Isolation And Transgenic Phenotype Analyses Of PtAUX1 Gene Of Populus Tomentosa Carr.

Populus is a widely cultivated species in the world. They are viewed as model systems in plant biology. Populus is the third plant species which is sequenced after Arabidopsis and rice. And it is also the first complete sequenced wood species. The sequencing of the Populus genome promises to enrich the biology knowledge of energy sources. The plan shows important practice value.In order to study the roles of PtAUX1 genes in poplar development, one grouped to Arabidopsis AtAUX1 genes were isolated from cambium materials using the PCR-based strategy combined with RACE methods. These genes are named asPtAUX1. By analyses of protein sequences, phenotypes of transgenic plants cultured in vivo and in vitro after confirmed by RT-PCR, PtAUX1gene was characterized.Using the bioinformatics software and database, analyzed the bioinformatics of the auxin influx carrier gene PtA UX1 and its encoded protein of Populus tomentosa, which was cloned previously. The result shown that the total length of the encoded area of PtAUX1 mature peptide is 1760 bp.This sequence included an open reading frame with the length of 1 431 b p, encoding 477 amino acid; PtAUX1 located on the cell membrane, there are totally 9 trans-membrane areas; in its second level structure, there include abundant alpha helix and randomly coil area, but only few beta strand area. Meanwhile, an Aa_trans conserved domain was discovered. A high consanguinity was found for PtAUX1 and 12 proteins. Among those, the closest relationship is to Pt×PtLAX1, with the consanguinity level of 97%.The best condition for mulberry leaf transformation was got and the complete mulberry genetic transformation system was established through the optimization of the five main factors which affect the mulberry genetic transformation result: the concentration of the Agrobaterium liquid, the time of infection, the time of co-culture, pH value of co-culture medium, the addition concentration of AS in co-culture medium. The most suitable leaf transformation system was got: GV3101 infected liquid concentration OD₆₀₀ is 0.2-0.3, infection time is 10-15min, co-culture time is 2-3d, pH value of co-culture medium is 5.4,and resistance selection be done immediately after co-culture.The auxin influx carrier gene PtAUX1was cloned into pET-28 a (+) expression carrier, and was inducted to expression in E.coli DH5α.Using the purified recombinant PtAUX1 protein as antigen, the PtAUX1polyclonal antibody was produced successfully. Through the Southern hybridization test,all the three positive plant systems canproduce strong hybridization signal, this mean the transformation of the auxin influx carrier gene PtAUX1to mulberry was successful.Result of shows PtAUX1 has a ubiquitous expression pattern (Fig. 5). PtA UX1 mRNA accumulation can be detected in young leaves, young shoots, immature roots, female and male organs. Such an expression pattern of PtAUX1 suggests PAT regulation in plant development events and cell differentiation is complex. But expression level exhibits very different, and the higher in root and in pistil, more low in young shoot and stamen.Its over-expression lines under a female cultivar background driven by 35S promoter announce a phenotype related flower development not exhibited in AtAUX1 mutants. Ovary-like structures each with 1～2 styles, but without a cup-like disc and a bract, can initial from regenerated transgenic bud tips in cluster, resembling an inflorescence. The histological dissection illuminated that an ovary-like structure has a womb and transmitting tissue inner its styles, but no ovules. PtAUX1expression pattern are ubiquitous and various in different organs, which is coincident with its candidate function in distributing auxin thorough a plant body as an auxin influx carrier. These results suggest that PtAUX1 or polar auxin transport has a substantial function in flower initiation.