| Perilla frutescens L.Britt,an annual herb in the Lamiaceae family,is a traditional Chinese medicine for both medicine and food in my country.Perilla leaves,perilla stems,and perilla seeds are all included in the 2020 Chinese Pharmacopoeia.Perilla has a variety of pharmacological activities,and its main chemical components are volatile oils,flavonoids,and phenolic acids.Ro Smarinic acid(RA)is the main phenolic acid component in perilla and also as one of the indicators for evaluating the quality of perilla.The research on the pathway of phenolic acids in perilla is the prerequisite for the quality control of perilla,the breeding of varieties,and the development of new resources.The hairy root culture system has the characteristics of short growth cycle,simple culture conditions,high and stable secondary metabolite content,etc.It is the preferred system for research on the pathway of perillic acid and also the large-scale production of perillic acid.Reliable system.In this study,the hairy root induction culture system and genetic transformation system of perilla were established,and the content of phenolic acids was determined.At the same time,the effects of different elicitors on the accumulation of phenolic acids were investigated.It provides a basis for analyzing the synthesis mechanism of phenolic acids in perilla and conducting research on quality control of perilla.The main findings are as follows:1.By investigating the infiltration time and co-cultivation time of Agrobacterium rhizogenes A4,R1000,C58C1 on perilla leaf discs,it is determined that the optimal induction conditions are Agrobacterium rhizogenes C58C1,15 min infusion,and co-cultivation for 3 days.The growth cycle of perilla hairy roots is 8 weeks.The hairy roots grow slowly in the first two weeks.The hairy roots grow in logarithmic growth period from 2 to 6 weeks.After the 7th week,the hairy roots grow in a stagnant period.2.The content of phenolic acids in the obtained 6 excellent monoclonal strains was determined.The results showed that the accumulation of 5 kinds of phenolic acid compounds in individual hairy roots is quite different.The P5 root system had the highest RA content,reaching 19.084 mg/g(dw),and the P4 strain had the lowest RA content,only 0.163 mg/g(dw),and the P4 strain did not contain caffeic acid ethyl ester.The content of caffeic acid,ferulic acid and ethyl caffeate in the P2 strain were the highest at 0.493,0.063,and 0.543 mg/g(dw),respectively;while the protocatechualdehyde was the highest in the P1 strain at 0.047 mg/g(dw).3.In this experiment,a genetic transformation system of perilla hairy roots was successfully established,and a transgenic line of perilla hairy roots overexpressing PHB,SmLAC20 and SmLAC28 genes was obtained.4.MeJA elicitor can affect the phenolic acid components of perilla hairy roots.When treated with MeJA elicitor for the 3rd day,the content of RA showed an upward trend,reaching the maximum value of 17.364mg/g(dw).However,at the third day after treatment.The content of caffeic acid showed a downward trend on the 6th and 9th days;the accumulation of caffeic acid gradually increased,and the maximum content reached 0.487mg/g(dw)on the 9th day after the treatment;the caffeic acid ethyl ester reached the maximum on the 6th day after the induction treatment 0.394mg/g(dw);the content of protocatechualdehyde and ferulic acid has no obvious change and the change is very Small.5.SA also has a certain effect on the phenolic acid components of perilla hairy root.After SA induction,the RA content reached the highest value of 20.919 mg/g after 9 days of induction;protocatechuic aldehyde,caffeic acid,ferulic acid,coffee The content of ethyl ester rose to the maximum on the 9th day of induction,which were 0.030,0.626,0.624,and 0.770 mg/g(dw),respectively. |
