| Glycyrrhiza uralensis Fisch is a medicine plant that belongs to the genus Leguminous Glycyrrhiza linn.It is used as a traditional Chinese herbal medicine for suppressing cough and relieving spleen.Liquritin is one of the two indexes for quality evaluation for licorice in the Pharmacopoeia of the People's Republic of China.At present,the low content of liquiritin is commonly found in artificially cultivated licorice,which can not meet the standard of medicine requirement.To reveal the function of key genes in the synthesis pathway of liquiritin is to be an important prerequisite for quality breeding of licorice.Synthesizing liquiritin through hairy root culture is also an important way to alleviate the contradiction between supply and demand of liquiritin.In this paper,chaconne isomerase gene was induced into hairy roots mediated by Agrobacterium rhizogenes.The effects of strain type,infection time and co-cultivation duration on the hairy roots transformation rate were investigated.Root fresh weight and liquiritin content were selected as screening indicators of excellent monoclonal roots;basic medium selection,different pH value treatment and YE elicitor treatment were performed to establish an efficient culture system.The main research findings were as follows:1.Induction and Identification of CHI-OE Hairy Roots of Glycyrrhiza uralensis Fisch: Agrobacterium rhizogenes K599 and Aqua1 were used to infect the cotyledonary nodes and the infestation time and co-cultivation duration was investigated.The results showed that the induction rate and the transformation rate of A.rhizogenes K599 were higher than that of Aqua1,which was 86.5% and 76.2%,respectively.The suitable time for Agrobacterium to invade the explants was 20 min,and the co-culture time after infection was 3 days.Compared with Aqua1,hairy roots induced by K599 initiated 1-2 days earlier,grew faster,stronger and branched more.2.Screening of excellent monoclonal roots: Screening of positive hairy roots was carried out based on fresh weight and liquiritin as indicators.Most of the roots grew slowly or even stagnant after subculture,and 8 root lines were screened for stable growth and higher liquiritin contents than GUS-OE control roots were detected followed HPLC methods.Among them,C11 roots grew fastest with a maximum fresh weight of 4.73 g and liquiritin content of 0.054%,which was 2.3 times that of the control,while C44 had the highest liquiritin content,0.059%,2.6 times that of the control.qRT-PCR results showed that the relative expression levels of CHI genes in root C44 and C11 were higher,19 and 17 times that of the control,respectively.3.Optimization of the culture system: Using C44 as a material,the basic medium,pH value,the concentration of the inducer,and the addition time of the inducer were used to analyze the fresh weight of hairy roots and the yield of liquiritin.The results showed that the growth rate of C44 root was the fastest in 1/2MS liquid medium,the fresh weight was 4.79 g,the proliferation multiple was 15.96 times,and the content of liquiritin was 0.059%;the growth rate of hairy roots and the accumulation of liquiritin at different pH values.The highest content in hairy roots was 0.058% at 5.8,fresh weight was 4.91 g,and the proliferative multiple was 16.37 times;the effect of YE concentration and time on the growth rate of hairy roots was not significant,but the treatment of 0.1 mg/mL and adding on the 10 th day significantly increased the content of liquiritin,1.2 times over that of the control.In this study,a hairy root induction system of the chalcone isomerase overexpression gene of licorice was established,and the fine hairy root system C44 with higher growth rate and glycyrrhizin content than the control was screened,and a suitable culture system was established.This result will lay the foundation for the synthesis of liquiritin and the study of the functions of liquiritin metabolism-related genes. |
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