Full Length CDNA Cloning And Preliminarily Functional Identification Of CaHB3 And CaCBSX3

The growth and development of plants and response(resistance)to biotic and abiotic stresses in the natural environment are always controlled tightly by the signal networks.The identification of structure and function about the important proteins,which regulate the signal networks,and analysis of the regulation signal networks are the important basical researches.And these researches are used to clarify the growth and development of plants and the adaptive mechanism to the environment.The research field is extremely active in the international biology study nowadays.In my research,two genes,related with the plant stresses,cDNA have been got from the cDNA library of the pepper,and the structure of the genes is studied and analysised preliminary.The over-expression vectors of two genes are restructured and transgenic tobacco is builded.Besides,the subcellular localization and transient expression in leaf of two genes are studied.The researches are the foundation of the analysis of the anti-signal networks in plants.The major study results follow:1)The random clones of the pepper cDNA library have been sequenced,and lots of clone sequences are obtained.The cDNA of two genes-CaHB3 and CaCBSX3-are obtained and studied,which are related to the plant anti-stress response.There are 320 amino acid residues.in the open reading frame of CaHB3,including the WFQNRR conservative sequence and leucine zipper.Due to the reason,CaHB3 belongs to the class I of HD-Zip transcription factor family.Expression production of CaHB3 is in the nucleus and CaHB3 has the basical feature of transcription factor.According to the high identity with ATHB3 in Arabidopsis thaliana,so it is named as CaHB3.The CBS domain protein is discovered firstly in pepper.There are 204 amino acid residues in the open reading frame of CaCBSX3,containing a pair of CBS domains.Because the protein has high identity with ATCBSX3,it is named as CaCBSX3.2)The over-expression vector,GFP vector and VIGS vector are restructured,and these vectors are transduced into the agrobacteriums.The over-expression vector is used to build the transgenic tobacco plants,and transgenic TO generation plants and its T1 generation strains are obtained.In the further analysis the phenotypic effects of CaHB3 and CaCBSX3 expression in tobacco,these researches are the foundation of the genes functional identification.3)Using the gene gun method and transient expression injection,the subcellular localizations of two genes expression production are researched and identified The results are that CaHB3 expression production is in the nucleus and guard cell,and CaCBSX3 expression production is on the plasma membrane.The transient expression of CaHB3 and CaCBSX3 can active the cell necrosis in pepper leaves and make the Electrical conductivity of pepper leaves rise.These results imply that CaHBS and CaCBSX3 may have participated in the plant immune response.4)With the chromosome walking method,the promoter DNA fragment of the CaHB3 is obtained and the length is 1280 bp.While it contains some common elements,like CAAT box and TATA box,there are also the special aci-acting elements in the gene promoter,like W box,which are related with the plant defensive response.Besides,there are cis-acting regulatory element involved in light responsiveness,ERE,GARE-motif and ABRE.This supports the experiment result of CaHB3 transient expression that CaHB3 may have participated in plant defensive response regulation.The above research results lay the important foundation for the further illustration of two genes' function and molecular mechanism in pepper immune and defensive response.