The Research On Root Specific Expression Of Peanut RS Gene For Producing Resveratrol

Resveratrol(Res)is an important phytoalexin that widely exists in Polygonum cuspidatum(giant knotweed),Vitis vinifera(grape),Arachis hypogasa(peanut)and other plants,and is particularly rich in the grape skins and peanut roots.It has a variety of biological activity.It is a natural antioxidant and can reduce blood lipids,inhibit platelet aggregation,anti-cancer,anti-inflammatory,anti-radiation,anti-aging,prevent cardiovascular disease and so on.It is known as the green anti-cancer drugs together with paclitaxel.Because of its deficiency supply from plants in nature,the use of biotechnology is expected to produce resveratrol abundantly.Resveratrol synthase(RS)is the key enzyme of resveratrol biosynthesis.In this study,we used tabacum root-specific expression promoter to drive the expression of peanut resveratrol synthase gene in hairy root of Nicotiana benthaviana and peanut.Using the hormone-free dependence and the rapid growth of hairy root,we got a large amount of hairy root through liquid culture to produce resveratrol.Results are as follows:1.Using plant expression vector pBI121 for the transformation of Agrobacterium thizogeney(A.r)and transferring them into Nicotiana benthaviana through A.r,we find that A.r can mediate genes carried by exogenous plasmid vector into the genome of Nicotiana benthamiana and express the gene in hairy root.Then we establish a complete method of the agrobacterium rhizogenes-mediated genetic transformation of Nicotiana benthaviana,which respectively obtained some positive hairy root lines of NtR12::RS and NtR2::RS.At last we determine the yield of resveratrol in hairy root and find that the highest yield is 2.12 ?g/g(FW)which is 4 times of the control.The result demonstrate that A.r can mediate its own genes relating to hairy root and foreign genes into foreign plants.2.We establish the liquid culture system of hairy root by researches of liquid culture system of hairy root.That is using 50 mL MS medium,28?,and 120 r/min.We can get 2.5 g hair root by culturing for 2 weeks.At the same time,we explore the influence of the induction of NaAc on the yield of resveratrol in this hairy root liquid culture system of Nicotiana benthaviana.According to the result,10 p,M NaAc treatment can effectively improve the resveratrol content in culture medium,and the total output of resveratrol increased 1 time.3.We explore the influence of different factor on the induction rate of explants by the transformation of peanut(Xinhui)through A.r for the establishment of resveratrol production of peanut hairy root.Then we find that Henan(A.r),concentration of bacteria suspension(OD600)0.5,co-culturing for 3 days,young leaves of peanut and B5 medium have advantages for the transformation of explants and the production of hairy root.Using Henan(A.r)carrying 35S::GUSA for the transformation of peanut leaf,we establish the complete system of hairy root induction of peanut and verify that the exogenous plasmid vector carrying genes can be transferred into peanut and expressed in hairy root.Furthermore we respectively obtain 2 positive hairy root of peanut lines of NtR12::RS and 3 positive hairy root of peanut lines of NtR2::RS,and we analyse the yield of resveratrol in peanut hairy root and find that the highest yield is 80.99 ?g/g(FW)which is 5 times of the control.The result demonstrate that root specific promoter-NtR2,NtR12-can improve the expression of RS in peanut root to synthesize resveratrol.4.We establish the liquid culture system of peanut hairy root by researches for increasing the production efficiency of resveratrol.That is 1/2 MS medium,28?,and 120 r/min.We can get 2 g hairy root by culturing for 2 weeks from 50 mL culture system.The induction of NaAc is able to increase the yield of resveratrol in hairy root,but MeJA can not work.20 ?M NaAc treatment for 24 h can effectively improve the resveratrol content in liquid culture medium system,and the output of resveratrol increased 0.8 time(141.03 ?g/g).NaAc can not only stimulate the secretion of resveratrol,but it makes the yield of resveratrol in transgenic hairy root to be 8 times of wild root of peanut.In short,this study firstly makes use of root specific expression promoter to drive peanut RS to express in hairy root of peanut or heterologous and establish the production technology system of resveratrol by transgenic hairy root.This paves the way for the production of resveratrol with high efficiency.Further in-depth study helps to produce and make use of resveratrol in large-scale.