烤烟成熟期淀粉代谢关键酶活性与基因表达研究

Activities of key enzymes related with starch metabolism and gene expression in flue-cured tobacco at maturity

[目的]探讨烤烟成熟期淀粉积累状况与淀粉代谢关键酶活性及其基因表达的关系,明确烤烟成熟期淀粉代谢的分子调控机制,为优质烟叶生产提供理论依据.[方法]以不同淀粉积累型烤烟品种秦烟96和豫烟6号为材料,测定成熟期烟叶淀粉含量及比例(直链淀粉、支链淀粉)和淀粉代谢关键酶活性,并对其关键酶基因(NtAGPase、NtGBSS Ⅰ、NtSSS、NtSBE、NtDBE、Nta-amylase和Ntβ-amylase)进行实时荧光定量PCR分析.[结果]在烤烟发育成熟期,烟叶淀粉、直链淀粉和支链淀粉含量呈先升高后降低的变化趋势,烟叶生理成熟期(移栽后85 d)其含量最高;随着烟叶的成熟,烟叶中支链淀粉所占比例逐渐增加.烤烟成熟期AGPase、GBSS Ⅰ、SSS、SBE和DBE活性呈先升高后降低的变化趋势,而烟叶淀粉酶活性则随着烟叶的成熟先降低后升高,烟叶过熟时其活性显著升高;与烟叶淀粉代谢关键酶活性变化趋势不同,其相关基因表达量变化差异较大.烟叶打顶至适熟阶段,AGPase和GBSSⅠ活性与烟叶直链淀粉含量相关性显著,对烟叶直链淀粉的积累贡献最大,NtGBSS Ⅰ对直链淀粉代谢起主要调控作用;SSS、SBE和DBE活性对烟叶支链淀粉积累起重要作用,NtSBE和NtDBE对烟叶支链淀粉代谢起主要调控作用.当烟叶由适熟至过熟时,淀粉酶(a-淀粉酶、β-淀粉酶)主要参与烟叶的淀粉代谢活动.同一生态环境和栽培条件下,秦烟96淀粉积累较多,具有较高的支链淀粉比例.[结论]烤烟碳水化合物积累以淀粉为主,AGPase和GBSS Ⅰ是影响直链淀粉积累的关键酶,SSS、SBE和DBE对支链淀粉积累有重要作用,SBE和DBE活性的差异可能是造成烟叶直链淀粉与支链淀粉比值不同的重要原因.

【Objective】This study explored the relationship between starch accumulation status,key enzyme activity and gene expression during the mature period of flue-cured tobacco to understand the molecular regulation mechanism of starch metabolism and provide theoretical basis for the production of high quality tobacco.【Method】Starch accumulating tobacco varieties of Qinyan 96 and Yuyan 6 were used to measure the starch content and ratio(amylose,amylopectin)and key enzyme activities of starch metabolism in flue-cured tobacco at maturity.The key enzyme genes(NtAGPase,NtGBSSⅠ,NtSSS,NtSBE,NtDBE,Ntα-amylase and Ntβ-amylase)were analyzed by real-time quantitative PCR.【Result】At the maturity stage of flue-cured tobacco,contents of starch,amylose and amylopectin in tobacco leaves increased first and then decreased with the highest values at the physiological maturity of tobacco leaves(85 d after transplanting).With the maturing of tobacco leaves,the proportion of amylopectin in tobacco leaves gradually increased.The activities of AGPase,GBSSⅠ,SSS,SBE and DBE in flue-cured tobacco increased first and then decreased.The activity of amylase in tobacco leaves decreased firstly and increased significantly at overripe stage.Different from the key enzyme activities of tobacco starch metabolism,the expression of related genes varied greatly.From the topping to proper maturity,the activities of AGPase and GBSSⅠwere significantly correlated with amylose content,and contributed the most to the accumulation of amylose in tobacco leaves.NtGBSSⅠplayed a major role in the regulation of amylose metabolism.The activities of SSS,SBE and DBE enzymes played an important role in the accumulation of amylopectin in tobacco leaves.NtSBE and NtDBE played a major role in the regulation of amylopectin metabolism in tobacco leaves.From proper maturity to overripe stage,amylases(α-amylase,β-amylase)mainly involved in the starch metabolism of tobacco leaves.Under same ecological environment and cultivation conditions,Qinyan 96 accumulated more starch and had higher amylopectin proportion.【Conclusion】Flue-cured tobacco accumulated carbohydrate predominantly as starch.AGPase and GBSSⅠwere key enzymes affecting amylose accumulation.SSS,SBE and DBE played an important role in amylopectin accumulation.The difference in enzyme activities between SBE and DBE may be an important cause of the difference in ratio of amylose to amylopectin in tobacco leaves.