The Cloning Of The NtWRKY-R1 Promoter And The Response To IAA And JA

Nicotine is a kind of secondary metabolite which was synthesis in tobacco plant roots.Nicotine concentration of the leaves increased rapidly by topping in field experiments of tobacco plant.Previously we proposed that the lateral root proliferation emerged by topping which was nicotine synthesis location,the main reason was that changing the balance of endogenous hormone(IAA)through removing the shoot apex and adjacent younger leaves of tobacco plant,and inducing JA signal substrate through topping damage stimulating enhanced capability of nicotine synthesis.In order to explore the relationship between IAA/JA and the ability of nicotine synthesis in root,we screened WRKY from SSH cDNA differences in expression library in the roots of befor and after topping of tobacco plant,and cloned it,which named NtWRKY-R1.The subject of this study is that cloning the NtWRKY-R1 promoter and investigating NtWRKY-R1 response to IAA,JA.The results as follows:1.After excision of apical meristem and adjacent younger leaves of DR5::GUS transgenic arabidopsis plant,GUS histochemistry staining at different time showed the color burn in root tip over time.Indicating that topping induced IAA consentration increasing in roots.2.Cloned the NtWRKY-R1 promoter using Genome-Walking technique which was analysised by PlantCARE databases on line,a variety of cis-elements were obtained including AuxRR and GARE-motif.Speculating NtWRKY-R1 would participate in IAA and JA signal pathway.3.Constructing pCAMBIA1391--386UTR117-GUS?pCAMBIA1391-793UTR117-GUS pCAMBIA1391--1387UTR117-GUS?pCAMBIA1391--1803UTR117-GUS fusion expression vector(35S-GUS is positive control).These vectors were transferred to BY-2 and tobacco plant,obtaining transgenic strain.4.Detecting the optimal reaction time of GUS activity in transgenic BY2 was 30min,and optimal concentration of 2,4-D and MeJA in the medium of culture transgenic BY2 was 0.5?m and 25?m respectively.After different fragment of NtWRKY-R1 promoterin in BY-2 were treated using MeJA and 2,4-D respectively,GUS activity were detected at different time.The data showed that GUS activity in-386UTR117 BY-2 was 3.8 times higher and-1387UTR117 BY-2 was 3 times higher than control in 0.5?n 2,4-D after 24h;and-1387UTR117 BY-2 in 25?m JA was 2 times higher than control.When 25?mMeJA and 0.5?m2,4-D were added to the medium of culture,the GUS activity of-1387UTR117 BY-2 decreased 50%than that in 0.5?m2,4-D and 30%than that in 25?mMeJA.5.In order to verify the response of NtWRKY-R1 promoter to 2,4-D and MeJA,the root tip GUS activity of-1387UTR117 transgenic tobacco were detected after spraying leaves and pouring roots with 25pm MeJA and 0.5?m 2,4-D respectively for 24h.The detecting data of GUS activity showed that the change tendency of data was same as that in transgenic BY2.The results of transgenic tobacco plant in 25 ?m MeJA and 0.5?m 2,4-D simultaneously was same transgenic BY2,too.Suggesting that the cross-talking signal between IAA and JA after tobacco plant topping.6.To explore the function of NtWRKY-R1 trascription factor in signal trasducing,phosphorylation modification of NtWRKY-R1 in the root tip of befer and after topping was detected by western blot.The results showed that the function of NtWRKY-R1 trascription factor was operated by phosphorylation.The NtWRKY-R1 responsed to cross-talking of IAA and JA signal pathway,but more efficient to JA.The performing function of NtWRKY-R1 depended on phosphorylation modification.Suggesting that NtWRKY-R1 transcription factor is a component of signal pathway in root,which responsed topping damage in shoot.