Studies On The Plant Gene Expression System Controlled By Heat Shock And Tetracycline

It is important to construct a strict, high efficient, sensitive, special and induced gene expression system in that the gene can be regulated precisely and conveniently and plays an important role in developmental research. At present, there are some gene regulation systems, such as temperature inducement, phytohormone inducement (ABA,IAA,Auxin), light inducement, adversity inducement, metal salt ion and so on. However, all of that inducement has a shortcoming which is induced pluralism. Currently, tet-regulation-systm which mainly includes two types of tetracycline transcriptional activator (tTA) and reveres tetracycline transcriptional activator (rtTA) is broadly applied. There exists in some disadvantage that it is the high level of leaking when shut, constructing mixed promoter is low efficiency, high concentration of tet is provided with poison to cell or plants. Meanwhile, though the regulation of rtTA is not induced, rtTA and tetO still have appetency to induce the gene expression. In the light of this, this study take use the principle of tTA system playing, expected to make expression regulation become more strict, high efficient, sensitive and universal. The result as follow:1, Connect TetO which contained four tetracycline operon elements to the vicinity of mini-CaMV35S promoter(-46bp mini 35S promoter), gained the artifical promoter Om35S fragment which contained four tetracycline operon elements, then cloned it into pSAU2006 vector to obtain the subclone vector pU-Om35Sgus with the code section of GUS and the ender Tons. The pC-Om35Sgus that can control the expression of GUS gene including the artificial promoter of Om35S was acquired by the restriction enzyme cutting the "Om35S-GUS" fragment and inserted it into the plant expression vector pVCT2020. Where after, we transformed the plant expression vector pC-Om35Sgus into Agrobacterium tumefaciens EHA105 by freezing and melting, and took it to infect the tobacco leaf. In the end, transient expression of the GUS gene was measured by histochemical method; the result indicated that Om35S-GUS of tobacco leaf was no the expression of GUS under the condition of normal tissue culture. That is to say, the artifical promoter Om35S took on the strict expression.2, From the site #157, #238, #273 or #404 amino-acid residue starts remaining delete the genomic sequence's N-terminal of AtHSF1a, and splicing this C-terminal genomic sequences to the gene tetR of heat shock tetracycline repressor protein, gained fused gene fragment RHSFC157, RHSFC-238, RHSFC273 and RHSFC404, then cloned this gene fragments into pSAU2006 vector, construction four middle vector p35S-RHSFC157, p35S-RHSFC238, p35S- RHSFC273 and p35S-RHSFC404 whose fused gene fragment were controlled by 35S promoter, through restricted enzyme cutting four vectors could gained four expression gene fragments: 35S-RHSFC157-Tnos, 35S-RHSFC238-Tnos, 35S-RHSFC273-Tnos and 35S-RHSFC404-Tnos. Meanwhile we also from restricted enzyme cutting pU-Om35Sgus vector gained "Om35S-GUS" fragment, then recombination this fragment with above-mentioned one in four gene fragments into the plant expression vector pVCT2020, gained 35SC157-Om35Sgus, 35SC238-Om35Sgus, 35SC273-Om35Sgus and 35SC404-Om35Sgus four regulation systems of tetracycline. In the end, we transformed the four plant expression vectors into Agrobacterium tumefaciens EHA105 by freezing and melting, thereafter, took it to infect the tobacco leaf. Whereafter, transient expression of the GUS gene was measured by histochemically method; The result indicated that at the temperature of 35℃and under the condition of no Tetracycline, all the expression live of GUS was very strong. On the contrary, there was no signal in the cultrue medium including 1mg/L Tetracycline. It showed that the four regulation systems all appeared function and strict regulated by the negative regulation of tetracycline regulation system.3. The 35S promoter of 35SC157-Om35Sgus was replaced for hot promoter HSP70m. We obtained a new plant expression regulation system 70mC157-Om35Sgus which regulated by double inducement and regulation. Likewise, transient expression of the GUS gene was measured to analysis the function of this system, The results shows: the expression of the GUS gene was no expression when there was no Tetracycline as well as below 25℃, the expression live of GUS was strong on the condition of dealing with no Tetracycline and above 28℃, among the total, the expression live of GUS was most weak in 28℃and most strong in 35℃. In addition use the cultrue medium including 1mg/L Tetracycline treatment the most expression live of GUS tobacco leaf difference times, there was not signal in the cultrue medium including 1mg/L Tetracycline after four hours. The result indicated that the plant expression regulation system 70mC157 -Om35Sgus strict regulated by temperature and Tetracycline, besides have a sensitive inducement.