Cloning Of SpsLAS2 And SpsLAS3 In Salix Psammophila,Characterization Of Its Tissue Expression And Construction Of Expression Vector

The development of meristem will affect plant morphogenesis.The plant morphology will greatly affect the comunity configuration and landscape effect of garden plants.To promote the molecular directed breeding and study the regulation genetic mechanism of Salix psammophila.We clon the SpsLAS2 and SpsLAS3 gene from the Salix psammophila and analyzed the bioinformatics and tissue specific expression of it.Constructing the overexpression vectors of 35s::SpsLAS2 and 35s::SpsLAS3.We used the sitefinding-PCR to clon the 5' upstream regulatory region of SpsLAS2 and analyzed the sequence of it.1.The coding sequence of SpsLAS2 and SpsLAS3 gene was obtained by homologous cloning method.The similarity of SpsLAS2 and SpsLAS3 gene sequences got to 96.69%,and the amino acid sequences similarity got to 97.6%.The sequence lengths of SpsLAS2 and SpsLAS3 were 1248 and 1254,coding 415aa and 417aa,respectively.The amino acid sequences have five typical domains of GRAS gene family and belong to the LS subfamily.2.Homologous protein sequence alignment and phylogenetic tree construction showed that the protein sequences of SpsLAS2 and SpsLAS3 were most closely related to Salix purpurea and Populus trichocarpa.But the homology difference between SpsLAS2,SpsLAS3 and herbaceous plant is great.3.Tissue-specific expression results of the SpsLAS2 and SpsLAS3 gene.The results showed that the SpsLAS2 gene's highest expression is the leaf axil,and then decreased respectively from the phloem,mature leaves,stem,root,lateral apical bud,bud.The SpsLAS3 gene's highest expression also is the leaf axil,and then decreased respectively from the mature leaves,lateral apical bud,bud,root,stem,phloem.4.The two genes' overexpression vectors were constructed by the reaction of the LR which between enzyme digested product and pMDC32 overexpression vector.After screening on LB medium containing Kana,colony PCR and sequencing showed that SpsLAS2 and SpsLAS3 genes were successfully constructed into the overexpression vector.5.The genomic DNA of Salix psammophila was used as a template.SiteFinding-PCR was used to clone 501 bp of the 5' upstream regulatory region sequence from the genome of Salix psammophila.Using SOGO and other online software to predict and ana lyze the sequence,it was found that there was a possible promoter in the 5' regulatory region of gene.In addition to the basic promoter element CAAT-BOX,there are some typical photoresponse elements,hormone response elements and homeotropic elements related to stress induction.It is concluded that the expression of SpsLAS2 gene may be regulated by light,hypothermia and gibberellin.