Studies On Cloning,expression And Characterization Of Phytochrome Interacting Factor CoPIF3 In Camellia Oleifera

Phytochrome interacting factors(PIFs)belong to the b HLH(basic helix-loop-helix)transcription factor subfamily,and play an important role in plant light signal response,growth and development,and stress response.Camellia oleifera(Camellia oleifera Abel.)is a unique high-quality woody oil resource in our country.Its main product,Camellia oil,has rich nutritional value and high economic and social benefits.It plays an important role in alleviating the shortage of edible oil and ensuring the safety of grain and oil.Studying the gene functions of the PIFs family in Camellia oleifera is helpful to reveal the light signal response mechanism,and is of great significance to the research of high light efficiency molecular breeding of Camellia oleifera.In this study,based on the transcriptome data of Camellia oleifera,using the Camellia oleifera ’Guoyou 13’ as the test material,a phytochrome-acting factor(PIF3)gene in Camellia oleifera was cloned by RT-PCR technology,and the gene was analyzed by subcellular localization,transcriptional self-activation,tissue-specific expression analysis,and prokaryotic expression analysis.The main findings are as follows:(1)A phytochrome interacting factor gene was cloned from Camellia oleifera ‘Guoyou13’.The open reading frame of this gene is 2169 bp,which encodes 722 amino acids and the gene is named Co PIF3.Amino acid sequence analysis showed that the C-terminus of PIF3 contains a b HLH domain,and the N-terminus contains an APB phytochrome B binding domain and an APA phytochrome A domain.It belongs to the b HLH transcription factor family.The Co PIF3 promoter sequence fragment(1248bp)was cloned successfully.The prediction of cis-acting elements showed that there are TATA-box,CAAT-box,light-responsive elements,environmental stress cis-acting elements,etc.And the light-responsive elements include AEbox,CAG-motif,G-Box,GT1-motif and LAMP-element.(2)Real-time fluorescence quantitative PCR analysis showed that Co PIF3 gene can be expressed in new shoots,annual leaves,petals,pollen and seeds,and the expression levels in different tissues are different.The expression status is annual leaves>new shoots>seeds >petals> pollen,the relative expression is the highest in annual leaves.(3)The yeast self-activation detection showed that Co PIF3 protein has transcriptional self-activation activity.(4)Western Blot detection results showed that the Co PIF3 protein was expressed in E.coli BL21,which was consistent with the predicted size.