Study On The Function Of Transcription Factor NtBRC1b In Nicotiana Tabacum

The branch development of plants plays a very important role in the growth morphology of plants.This process involves the establishment of morphology and the distribution of nutrients.The branching of the plant originates from the production of axillary buds,and a large amount of nutrients are consumed by the growth of axillary buds.As one of cash crops,tobacco(Nicotiana tabacum)is also an important model plant.Studying the function of tobacco branch genes not only benefits our understanding of the branching mechanism,but also has important economic value.It can effectively reduce the branches of tobacco in cultivation process and produce high-quality tobacco leaves.According to incomplete statistics,the annual cost of purchasing pesticides that inhibit axillary buds is as high as several billion yuan in tobacco areas across the country.Therefore,the preparation of tobacco materials with little or no axillary buds is of great significance to tobacco agriculture.Current research shows that the TCP transcription factor family plays an important role in the regulation of plant branches.The BRANCHED1(BRC1)gene belonged to TCP family,which is involved in the development of axillary buds in Arabidopsis thaliana,Solanum lycopersicum,Pisum sativum and other plants.However,it is unclear whether BRC1 plays a role in the development of tobacco axillary buds.In this study,the Honghua Dajinyuan Tobacco was used as the research object,and identificated two homologous NtBRC1b genes to AtBRC1 in Arabidopsis,which is NtBRC1b-1 and NtBRC1b-2.Further,CRISRP/Cas9 and other experiment were applied to explore the effect of NtBRC1 on the growth and development of axillary buds in tobacco,and then a double luciferase reporting experiment and yeast one-hybrid experiment were used to study whether NtBRC1b has a regulation role to 9-CIS-EPOXICAROTENOID DIOXIGENASE 3(NtNCED3),that is related to abscisic acid synthesis.The main results obtained in this research were as follows.1.Identification of BRC1b in tobacco using bioinformatics and TA clone.BRC1 protein from A.thaniana was blasted with tobacco genome,which obtained four highly similar protein sequences in tobacco,named as NtBRC1a-1,NtBRC1a-2,NtBRC1b-1 and NtBRC1b-2,respectively.Further two genes(NtBRC1b-1 and NtBRC1b-2)were cloned from tobacco genome.Their coding sequences from TA clone were a little different compared with the sequence in tobacoo genome database.Gene structure analysis showed that NtBRC1b-1 and NtBRC1b-2 had two and one exon,respectively.Amino acid multiple sequence alignment showed that NtBRC1b-1/2 had TCP domain.Phylogenetic tree showed Nt BRC1b-1 and NtBRC1b-2 are clustered with BRC1b of tomato and potato.Tissue expression profiling analysis showed that both of NtBRC1b-1 and NtBRC1b-2 specifically expressed in axillary buds and leaves with high level compared to other tissues.The results of subcellular localization showed both of NtBRC1b-1 and NtBRC1b-2 were localized in the chloroplast and cytoplasm.2.Function study of NtBRC1b using CRISPR/Cas9Combined topping experiments and RT-PCR method,we found the axillary buds grew rapidly after topping in wide-type tobacco,and the expression level of NtBRC1b-1 and NtBRC1b-2 decreased significantly,which implied the Nt BRC1b gene is a negative regulator of axillary bud growth and development.Further NtBRC1b gene was knocked out using CRISPR/Cas9 gene editing technology,and then obtained mutant individuals successfully.After topping,the NtBRC1b-1 or NtBRC1b-2 mutant showed longer length of axillary buds compared to wild-type tobacco,and the length of axillary buds of double genes(NtBRC1b-1 and NtBRC1b-2)knockout plants was longer than that of single gene(NtBRC1b-1 or NtBRC1b-2)knockout plants.In addition,the axillary buds of double mutant plants grew faster than single gene knockout plants and wild type.Comprehensive analysis shows that tobacco NtBRC1b has an inhibitory effect on the growth of axillary buds.In order to prepare tobacco material with little or no axillary buds after topping,we abtained overexpression transgenic line of NtBRC1b through vector constrction and genetic tranformation,which laid the foundation for subsequent functional analysis and molecular breeding.3.The regulation of NtBRC1b to NtNCED3Considering that BRC1 is involved in the regulation of abscisic acid(ABA)synthesis,we further analyzed the regulation of tobacco NtBRC1b on the transcription of ABA synthase gene NtNCED3.RT-PCR analysis showed that the expression of tobacco Nt BRC1b and NtNCED3 genes was up-regulated by ABA.Cloning obtained the promoter of NtNCED3,and a double luciferin reporting system showed that NtBRC1b promoted the activity of NtNCED3 promoter.In addition,a yeast one-hybrid experiment showed that NtBRC1b could not interact with promoter of NtNCED3.Comprehensive analysis shows that NtBRC1b is regulating the transcription of NtNCED3 gene positively,however,which regulate NtNCED3 gene through indirect regulation way.Considering the results of bioinformatics,RT-PCR and other experiments,we cloned and identified two genes(NtBRC1b-1 and NtBRC1b-2)that was highly expressed in axillary buds and leaf.After topping,the expression level of NtBRC1b gene decreased with longer length of axillary buds in wide-type tobacco.Considering CRISPR/Cas9,the mutant of NtBRC1b showed longer length and more quickly growth speed in axillary buds,when compared to wide-type tobacco.The above results demonstrated NtBRC1b was a negative regulatory factor participating in axillary bud growth in tobacco.In addition,the NtBRC1b transcription factor can initiate the expression of the downstream NtNCED3 gene,but does not directly bind to this gene.This study provides a new clue for the molecular regulation mechanism of tobacco axillary buds.