Enhanced Accumulation Of Solanesol In Hariy Root Of Nicotiana Tabacum Following Elicatation

Objective:In addition to manufacturing cigarettes,tobacco is an important raw material of solanesol,a drug precursorof preventing cardiovascular diseases?coenzyme Q10?.The content of solanesol in tobacco leaves was 0.3%-3%.Because tobacco leaves contains abundant of secondary metabolites,it is difficult to extract high purity solanesol.Hairy roots grow rapidly and can synthesize secondary metabolites.In this study,four types of Agrobacterium rhizogenes strains were used to infect tobacco leaves to induce hairy roots,Using elicitors as regulators to study the growth of tobacco hairy roots and solanesol accumulation,we explored suitable strains for tobacco hairy root induction and screened the tobacco hairy root growth and solanesol accumulation Induce better elicitors.Methods:In this study,there are four Agrobacterium rhizogenes strains were used to screen tobacco hairy roots using tobacco sterile leaves.We went on optimizing the initial inoculation amount and basic mediums in order to obtain the tobacco hairy root with high growth rate and high solanesol content.The Agrobacterium rhizogenes gene rolB was detected by PCR.In order to improve the growth of tobacco hairy roots and the content of solanesol,we added 5?M,10?M,20?M,50?M H₂O₂ and 10?M,50?M,100?M and200?M SA in basic medium at 0,9d and 18d,respectively.Finally,the expression level of the key enzymes of solanesol biosynthesis were detected by qRT-PCR.Results:1.Four Agrobacterium rhizogenes strains?R1601,A4,LBA9402,15834?were used to infect tobacco leaf.A total of 276hairy root lines were successfully established andthe highest transformation efficiency was 97.6%using strain LBA9402with OD=0.6.According to the growth and main secondary metabolites,we selected 15 hairy roots with rapid growth and more lateral lines for further study.Among them,hairy root line 2262produced up 9.441±0.388 gdry weight.The levels of nicotine,chlorogenic acid and isolanesol were27.84±2.28 mg/g,8.494±0.499 mg/g,0.687±0.034 mg/g,respectively.2.In order to optimize the culture conditions,the effects of initial inoculation amount and basal medium on the growth of tobaccohairy root were considered.The optimum conditions for growth of hairy roots were as follows:when the inoculation amount was 200the hairy roots grownrapidly.The growth of tobaccohairy roots reached 12.0±0.25g/Lwhen B5 was used as basal medium.3.Elicitors which were added to the culture had a stimulating effect on the tobacco hairy root growth and solanineaccumulation.The induction effect of H₂O₂ was better than that of SA.comparisingwith control,the dry weight of the hairy root was increased by 15.2%on the 9^th day and at 20?M H₂O₂.The yield of solanesol reached 55.068 mg/L±6.186mg/L,which was 4.2 times of that of the control.We also studied the expression of the key enzymes of solanesol biosynthesis,FPS,DXR and IPI after H₂O₂ addition.The results showed that the highest expression was 16 h after H₂O₂ addition,which resulted in the enhancement of solanesol.Conclusion:The strain LBA9402 was suitable strain for induction of tobacco hair root.When the inoculation amount was 200,the hairy roots grew rapidly on B5 medium.H₂O₂ could significantly induce the solanesol content.Adding 10?M H₂O₂,the yield of solanesol was4.2 times higher than that of the control at 9 days.The key genes of solanesol biosynthesis were induced.