Influence On Root Development In Arabidopsis By ?GlcY And Construction Of AGPs Gene Interference Vectors And The Genetic Transformation In Brassica

Arabinogalactose protein(AGPs)is a kind of glycosylation protein molecules with very rich hydroxyproline/proline.AGPs might be involved in the regulation of somatic embryo development,cell proliferation,cell expansion,elongation,but also may be involved in programmed cell death,development and other processes of embryonic gametophyte.1.In this paper,Brassica napus L.is used for the study.Select Arabidopsis the same as the cruciferous and use the sequence which has been determined.And choose two AGP-related genes AGP3,AGP8.By existing literature and the sequences that researchers have measured to construct rapeseed RNA interference vector.Cut the PCR products with SpeI/SacI and clone them to pTCK303 carrier.After sequencing is right,cut with BamHI/KpnI to clone and finish the constructing.While making Brassica napus L.seeds germinate and preparing engineering infecting strain,transfer the entire RNA vector into agrobacterium EHA105 respectively by the freezing and thawing method.After that,convert them into rapeseed petioles,and cultivate the callus to take root.2.At the same time,this experiment studied the effects on root growth and development in Arabidopsis thaliana by ?Glc Y.Culture the seeds respectively in 1/2 MS medium,1/2 MS+10?M ?G1cY medium,1/2 MS+ 10?M ?GlcY medium,1/2 MS+ 10?M ?GlcY>1/2MS medium.And we can find that:(1)As growing,the root treated by ?GlcY will present red color by the reaction between?GlcY and AGPs on the root epidermal cell walls.Compared to the control,?GlcY significantly inhibit the growth of roots.After culturing the roots back into 1/2 MS medium again,this phenomenon will develop and back to normal.Further cytology observation shows that the reaction between PGlcY and AGPs lead to smaller root elongation cells with the obvious expansion of the epidermal cells.(2)The expression of PIN 1 in the control arabidopsis root vascular cells can be detected in great quantities,and orientate in the base of cells.But very tiny distribution can be detected in the roots treated by ?GlcY.That shows ?GlcY hinders the distribution of PIN 1 to play a role in the polar transport of auxin to roots.The intensity of fluorescence will rebound after culturing the roots into 1/2 MS medium again.And the content of PIN1 will increase.(3)The expression of DR5 in the control arabidopsis root meristem cells can be detected,while it will enhance by treating with ?GlcY.This phenomenon will develop back to normal,after culturing the roots back into 1/2 MS medium again.These results demonstrated that?GlcY can cause excessive protein aggregation of auxin in the meristem part of roots to inhibit the growth of roots.(4)PIN2 locates on the top of plasma membranes in the epidermal cells and epithelial cells of roots.It shows under the action of PIN2 the auxin transports up from the base of the roots to the superjacent cells to maintain the growth and development.But the expression of PIN2 will reduce or disappear after treating by ?GlcY.That shows the reaction between?GlcY and AGPs changes the polarity orientation of PIN2 to hinder the polarity of auxin and influence the formation of epidermal cells pattern with the occurrence of epidermal cells.But the block is reversible after culturing the roots back into 1/2 MS medium again.