Functional Analysis Of NtHIN1 In Response To Tobacco Topping

Tobacco is an important cash crop,and topping(removing inflorescences and adjacent young leaves)is an important cultivation measure to improve tobacco yield and quality.Topping can not only change the changes of endogenous hormones in plants,such as IAA,ABA,JA,and other hormones,thereby regulating the growth and development of tobacco,but also promote the synthesis of secondary metabolites such as nicotine in tobacco.Studies have found that topping can significantly increase the expression of Nt HIN1 gene in tobacco roots and leaves,indicating that this gene is involved in the process of tobacco response to topping.In this paper,the Nt HIN1 gene was cloned and its function in response to tobacco topping was studied using overexpressed and silenced transgenic tobacco plants as materials.The main results are as follows:1.The Nt HIN1 gene was cloned from tobacco and found to have a total length of830 bp,a CDS region of 690 bp,encoding 229 amino acids,and a conserved domain LEA-2 at 101-203,indicating that the Nt HIN1 protein belongs to the LEA2 family.QPCR analysis showed that Nt HIN1 gene was expressed in roots,stems,and leaves of tobacco during the entire growth period,but the highest expression level was found in roots and the lowest expression level was found in leaves.2.The promoter of Nt HIN1 gene was cloned and analyzed for cis acting elements.It was found that in addition to common elements in plants,there were also cis acting elements related to plant hormones,abiotic stresses,and defense responses such as drought,high salinity,IAA,ABA,and ethylene in the promoter sequence.The research results showed that these hormones,abiotic stresses,and reactive oxygen species can promote the expression of Nt HIN1 gene,This indicates that topping can regulate the expression of Nt HIN1 gene through hormones and reactive oxygen species.3.In order to study the function of Nt HIN1 gene,overexpression and silencing expression vectors of Nt HIN1 gene were constructed,and transgenic tobacco was obtained by Agrobacterium tumefaciens mediated leaf disc transformation.The expression levels of nicotine synthesis genes,ABA synthesis related genes,active oxygen scavenging genes,and disease resistance related genes in overexpressed transgenic tobacco plants were higher than those in wild type tobacco plants,while the expression levels in silent transgenic tobacco plants decreased,indicating that Nt HIN1 gene may be involved in nicotine synthesis,ABA synthesis,active oxygen scavenging,and disease resistance processes.4.In order to explore the function of Nt HIN1 gene in response to stress,it was found after drought treatment on transgenic and non transgenic tobacco that the content of reactive oxygen species and malondialdehyde in overexpressed transgenic tobacco plants was lower than that of wild type tobacco,but higher in silent transgenic tobacco plants than that of wild type tobacco;The activities of antioxidant enzymes such as SOD,POD,APX,CAT,GR,and proline content in overexpressed transgenic tobacco plants are higher than those in wild-type tobacco plants,while the reverse is true in silent transgenic tobacco plants,indicating that Nt HIN1 gene is involved in the stress resistance process of tobacco.Since topping can promote the expression of this gene,it indicates that it can improve the resistance in tobacco topping response.In summary,Nt HIN1 is a gene that responds to tobacco topping.Topping can promote the expression of the Nt HIN1 gene through IAA,ABA,JA,and H2O2.The Nt HIN1 gene is involved in the process of topping promoting nicotine synthesis and enhancing tobacco resistance.