Role And Regulation Of NtHB6 On Nitrogen Flow Direction And Early Flowering In Tobacco(Nicotiana Tabacum) Leaves

Tobacco is a solanaceous crop with leaves as the main economic part.The flow of nitrogen in tobacco leaves determines the composition of its nitrogen compounds,and the nitrogen compounds in tobacco leaves are closely related to the quality of flue-cured tobacco.The nitrogen transported from tobacco to tobacco leaves is stored in different cell structures of leaf cells and some free nitrogen-containing compounds.This distribution method determines the strength of leaf photosynthesis,affecting the toughness of the leaves and the chemical defense strength,so the study The flow of nitrogen in tobacco leaves is of great significance,and it has also become one of the hot spots in current plant science research.In the early stage of this study,Ultra Performance Liquid Chromatography-Quadrupole-Time Of Flight-Mass Spectrometry(UPLC-Q-TOF-MS)and Gas Chromatography-Mass Spectrometry(GC-MS)technology to establish an analysis system for nitrogen compounds in tobacco leaves.Using RNA sequencing(RNA-Seq),NtHB6 was selected from candidate genes that control the nitrogen distribution flow of tobacco leaves under different cultivation measures.In this study,NtHB6 gene sequence,subcellular localization and transcriptional activity were analyzed;NtHB6 overexpression vector,interference vector and CRISPR/Cas9 gene editing vector were constructed and genetically transformed into flue-cured tobacco variety K326.Through the phenotypic identification of tobacco transgenic plants and the comparative analysis of the transcriptome of NtHB6 overexpressing plants and control plants,a preliminary analysis of the regulatory mechanisms of NtHB6 in regulating tobacco drought resistance,leaf nitrogen flow direction and early flower phenotype was conducted.The main findings are as follows:1.Construction of NtHB6 transcription factor expression vector,subcellular localization and transcription activityThis study cloned the coding sequence(CDS)of NtHB6 gene from tobacco,with a total of 1026 bp.The pEarley Gate101-HB6 overexpression vector,RNAi-p FGC5941M-HB6 interference vector and CRISPR/Cas9-HB6 gene editing vector were constructed.In this study,pEarley Gate101-HB6 overexpression vector was used to detect the subcellular localization of NtHB6 protein.After the EYFP enhanced yellow-green fluorescent tag carried by the over-expression vector was expressed in tobacco protoplasts,it was observed by laser confocal microscopy to coincide with nuclear fluorescence,proving that NtHB6 transcription factor is the nuclear localization.In this study,the binding motifs of At HB6 and At HB16 in Arabidopsis were repeated in tandem,and a dual luciferase reporter gene vector was constructed,which was co-transformed with pEarley Gate101-HB6 over-expression vector to express B.nicotiana leaves for transient expression.Box to determine its dual luciferase activity.The results showed that NtHB6 transcription factor has transcriptional activation activity and can be combined with the binding motifs CAATCATTAAT and TAATAATT of Arabidopsis At HB6 and At HB16,so that the luciferase reporter gene on the carrier is expressed,thereby enhancing the activity of LUC luciferase.The ratio of LUC/REN is significantly increased.2.Preliminary study on the mechanism of NtHB6 transcription factor regulating tobacco drought resistanceThe drought stress treatment experiment of potted soil cultivation found that overexpressing lines were significantly higher than other types of tobacco in terms of photosynthetic rate,stomatal conductance,intercellular CO2 concentration and transpiration rate.The PEG-treated medium drought stress test results showed that the overexpression line and K326 line had the largest phenotype difference in the control group.Through the RNA-Seq analysis of the indoor tobacco plants,three genes TCTP,TPC1 and CCR2 were screened out.It is speculated that NtHB6 may directly or indirectly regulate the expression of the above three genes,thereby regulating the adaptation process of tobacco drought stress.3.The function of NtHB6 in regulating the nitrogen flow of tobacco leavesNtHB6 overexpression plants and K326 plants were subjected to nitrogen treatment hydroponic test.The results showed that the nitrogen content had a regulatory effect on the expression of NtHB6 in tobacco;the SPAD value of the NtHB6 overexpressing plants in the nitrogen-rich treatment group and the CK group was significantly higher than that of the K326 plant.In order to find the genes involved in regulating the nitrogen flow of tobacco leaves,this study conducted RNA-Seq on the transgenic and K326 plants in field and indoor cultivation,respectively.The results show that NtHB6 is directly or indirectly involved in regulating the synthesis,modification and metabolic processes of different amino acids in the tobacco leaf nitrogen flow pathway.Metabolomic analysis results showed that there were significant differences in the content of alkaloids,amino acids,organic acids and other compounds in the NtHB6 overexpression plant and the K326 plant.Among them,the NtHB6 overexpression plants of 4 kinds of alkaloids(nicotine,nornicotine,pseudo-equisetine,neonicotinoid),caffeic acid and glutamine were significantly higher than the K326 plants.In addition,through scanning electron microscopy observation,it was found that the secretion between the leaf epidermal cells of the NtHB6 overexpressing plants was significantly more than that of the K326 plants.The natural aphid test results showed that the NtHB6 gene editing plants was susceptible to aphids,the K326 plants was not susceptible to aphids in the early growth stage,and the NtHB6 overexpression plants was not susceptible to aphids.4.The function of NtHB6 in regulating the occurrence of tobacco early flowering and reproductive system developmentThe early flowering of NtHB6 overexpression tobacco was first discovered during the pot planting of T0 generation,and then verified in the field cultivation experiment.From the comparison of the recorded flowering time of the transgenic plants and the K326 plant,it was found that the average flowering time of the transgenic tobacco plants was 6.5 days earlier than that of the K326 plant.63 genes related to flowering control were identified in the indoor tobacco transcriptome data,while 462 genes related to flowering control were identified in the field tobacco transcriptome data.Compared with field tobacco,indoor planting tobacco flowering time is 14-20 days earlier.In indoor planting tobacco,the average flowering time of NtHB6 overexpression plants is only 3.1 days earlier than that of K326 plants.It is speculated that the reason for the difference is that the regulation of flowering time by NtHB6 may also be affected by environmental factors such as photoperiod,light intensity,blue light and circadian rhythm.5.Analysis of the downstream regulatory network of NtHB6 transcription factorIn this study,transcriptome data of NtHB6 overexpression plants and control plants cultivated in the field and indoors were used to screen downstream genes of NtHB6 from differentially expressed genes according to the binding motif in their promoter sequences.A total of 25 genes containing the binding motifs CAATCATTAAT and TAATAATT in the promoter were selected.It is speculated that NtHB6 may regulate the expression of these genes,and then regulate the synthesis of primary and secondary products of different nitrogen metabolism in tobacco leaves,and may also have a certain effect on carbon and nitrogen distribution.NtHB6 overexpresses transgenic plants.There are a large number of genes related to growth and flowering time in down-regulated expressed genes.NtHB6 may be a negative regulator of growth and flowering time.It can indirectly promote tobacco by reducing the expression of cell differentiation and flowering time inhibitors.The plants grow rapidly,increasing their biomass,and prompting the tobacco plants to flower ahead of time.In addition,its down-regulated expression genes also participate in the regulation of nitrogen metabolites to a certain extent,and regulate the nitrogen flow of tobacco leaves by regulating the aromatic amino acid family and amine metabolism.