Funtional Analysis Of NtILR3 In Response To Tabocco Topping

Tobacco topping removes the top advantage of tobacco plant,blocks the transfer of assimilates to reproductive organs,promotes distribution of photosynthate to leaves,increases the accumulation of dry matter in leaves,and improves the yield and quality of tobacco leaves.At the same time,topping of tobacco plants will induce a series of physiological response processes,such as the change of hormone balance,the promotion of root development,the increase of nicotine synthesis and so on.Tobacco b HLH(basic/helix loop helix,b HLH)transcription factor ILR3(IAA leucine resistant 3)regulates plant growth and development.Our previous results found that tobacco topping could significantly increase the expression of Nt ILR3,suggesting that it may be a gene in response to tobacco topping.In order to explore the role of this gene in tobacco topping response,the research group cloned Nt ILR3 gene from tobacco cultivar K326(Nicotiana tabacum L.cv K326)and analyzed its biological function.The main research results are as follows:(1)Nt ILR3 gene was cloned and analyzed by bioinformatics.Nt ILR3 gene belongs to the subfamily of tobacco b HLH family,having a total length of 1113 bp,including 5exons and 4 introns.The total length of the coding region is 708 bp and encodes 235 amino acids.Among them,76 to 133 are the conserved sequences of Nt ILR3,including the typical domain sequence of b HLH(basic helix loop helix DNA binding super protein family).(2)The promoter of Nt ILR3 gene was cloned.Through sequence analysis,it was found that in addition to the common cis acting elements in plant genes,there are many cis acting elements related to plant hormones and abiotic stress,such as ABA,Me JA,IAA and drought stress,indicating that Nt ILR3 may be involved in hormone signal transduction and abiotic stress response.(3)The p CAMBIA1302-ILR3 vector was constructed and transiently transformed into Benshi tobacco.It was found that Nt ILR3 was located in the nucleus by LSM880 laser confocal microscope.(4)Nt ILR3 has tissue-specific expression in different growth stages of tobacco,and it was found to be mainly expressed in tobacco roots.Topping,Me JA and IAA treatment can up-regulate the expression of Nt ILR3,and drought and ABA treatment can down-regulate the expression level of Nt ILR3,indicating that Nt ILR3 may participate in different signal pathways and play an important role in the process of plant growth and development.(5)It was found that there was a binding site of MYC transcription factor on the promoter of Nt ILR3 gene.The p MDC164-GUS-PNt ILR3 vector was constructed and transferred to the transgenic tobacco plant with Nt MYC1a-1300 by Agrobacterium transient transformation.The GUS staining results showed that Nt MYC1 a could regulate the expression of Nt ILR3 gene.(6)In order to study the function of Nt ILR3,the overexpression and silencing vector of Nt ILR3 was constructed,and the wild-type K326 tobacco plant was transformed by Agrobacterium tumefaciens leaf disk transformation.After the transgenic tobacco plants were verified at DNA and RNA levels,their molecular phenotype preliminarily shown that Nt ILR3 could participate in the regulation of tobacco topping on K metabolism,Fe metabolism,nicotine synthesis and tobacco growth and development.