Cloning And Expression Of The Transcription Factor Gene NtMYC2s Related To Nicotine Synthesis And SNP Analysis

Tobacco nicotianan tabacum is one of the main economic crops planted in China,and plays an important role in the national economy.Nicotine is a unique class of alkaloids in tobacco.On the one hand,it is widely used in medical,chemical and biological pesticide fields,on the other hand,the content of nicotine is also an important factor affecting the quality of tobacco leaves.Quality improvement is one of the important contents of tobacco breeding.How to shorten the selection period and accelerate the genetic improvement process are the main problems that need to be solved.The solution of these problems depends on the understanding and mastering of the genetic basis of tobacco related quality traits.In recent years,jasmonic acid signal transduction transcriptional factors such as MYC2,a key regulatory factor in the pathway,have been shown to be associated with nicotine synthesis.In this paper,a MYC2 transcription factor gene EST sequence related to nicotine biosynthesis pathway was screened from the tobacco transcriptome database constructed in our laboratory,and its cDNA was cloned.The differential expression and single nucleotide polymorphism(SNPs)in heterosis combinations with different nicotine content were analyzed in order to lay a foundation for speeding up the breeding process of tobacco quality improvement and the utilization of niacin heterosis.The results were as follows:1、The heterotic performance of nicotine content for two consecutive years in 2016 and 2017 showed that VA116 × Basma was a strong combination and VA116 × GDH88 was a weak dominant combination.Based on the related literature and the analysis of differentially expressed genes in the constructed tobacco transcriptome database,the target transcription factor gene MYC2 was screened.Based on the EST sequence of the gene,the full-length sequence of cDNA was cloned by PCR and named NtMYC2 s.The gene has a total length of 2633 BP,an open reading frame of 2046 bp,encoding 681 amino acids,and belongs to the bHLH family.Phylogenetic tree analysis showed that it was homologous to the MYC2 transcription factors of Arabidopsis,chicory,morning glory and soybean,and the highest homology with tomato and peppers of Solanaceae and chili,and the closest relationship.2、Real time fluorescence quantitative PCR analysis showed that the NtMYC2 s gene was expressed in a large number of tobacco leaves and roots at 1 weeks after topping.It was suggested that the gene might be a positive regulator of nicotine synthesis.The expression of NtMYC2 s in the same period was significantly higher than that of the 5 key enzyme genes,and the relative expression of the gene was expressed in large quantities,both in the roots and in the leaves.The amount of NtMYC2 s was more than 40 times of the parents,but in the weak dominant composite material,the expression in the same period was lower than that in the strong dominant combination,and the expression in the leaves was lower,even lower than the average value of the parents.The relative expression of NtMYC2 s was significantly positively correlated with the relative expression of QPT and MPO genes in the 1 weeks before topping.The correlation coefficients were 0.97** and 0.95**,respectively,and the correlation coefficient was significantly correlated with ODC,and the correlation coefficient was 0.86*;the correlation was not significant with PMT and ADC,and the correlation coefficients were 0.74 and 0.79,respectively.1 weeks after topping,the relative expression of NtMYC2 s was significantly positively correlated with the relative expressions of ADC,PMT and MPO,and the correlation coefficients were 0.93**,0.93**and 0.99**,respectively,and the correlation coefficient was 0.89*,and the correlation coefficient was not significant with QPT,and the correlation coefficient was 0.72.The correlation between the expression of NtMYC2 s and the expression of some key enzyme genes varies greatly before and after topping.It is presumed that the transcription factor is likely to be associated with the specific gene of key enzymes and thus plays a regulatory role.However,this specific binding is not directly associated with a key enzyme group,and these key enzyme genes are also available.There can be interaction effects or competitive binding of NtMYC2 s transcription factors.3、SNP was detected by MEGA6 and Dna SP 5.0,and 39 SNP loci were detected,and the SNP frequency was 1 / 71,among which the SNPs occurred most in the intron region.There were11 synonymous mutations and 4 missense mutations in 15 SNPs in exon 15.The distribution of SNPs in different materials was analyzed,but the number of SNPs in single material was more than that of their parents,but the total number of SNPs was less than the total number of SNPs in the parents.in 4 positions where missense mutations occurAmong them,Va116×Basma had two strong heterosis,one was Va116 × GDH88,and the other was in Basma,one parent of strong heterosis.The results laid a foundation for the association analysis based on the internal SNP of theMYC2 gene.