of DBR2 Promoter to be further studied.