The Effect Of Key Enzyme Activity On The Formation Of Heterosis In Nicotine Synthesis Pathway

Nicotine is a kind of alkaloid unique to tobacco,and its content is a key indicator to measure the quality of tobacco and its cigarette products.In order to explore the relationship between related enzyme activities and metabolites in the nicotine synthesis pathway and heterosis,provide a basis for the genetic improvement and regulation of nicotine content.In this study,14 materials with large differences in nicotine content were selected as parents,using NC?genetic mating design,48 hybrid combinations were prepared,and the nicotine content of the tobacco leaves in the parents and hybrid combinations was measured at different growth stages of tobacco The heterosis performance of alkali content was analyzed,and the variation rule of nicotine content and heterosis at different growth stages was discussed,and the main period of nicotine heterosis performance was clarified;strong and weak dominant materials were screened,enzyme-linked immunoassay was used to determine the activity of related enzymes in the nicotine synthesis pathway,and quasi-targeted metabolomics(LC-MC)was used to determine the relative content of related metabolites in the nicotine synthesis pathway.The differences in metabolites were analyzed for the related enzyme activities in the nicotine synthesis pathway and the relationship between metabolites and nicotine content and heterosis.The main findings are as follows:1.The dynamic analysis of nicotine content shows that the overall change trend of the nicotine content of the hybrid combination is the same as that of the parent.The nicotine content shows an upward trend as the growth period is delayed.The nicotine content of the material varies greatly in different periods.The analysis of the dynamic changes of heterosis of nicotine content in different periods of six randomly selected hybrid materials shows that the average value of heterosis and heterosis performance of each hybrid combination in each period reached the maximum in 74 days after transplanting,indicating that tobacco Alkaline heterosis performance is better in thisperiod,so this period is selected as the key period of heterosis performance.During this period,the heterosis of Va116×Basma was strong,with a mid-parental advantage of52.85%;the heterosis of K326×Qinggeng was weak,with a mid-parental advantage of-5.62%,and the two combinations were selected as strong and weak.Representative combination,used to study the relationship between the formation of nicotine heterosis and key enzyme activities and metabolites.2.Analysis of enzyme activity and nicotine content showed that in combination K326×green stem,A622 enzyme activity was significantly positively correlated with nicotine content,and the other eight enzyme activities did not reach significant levels with nicotine content;in combination Va116×Bar In Sima,MPO and BBL enzyme activities were significantly negatively correlated with nicotine content,AO and A622 enzyme activities were significantly positively correlated with nicotine content,and PMT and nicotine content were extremely significantly positively correlated.A622 reached a significant level with nicotine content in both combinations,indicating that the nicotine content may be greatly affected by the enzyme activity of A622,that is,the nicotine content increases as the enzyme activity of A622 increases.The enzymatic activities of related enzymes in the nicotine synthesis pathway of hybrids are higher than those of their parents;The strongest enzyme activity is QS and ADC,MPO and BBL are higher,and the other enzyme activities are weaker;The enzyme activity in the combination of strong and weak advantages and the enzyme activity of their parents have reached a very significant difference.The most significant difference in relative expression of enzyme activity in the combination of strong and weak advantages is PMT,followed by BBL,QPT,PMT and QPT enzyme activity weakened in the combination of strong advantage,BBL enzyme activity in Increased in the combination of strong advantages..The correlation between enzyme activity relative expression and nicotinic content and heterosis value showed that PMT?QPT?BBL had the greatest correlation with nicotinic content and heterosis PMT,followed by QPT,and finally BBL.This indicates that the enzyme that has the greatest influence on heterosis in the nicotine synthesis pathway is PMT,followed by QPT.In addition,there is an interaction relationship between the relative expression levels ofenzyme activities,PMT and QPT have a very significant positive correlation,and the two work together to affect the performance of nicotine heterosis.3.Correlation analysis of metabolites in the nicotine synthesis pathway was performed.The results showed that the clustering heat map and PCA scores showed that the difference between the metabolites of the strong dominant combination and the parent was greater than that of the metabolites of the weak dominant combination and the parent.The metabolic products differ greatly in the combination of strong and weak advantages: putrescine,ornithine,N-methylpyrrolidine salt,aspartic acid,NAMN(nicotinic acid single nucleotide),agmatine,among which Amine,ornithine,N-methylpyrrolidine salt,agmatine metabolite content increased in the strong advantage combination,aspartic acid,NAMN(nicotinic acid single nucleotide)metabolite content in the strong advantage combination reduce.Correlation analysis of the relative expression of different metabolites in the combination of strong and weak advantages,nicotine content,and heterosis value shows that the relative expression of the metabolite NAMN(nicotinic acid single nucleotide)in the nicotine synthesis pathway It has a very significant positive correlation with nicotine content and nicotine heterosis.Aspartic acid and ornithine are negatively correlated with nicotine content and nicotine heterosis,putrescine,N-methylpyrrolidine salt,guanidino Butylamine was positively correlated with nicotine content and nicotine heterosis,and none of them reached a significant level.Except for NAMN,the correlation coefficients of putrescine with nicotine content and nicotine heterosis reached the largest,0.74 and 0.64,respectively.This shows that the key metabolite affecting the formation of nicotine heterosis is NAMN(nicotinic acid single nucleotide),followed by putrescine.The above results indicate that the key enzyme affecting the formation of nicotine heterosis is PMT,followed by QPT;The key metabolite is NAMN(nicotinic acid single nucleotide),followed by putrescine;Quinolinic acid phosphate ribosyl transferase(QPT)catalyzes the formation of its substrate quinolinic acid to form NAMN(nicotinic acid single nucleotide)and putrescine-N-methyl transferase(PMT)catalyzes the formation of its substrate putrescine to N-Methyl putrescine is the twokey link in the formation of nicotine heterosis.The research results can provide a theoretical reference for the regulation of heterosis of nicotine content.