A Study Of The Technology Of Regulating Flue-cured Tobacco Nicotine Biosynthesis By RNAi

Flue-cured tobacco nicotine content in our country espically in upper leaf is too high,which has significant negative effects on Flue-cured tobacco’s sell,exploitation,production and people’s health.The agronomic measu res cannot effectively regulate nicotine synthesis at present,and transgenic tobac co has a major security problem.This study was based on RNAi technology,fir st we regarded PMT gene sequence as a template to construct an expression v ector containing IR(inverted repeat),then transformed the vector into RNase III deficient strain DY330 to acquire the engineered bacteria which expresses hpR NA,at last we collected hpRNA through cultivating engineered bacteria and spr ayed tobacco root with hpRNA to reduce the content of nicotine in tobacco.Th e main contents were as follows:1.Construction of engineered bacteria which expresses hpRNAWe extracted total RNA of tobacco and reversed transcription to o btain the cDNA of tobacco.We designed two pairs of primers according to P MT gene sequence,obtained two sequences by polymerase chain reaction whi ch have part of same sequence,then linked them to T vector to get the cloni ng vector containingIR,and the alignment result was 99%in accordance.By li nking the IR to expression vector pET-21a,the vector was transformed into ex pression strain and acquired the engineered bacteria at last.2 Optimization of fermenting conditions in engineering bacteria the results were that when induced concentration of IPTG was 0.4mM,OD600 of bacteria was 0.5 and Induction time is four hours,the yield of hpRNA is higher.3.pot experimentWe chose two group of tobacco which had the same growth vigour,one grou p we sprayed tobacco root by hpRNA and another is blank control.Three days later,we extracted total RNA of tobacco and reversed transcription,which found that the expression of PMT gene had been slowing down accoding to t he control line.We determined the content of nicotine in tobacco about forty days later and we found that the content of nicotine in conduct group is low er to the control set.4.field experimentWe select two fields which have the same natural condition,one is treatment group and another is control group.we first sprayed tobacco root with hp RNA extracted from 10ml bacteria liquid when the tobacco was in spherical plant stage,and then we do it again every 20 days.After baking we determi ned the nicotine content in each part,the results show that different parts of tobacco nicotine content were decreased and of which the middle leaves have the most decrement about 1%.