| Narcissus tazetta var.Chinensis is an important Chinese New Year Eve Festival-Flower with high ornamental and economic value.The mechanism of flower development is one of the important topics in the study of flower development.MADS-box genes play an important role in regulating plant growth and development.AGL6 gene belongs to MIKC family of MADS-box gene,which regulates flower organ development and flower induction.TFL1 gene is the characteristic gene of inflorescence meristem,and participates in the regulation of flower induction.Chinese narcissus is a typical high temperature induced flowering plant,and its molecular mechanism is poorly understood.In this study,we preliminarily studied the mechanism of NtAGL6,a MADSbox gene of AGL6,and analyzed the function of TFL1 gene of Chinese narcissus.The main research contents and results are as follows:1.Cloning and analysis of NtAGL6 gene promoterThe 803 bp upstream of NtAGL6 initiation codon was cloned from the genomic DNA of ‘jinzhanyintai' using genome walking method.It contains 9 core elements TATA box and 17 enhancement elements CAAT box,as well as many functional elements related to development regulation and environmental response.The Ca MV35 S promoter in plant expression vector PBI121 was replaced by NtAGL6 promoter sequence,and the promoter expression vector p BI121-NtAGL6QDZ::Gus was successfully constructed.The results of transient expression in tobacco leaves showed that NtAGL6 promoter can activate the expression of reporter gene and has promoter activity.High temperature and GA3 induction can activate the expression of GUS gene.Transient expression of the promoter in different tissues and organs of Chinese narcissus showed that the promoter was expressed in petals,pistils,corona and bulb plates.2.Genetic transformation of Chinese narcissus by NtAGL6 RNAi vectorThe specific 500 bp sequence of NtAGL6 was selected.The primers were designed according to the cutting site of p TCK303 and the sequence of NtAGL6.After PCR amplification,the NtAGL6 fragment with the required cutting site was obtained.The positive and negative gene fragments were respectively connected to the vector p TCK303 to construct the interference expression vector p TCK303-Ri NtAGL6.The‘jinzhanyintai' and ‘baiye' was transformed through agrobacterium-mediated transformation,resistant buds were obtained.3.Preliminary analysis of the mechanism of NtAGL6 in Chinese narcissuspHIS2-FT1 Pro and p GADT7-AGL6 were constructed using In fusion technology.The results of yeast single hybrid showed that NtAGL6 was not directly combined with the Nt FT1 promoter of Chinese narcissus.p GADT7-NtAGL6,p GADT7-NtAP1,p GADT7-NtAG,p GADT7-NtAP3,p GADT7-Nt SOC1,p GADT7-Nt SVP,p GADT7-Nt FT1 and p GBKT7-NtAGL6 were constructed.The results of yeast two hybrid showed that NtAGL6 can interact with NtAP1,NtAG,NtAP3,Nt SOC1,and Nt SVP,and it can form homologous dimer by itself,but not with Nt FT1.The expression vectorsp SAK277-Nt SOC1,p SAK277-NtAGL6 and p GREEN?0800-FTQDZ-LUC were constructed.The results showed that Nt SOC1 and NtAGL6 did not further enhance the activity of Nt FT1 promoter.4.Cloning and expression pattern of TFL1 gene in Chinese narcissusThe TFL1 gene of Chinese narcissus were cloned by RT-PC using‘Jinzhanyintai' as material.Two genes were obtained in this experiment and named Nt TFL1-1 and Nt TFL1-2.The obtained gene sequence was analyzed by bioinformatics,the subcellular localization and expression pattern were carried out.Bioinformatics analysis showed that the ORF of Nt TFL1-1 was 501 bp,encoding 166 amino acids,and the ORF of Nt TFL1-2 was 525 bp,encoding 174 amino acids,all of which had conserved domains of the TFL1 family.Nt TFL1-1 and Nt TFL1-2 were localized in the cytoplasm.The results of q RT-PCR analysis showed that the expression of Nt TFL1-1 and Nt TFL1-2 genes was lower in the early stage of flower bud differentiation and inflorescence primordium formation and higher in flower primordium formation.The expression of Nt TFL1-1 and Nt TFL1-2 in roots was significantly higher than that instems,leaves and mature flowers. |
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