Cloning And Expression Analysis Of NnAP1 Gene From Nelumbo Nucifera

Lotus(Nelumbo nucifera Gaern)is a member of the family Nelumbonaceae and NelumboAdans,and one of the aquatic perennial herb flowers.It has huge flowers and floral light,and is China's major aquatic ornamental plants.Using genetic engineering technology to cultivate new varieties of flowers,has been the research focus of ornamental horticultural plant breeding.At present,many ornamental plants floral developmental genes have been cloned,but few studies have been reported on floral developmental genes of Nelumbo nucifera Gaern.In this study,the cDNA sequence fo API has been cloned from the Nelumbo nucifera'Hongxia' by RT-PCR and RACE.And the bioinformatics has been analyzed.Then we using the relative quantitative PCR method,analyzed the expression of genes in different organs API characteristics and different developmental stages.Moreover,we constructed the plant expression vector of API.The main results are as follows:1.Cloning and analysis of the full length cDNA of API from the lotus by RACE and RT-PCRWith the basis of known sequence,we designed both inside and outside 5'RACE primers to amplify,obtained the 5' sequence,and the full length cDNA of AP1 is 1141 bp,it contains a largest ORF that is 7953bp encoding 264 amino acids,The accession number was KF361315 in GenBank,It was named NnAP1.Sequence analysis showed that the gene containing a MADS-box and K domain,and belongs to MADS-box gene family.When the NnAP1 gene was blasted with API homologous genes in GenBank,found that they had high similarity.Phylogenetic tree analysis revealed that NnAP1shared the same ancestor.2.Using the relative quantitative PCR method and expression analysisWe designed the quantitative PCR primers,and the lotus of 18S-rRNA was as a reference gene,expression analysis of floral development gene AP1 was measured.The results that different gene has different expression patterns.Quantitative real-time PCR analysis showed that NeAP1 gene was expressed in various organ in different developmental stages,it had the highest expression in bloom and had trace expression in young stage and flower withering stage.It suggested that NnAP1 should play a role in flower development process.3.Construction of AP1 gene expression vectorUsing enzyme digestion,the plant expression vector of AP1 gene was was constructed successfully.First we constructed AP1-T vector,then done double digestion,the AP1-T vector and pCAMBIA1301 vector both with the Nco I restriction sites were connected,we obtained pCAMBIA 1301-AP1 carrier preliminary.