Cloning And Functional Identification Of DELLA Protein PlGAl1 Genes Promoter Of The Paeonia Lactiflora

Paeonia lactiflora is perennial herbaceous ornamental plant which is also an important landscape plant in Northeast China.However,the seed of Paeonia lactiflora has the typical characteristic of the upper and the lower hypocotyl double dormancy,its growth cycle can last for half a year.Therefore,the germination rate is low in the process of seed reproduction owning to the dormancy can not be broken so that it has a severe effect on seed-breeding production.Gibberellin(GA)as the most important hormone in plants can promote seed germination and hypocotyl elongation which make DELLA protein family gene PlGAl1 play an important role during seed germination,but the PlGAl1 gene promoter regulates the transcriptional expression of genes.In this study,I use the hybrid seeds of Paeonia lactiflora seeds as the material.On the basis of cDNA 1872 bp full-length sequence of DELLA protein PlGAl1 gene which is related to dormancy germination of Paeonia lactiflora.I took advantage of chromosome walking technique and successfully cloned promoter sequence of Paeonia lactiflora DELLA protein PlGAl1 gene 861 bp.The main results are as follows,1.The quality of DNA extracted from different tissues of Paeonia lactiflora are different.I test uality,concentration,yield and PCR amplification of the obtained genomic DNA by Nanodrop 2000 micro spectrophotometer.The experimental result indicated that the OD260/OD280 values of seeds,tissue culture leaves and mature leaves were at 1.80-1.90 in the genomic DNA obtained from 7 tissues.Among them,the quality,yield and concentration of DNA extracted from tissue culture leaves was the highest,and PCR amplification was also the best.The second are seeds and mature leaves.The worst is the seed coat.2.Compared the similarity of cDNA sequences of DELLA genes from other species which has published on NCBI.According to the homology sequence of DELLA protein PlGAl1 gene,two downstream primers are designed,I take Paeonia lactiflora genomic DNA as a template and clone the 5 'flanking sequence of DELLA protein PlGAl1 gene by using Chromosome walking technique,so I obtained upstream of DELLA protein PlGAl1 gene which includes CDS area and DNA fragment of 1047 bp.Compared with NCBI database sequence,I found that 861 bp of the PlGAl1 gene promoter sequence was cloned for the first time.3.Through promoter prediction software PlantCARE,I found that a lot of T/A basic group appear near initiation codon of promoter and some gibberellin responsive elements are also exist in here such as GARE-motif,TATC-box,P-box and some light responsive elements including GAG-motif,I-box,G-box,TCT-motif,MNF1,Spl,sbp-CMAlc,also found that such as AAGAA-motif,ARE,TCA-element,TGACG-motif,Skn-1,motif,as-2-box,circadian and other cis-acting elements.4.DELLA protein PlGAl1 gene promoter PlGAl1p was cloned by PCR technology.Established GUS fusion expression vector PlGAl1p-GUS by using promoter fragment PIGAIlp to replace 35S strong promoter in pCAMBIA1301 and transferred into Agrobacterium tumefaciens LBA4404.That is to say,the can build the foundations for understanding the internal regulation mechanism of seed germination and giving exogenous gene an accurate positioning in Paeonia lactiflora seeds,and it is of great significance to promote the germination of Paeonia lactiflora seeds and other species.