A Research Of Cloning And Expression Of PagD53 Gene And Its Interaction With PagD14 Gene In Poplar

Poplar is an important industrial timber and afforestation tree species in China.It is particularly important to improve the productivity of poplar timber and to cultivate poplar varieties of good quality.Light energy is the primary energy source for plants to accumulate biomass.Canopy is a place where trees intercept light energy for photosynthesis.Therefore,canopy structure and canopy density are important factors that determine the wood yield of stand.The crown structure and canopy density of poplar are mainly determined by branch development.In woody plants,branching mainly refers to the development and regulation of lateral branches.Side branches are developed from axillary buds at the leaf axils.The development of side buds and side branches is a complex physiological process.The process is regulated by various factors such as plant heredity,hormone regulation,growth environment and nutritional conditions.Among them,plant hormones play an important role in the regulation of branching development.It has been reported that Strigolactone?SLs?plays an important role in the regulation of plant branch development.In plants,besides D14protein,the receptor that can receive the signal of scyllactone also includes F-box protein D3and Clp protease family protein D53,which are involved in the signal regulation of SLs through the interaction and formation of complex.In this paper,84K?Ppulus alba×P.glandulosa?was used as the experimental material to extract the total RNA and the cDNA obtained by reverse transcription was used as the template.The primers were designed according to the D53 gene number?Potri.009G046700?in the JGI database,and the PagD53 gene was cloned to construct a recombinant expression vector for PagD53,including:35S::PagD53,PBI121-PagD53-GFP,pGBKT7-PagD14,pGADT7-PagD53,PSPYNE-PagD14,pSPYCE-PagD53.The primers were designed according to the D53 gene number?Potri.009G046700?in the JGI database,and the PagD53gene was cloned to construct a recombinant expression vector for PagD53,including:35S::PagD53,PBI121-PagD53-GFP,pGBKT7-PagD14,pGADT7-PagD53,PSPYNE-PagD14,pSPYCE-PagD53.Among them,35S::PagD53 is used for Agrobacterium-mediated infection of 84K plants;PBI121-PagD53-GFP is used for tobacco subcellular localization;pGBKT7-PagD14 and pGADT7-PagD53 are used for yeast double hybrids;Two-photon interaction.Based on the above experiments,the function,subcellular location and interaction protein of PagD53 gene were studied,and the function and mechanism of PagD53 gene in poplar branching development were studied and draw the following conclusions:1.The PagD53 gene of poplar was cloned,the gene length is 3345bp,encoding 1114amino acids,the protein of PagD53 is predicted,its molecular weight is 121943.92 Da,the theoretical isoelectric point?PI?is 6.45,the molecular formula is C₅₃₀₁H₈₄₉₄N₁₅₁₀O₁₆₈₃S₅₁,and the total number of atoms is 17039,the fat coefficient is 80.85,the total average hydrophilicity?GRAVY?is-0.313,and it is predicted to be a hydrophilic protein.2.The expression of PagD53 in 84K different tissues was detected by fluorescent quantitative PCR.The results showed that:the order of expression of PagD53 in different tissues from high to low was:bud,root,stem,leaf,and the relative expression of bud was2.548,About 6.67 times the root.3.The overexpression vector PBI121-PagD53 was constructed,and 84K poplar was transformed by Agrobacterium-mediated method.The positive seedlings screened by Kana were tested and identified by gel electrophoresis,and finally 4 transgenic lines were obtained.4.We constructed the PBI121-PagD53-GFP expression vector and carried out subcellular localization through transient transformation of tobacco.The results showed that PagD53 was located in the nucleus.5.The pGBKT7-PagD14 and pGADT7-PagD53 expression vectors were constructed and transformed into yeast AH109.After self-activation detection without self-activation,the co-transformed yeast can be transferred into SD/-Ade/-His/-Leu/-Monoclonal growth on the Trp four-defect plate,indicating that PagD53 protein and PagD14 protein interact in yeast6.We constructed the pSPYNE-PagD14 and pSPYCE-PagD53 vectors,then we using Agrobacterium carrying recombinant plasmids to infect tobacco leaves.Yellow fluorescence was observed in tobacco mesophyll cells after confocal microscopy.It shows that PagD53 and PagD14 exist in plants Interaction phenomenon.Furthermore,it is speculated that PagD53and PagD14 may jointly regulate the signal transmission pathway of SLs,thereby affecting the branching development of poplar.