Cloning And Expression Analysis Of APETALA3 And LEAFY Gene In Lotus

APETALA3(AP3)genes which belong to B functional genes in MADS-box genes,play important roles in the formation of flower organ and reproductive development.The characteristics of the stamens is decided by AP3 or PI interaction with AG.LEAFY(LFY)genes are a class of inflorescence meristem identity gene which play important roles in the process of flowering.In this study,partial sequences of AP3 gene was cloned from Nelumbo nucifera 'Hongxiamantian'by reverse transcriptase-polymerase chain reaction(RT-PCR).Through expression patterns of AP3 and LFY gene in different growth periods and different organizations,the regulatory effect from flowering gene to floral organ formation is speculated,the molecular regulation mechanism of flower organ development of lotus is lay a good foundation for the further study,lotus molecular breeding is provided theory basis.The mainly results were as follows:(1)By using RT-PCR method,partial cDNA sequences of LFYwere cloned from lotus.The sequences were 430bp long,encoded a protein of 143 amino acids and named NnAP3.Sepquence analysis showed that the deduced amino acid sequence shared 73%,73%,73%,72%,72%,71%and 71%identity with that of AP3 of Magnolia championii,Magnolia grandiflora,Magnolia wufengensis,Magnolia praecocissima Magnolia baillonii,Magnolia figo,Liriodendron tulipifera.Phylogenetic tree analysis also indicated that lotus had the closest genetic relationship with Platanus occidentalis and magnoliaceae plants such as Magnolia championii,Magnolia grandiflora,Magnolia wufengensis,Magnolia praecocissima,.(2)The proteins AP3 genes encode are speculated its molecular mass of 16 728.6Da,molecular formula for C719H1150N222O231S4,theory of isoelectric point of 6.33.Basic amino acid which has 27 accounted for 18.9%of total amino acids;acidic amino acids which has 23 accounted for 16.1%of total amino acids;hydrophobic amino acids,which has 47 accounted for 32.9%of total amino acids;polarity is not charged amino acids which has 46 accounted for 32.2%of total amino acids.In the secondary conformation of the protein,spiral structure,folding structure and line structure appear alternately.Spiral structure most,accounted for 51.05%;folded structure second,accounted for 43.36%;linear structure least 5.59%.AP3 genes from Lotus belong to part of the conserved domain database of K-box gene family.(3)Using Real-time quantitative PCR and RT-PCR Semi-quantitative technology,tissue specific expression of lotus NnAP3 genes are researched with Lotus 18SrRNA as internal gene.Real-time quantitative PCR analysis indicated that the AP3 was expressed in the flower of bud stage and full bloom which has a high relative transcript level.And it was hardly expressed in the root,stem and leaf in the different periods.Semi-quantitative analysis show AP3 transcription product accumulation was no detected in roots,stems,leaves,only exist in the flowers.(4)Using Real-time quantitative PCR and RT-PCR Semi-quantitative technology,tissue specific expression of lotus LFY genes are researched With Lotus 18SrRNA as internal gene.Real-time quantitative PCR analysis indicated that the LFY was ubiquitously expressed in the root,stem,leaf and flower in the different periods.These are expressed organ growth requires the presence of LFY in root,stem,leaf and flower.Semi-quantitative expression analysis show LFY transcription product accumulation was detected in different periods.And the highest expression was detected in the bud period.