A Preliminary Study On The Function Analysis Of Nt21 And Nt92 Gene In Nicotiana Tabacum

Nicotiana,is one of the most important economic crops in our country and the world,but also an important model of molecular biology and cell genetic research.Tobacco is covered with epidermal hair,which is the first line of defense against biotic and abiotic stress.According to whether has glandular body,epidermal hair can be divided into secretory glandular trichome and no secretory of epidermal hair,glandular trichome is the main place for the occurrence of secondary metabolism in tobacco.The secretions of tobacco glandular trichome which is important precursors of tobacco flavoring substances,not only has high economic value?spices,cosmetics,drugs,insecticides,etc.?,but also is one of the most important factors which affects the quality of tobacco?aroma,smoke,stress resistance?.At present,there are few reports on the genomic information and function of glandular trichome.Therefore,it has a very important scientific value and practical significance to isolate,clone and study deeply biological function of genes about secretion amd stress from glandular trichome in tabacco,and finally to cultivate the high secretion,stress resistance,low toxicity of new varieties of tobacco.On the basis of previous high-throughput sequencing,the function of Nt21 and Nt92,which are highly expressed only in secretory glandular trichome,were studied in detail.The main results are as follows:The Nt21 gene encodes 320 amino acids and contains two highly conserved MYB domains,belonging to the R2R3-type MYB transcription factor.Nt21 protein is located in the cytoplasm and has self-activating activity.However,neither the N-terminal?1aa-112aa?nor the C-terminal?113aa-320aa?of the Nt21 protein was self-activating,suggesting that the activation domain of the Nt21 protein was destroyed.There are many cis-acting elements associated with hormone response,stress,and a root-specific element expression in the upstream of the Nt21 gene;ABA treatment of wild-type seedlings to be found Nt21 genes upregulated between 2h and 12h,the highest expression levels at 4h,indicating Nt21 responses to ABA and may be involved in stress response.Nt21 expresses differently in tissues,the highest GUS activity and the highest expression level were in young roots.In addition,overexpression of Nt21 resulted in the up-regulation of CYC and CYP expression,which is opposite to supression of Nt21.And found that the surface of Nt21 overexpressing plants had similar granular formation on the leaves.At the same time,DGE sequencing revealed that gene₇9205,which was highly homologous to the geranyl linalool synthase gene TPS4,was significantly down-regulated in Nt21 overexpressing plants.Therefore,it is a preliminary view that overexpression of Nt21 resulted in a significant down-regulation of gene₇9205 expression,leading to the hindrance of geranyl linalool synthesis.Therefore,the terpenoid precursor GGPP was enriched and the expression of CYC and CYP were up-regulated,cembrane compounds increased,secondary metabolic activity enhanced,resulting in secretion enriched to form particulate deposits on blade surface.The Nt92 gene encodes 316 amino acids and contains one highly conserved DUF868 domains,belonging to the DUF868 family.Nt92 protein is located in the cytoplasm and has no self-activating activity.There is a large number of cis-acting elements associated with hormone response and stress in the promoter region of Nt92.The expression of GUS was detected in the tissues and organs of PNt92::GUS transgenic plants,and GUS activity was the highest in mature leaves,which was consistent with the results of qRT-PCR.Compared with the wild type,Nt92 overexpressing plants and Nt92 RNAi plants had no significant difference in phenotype.DGE sequencing revealed that many genes related to stress response and membrane transport were differentially expressed in Nt92 overexpressing plants,suggesting that Nt92 is likely to be closely related to stress response and transport of intracellular substances such as protein.