Cloning And Functional Analysis Of CCR Gene Promoters In Zea Mays

People are concerning about whether exogenous gene in plant could realize the specific tissue expression or not.The promoter as one of the molecular components that can control exogenous gene expression in specific tissues or organs,occupies the important research significance.Generally used by people at present,is the constitutive promoter,such promoter can drive exogenous genes highly expressed in various parts of the plant,but also caused the unnecessary waste of nutrition and plant cell excessive load.Specific promoter can regulate the expression of target gene in the specific tissue site well to avoid this problem.Therefore,the study of specific promoter not only of great significance to improve the quality of the maize,but also provides the reference for the study of control element of the genetic engineering.This topic on the basis of the results through bioinformatics,build the CCR gene promoter of maize,as well as its screening fragment expression vector.Transgenic rice plants were obtained through the agrobacterium mediated method,the expression of its features are analyzed.The main results were as follows:1.A 2145 bp gene promoter was isolated from the maize genome,which was named as CCR.We obtain three different length of fragment screening through the 5'-upstream region method,respectively named p CCR-1,p CCR-2 and p CCR-3,the fragment size respectively for 1650 bp,1126 bp and 548 bp.The four promoter fragments were connected with the dual expression vector p CAMBIA1301 containing the GUS reporter gene by double enzyme digestion method,and four new expression vectors were constructed.2.From promoter subsequence analysis related websites,the promoter in addition to the basic cis acting element such as TATA-box and CAAT-box,also contains four inducible elements: ARE,Box-W1,CGTCA-motif and MBS,four light responsive element: Box 4,Box I,G-box and Sp1,three organ expression element: ROOTMOTIFTAPOX1,POLLEN1LELAT52 and skn-1 motif.3.Application of Agrobacterium-mediated to transformed four new expression vectors into rice.Received 42 strains of rice transgenic plants,of which the CCR has 11 strains,CCR-1 13 strains,CCR-2 8 strains and CCR-3 10 strains.After detection by PCR and hygromycin,a total of 27 strains positive for plants.4.The GUS histochemical staining showed that CCR gene expression in root,stem,leaf,glume,but flowers and seeds,suggesting that CCR gene was the vegetative organ specific promoter.Deletion segments p CCR-1 expressed in leaf,glume,flowers and embryos,the rest parts do not express.p CCR-2 only expressed in leaves,and the rest of the tissue did not express,namely p CCR-2 as leaf specific promoter.p CCR-3 expressed in stems and glume,the rest parts were not express;...