Cloning and interacted protein identification of AGL12 gene from Lonicera macranthoides
MADS-box protein family are important transcriptional regulatory factors in plant growth and development.The AGAMOUS 12(AGL12)subfamily is believed to play an important regulatory role in the process of plant flowering transition.To explore the potential mechanism of AGL12 subfamily involved in regulating the flower development of Lonicera macranthoides,quantitative real-time polymerase chain reaction(qRT-PCR),prokaryotic expression and yeast two-hybrid techniques were used to analyze the expression pattern,protein expression,and protein-protein interaction pattern of LmAGL12 based on transcriptome data.The results showed that the LmAGL12 gene contains a 603 bp open reading frame(ORF),encoding 200 amino acids,and the encoded protein was stable and hydrophilic without a transmembrane region and signal peptide.Through homologous sequence alignment and phylogenetic analysis,it was confirmed that LmAGL12 protein belongs to the MADS-box protein family and is closely related to the AGL12 protein of Heracleum sosnowskyi and Daucus carota subsp.sativus.The LmAGL12 gene was cloned into prokaryotic expression vector pET-28a and the recombinant constructs were transformed into Escherichia coli BL21(DE3),which inducing the target protein successfully.The yeast two-hybrid results showed that LmAGL12 protein interacts with LmSVP protein,LmSOCl protein and LmAPl protein,respectively.The qRT-PCR results showed that LmAGL12 gene were differentially expressed in different development stages of flower bud,stem,and leaves of'Longhua'and'Baiyun'in L.macranthoides.The LmAGL12 gene showed the highest expression level in the middle stage of the'Longhua'floral bud;For the'Baiyun'variety,the relative expression level of LmAGL12 gene is the highest in the stem.This study cloned the LmAGL12 gene in L.macranthoides and analyzed it expression for the first time,enriching the research on flower organ development and providing a research basis for further exploring the molecular mechanisms of long bud stage and non-unfolded corolla in L.macranthoides,as well as for variety improvement.
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