玉溪、许昌烤烟烟叶中淀粉酶基因的表达及酶活性分析
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摘要
为分析河南、云南两地烤后烟叶中糖含量差异形成的原因,以许昌、玉溪成熟期烤烟中部烟叶为材料,通过基因芯片数据寻找与淀粉降解相关的差异基因。利用实时定量PCR和淀粉酶活性分析进行验证,并测定淀粉、总糖的含量(质量分数)。结果表明:获得4个与淀粉降解相关的差异基因,其中2个为α-淀粉酶基因、2个为β-淀粉酶基因,这4个基因在玉溪烟叶中的表达量明显高于许昌烟叶。玉溪烟叶中的α-和β-淀粉酶活性高于许昌烟叶。在玉溪、许昌成熟期中部烟叶中,淀粉含量分别为33.00%和35.00%,两者差异不大。烤前烟叶的总糖含量玉溪烟叶(2.10%)低于许昌烟叶(3.20%),而烤后烟叶的总糖含量玉溪烟叶(36.46%)高于许昌烟叶(20.60%)。表明影响烤后烟叶总糖含量的主要因素为淀粉酶,而不是烤前烟叶的淀粉积累量。
In order to identify possible causes behind the differences of sugar contents between cured tobacco leaves from Henan and Yunnan, starch degradation-related genes were identified on the basis of gene array data of middle tobacco leaves grown in Xuchang and Yuxi at mature stage. The gene array results were verified by qPCR and enzyme activity, and the contents of starch and total sugar were detected. The results showed that there were four starch degradation-related genes: two α-amylase genes and two β-amylase genes. The expressions of four amylase genes and the activities of α-and β-amylases were higher in Yuxi leaves than in Xuchang leaves. The starch contents in middle tobacco leaves from Yuxi and Xuchang at mature stage were 33.00% and 35.00%,respectively. And there was no obvious difference of starch content between Yuxi and Xuchang leaves. The total sugar content in Yuxi tobacco leaves(2.10%) before flue-curing was lower than that in Xuchang(3.20%).However, the total sugar content in cured Yuxi leaves(36.46%) was higher than that in cured Xuchang leaves(20.60%). The results indicated that amylase was a key influencing factor of the total sugar content in cured leaves,while the starch accumulation in tobacco leaves before flue-curing was not.
In order to identify possible causes behind the differences of sugar contents between cured tobacco leaves from Henan and Yunnan, starch degradation-related genes were identified on the basis of gene array data of middle tobacco leaves grown in Xuchang and Yuxi at mature stage. The gene array results were verified by qPCR and enzyme activity, and the contents of starch and total sugar were detected. The results showed that there were four starch degradation-related genes: two α-amylase genes and two β-amylase genes. The expressions of four amylase genes and the activities of α-and β-amylases were higher in Yuxi leaves than in Xuchang leaves. The starch contents in middle tobacco leaves from Yuxi and Xuchang at mature stage were 33.00% and 35.00%,respectively. And there was no obvious difference of starch content between Yuxi and Xuchang leaves. The total sugar content in Yuxi tobacco leaves(2.10%) before flue-curing was lower than that in Xuchang(3.20%).However, the total sugar content in cured Yuxi leaves(36.46%) was higher than that in cured Xuchang leaves(20.60%). The results indicated that amylase was a key influencing factor of the total sugar content in cured leaves,while the starch accumulation in tobacco leaves before flue-curing was not.
引文
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[12] YC/T 159—2002烟草及烟草制品水溶性糖的测定连续流动法[S].YC/T 159—2002 Tobacco and tobacco products—Determination of water soluble sugars—Continuous flow method[S].
[13] YC/T 216—2013烟草及烟草制品淀粉的测定连续流动法[S].YC/T 216—2013 Tobacco and tobacco products—Determination of starch—Continuous flow method[S].
[14]唐煌,张军杰,鲁黎明,等.四川三大烟区烤烟淀粉分解酶类及其基因表达差异分析[J].南方农业,2017,11(25):31-36.TANG Huang,ZHANG Junjie,LU Liming,et al.Analysis of starch degradation enzymes and their gene expression in flue-cured tobacco from three tobacco-growing areas in Sichuan[J]. South China Agriculture,2017,11(25):31-36.
[15]宫长荣,袁红涛,陈江华.烤烟烘烤过程中烟叶淀粉酶活性变化及色素降解规律的研究[J].中国烟草学报,2002,8(2):16-20.GONG Changrong,YUAN Hongtao,CHEN Jianghua.Studies on activity of amylase and degradation of starch and pigment in tobacco leaf during the process of curing[J]. Acta Tabacaria Sinica,2002,8(2):16-20.
[2]董维杰,张忠锋,窦玉青,等.烤烟烟叶淀粉含量影响因素及其与烟叶质量关系研究进展[J].广东农业科学,2015,42(8):11-16.DONG Weijie,ZHANG Zhongfeng,DOU Yuqing,et al. Research on influencing factors of flue-cured tobacco starch content and its relationship with tobacco leaf quality[J]. Guangdong Agricultural Sciences,2015,42(8):11-16.
[3] Weeks W W. Chemistry of tobacco constituents influencing flavor and aroma[J]. Recent Advances in Tobacco Science,1985(11):175-200.
[4]李婷婷,陆新莉,苟正贵,等.烘烤方式对烤烟蛋白酶和淀粉酶活性及化学成分的影响[J].山地农业生物学报,2018,37(3):69-73.LI Tingting,LU Xinli,GOU Zhenggui,et al. Effect on main enzyme activity and chemical composition of flue-cured tobacco by different curing methods[J].Journal of Mountain Agriculture and Biology,2018,37(3):69-73.
[5]艾复清,韩硕,翟欣,等.红花大金元烘烤过程中主要酶活性变化规律的研究[J].山地农业生物学报,2010,29(1):6-9.AI Fuqing,HAN Shuo,ZHAI Xin,et al. The activity fluctuation of several enzymes in flue-curing of Honghuadajinyuan[J]. Journal of Mountain Agriculture and Biology,2010,29(1):6-9.
[6]王怀珠,杨焕文,郭红英,等.淀粉类酶降解鲜烟叶中淀粉的研究[J].中国烟草科学,2005,26(2):37-39.WANG Huaizhu,YANG Huanwen,GUO Hongying,et al. Effects of additional amylase on degradation of starch in fresh tobacco leaves during flue-curing[J].Chinese Tobacco Science,2005,26(2):37-39.
[7] GONG Changrong,YUAN Hongtao,CHEN Jianghua,et al. Studies on amylase and degradation of starch and pigment of tobacco leaf during process of flue-curing[J]. Agricultural Sciences in China,2004,3(12):931-936.
[8]张松涛,杨永霞,滑夏华,等.不同生态区烟叶淀粉生物合成动态比较研究[J].中国烟草学报,2012,18(4):31-34,40.ZHANG Songtao,YANG Yongxia,HUA Xiahua,et al.Comparison of dynamic metabolism of starch biosynthesis in tobacco leaves from different ecological zones[J]. Acta Tabacaria Sinica,2012,18(4):31-34,40.
[9]王晶.不同生态区烟叶碳水化合物合成及积累规律的比较研究[D].郑州:河南农业大学,2013.WANG Jing. Research on the dynamic synthesis and accumulation regularity of carbohydrate on tobacco leaves of different ecological zone[D]. Zhengzhou:Henan Agricultural University,2013.
[10]宫长荣,袁红涛,周义和,等.烟叶在烘烤过程中淀粉降解与淀粉酶活性的研究[J].中国烟草科学,2001,22(2):9-11.GONG Changrong,YUAN Hongtao,ZHOU Yihe,et al.Studies on degradation of starch and change of activity of amylase of tobacco leaf during process of curing[J].Chinese Tobacco Science,2001,22(2):9-11.
[11]张慧,金立锋,徐国云,等.烟草慢阴离子通道蛋白基因NtSLAH1的克隆及表达分析[J].烟草科技,2018,51(3):1-6.ZHANG Hui,JIN Lifeng,XU Guoyun,et al. Cloning and expression analysis of slowly activating anion channel gene NtSLAH1 from Nicotiana tabacum[J].Tobacco Science&Technology,2018,51(3):1-6.
[12] YC/T 159—2002烟草及烟草制品水溶性糖的测定连续流动法[S].YC/T 159—2002 Tobacco and tobacco products—Determination of water soluble sugars—Continuous flow method[S].
[13] YC/T 216—2013烟草及烟草制品淀粉的测定连续流动法[S].YC/T 216—2013 Tobacco and tobacco products—Determination of starch—Continuous flow method[S].
[14]唐煌,张军杰,鲁黎明,等.四川三大烟区烤烟淀粉分解酶类及其基因表达差异分析[J].南方农业,2017,11(25):31-36.TANG Huang,ZHANG Junjie,LU Liming,et al.Analysis of starch degradation enzymes and their gene expression in flue-cured tobacco from three tobacco-growing areas in Sichuan[J]. South China Agriculture,2017,11(25):31-36.
[15]宫长荣,袁红涛,陈江华.烤烟烘烤过程中烟叶淀粉酶活性变化及色素降解规律的研究[J].中国烟草学报,2002,8(2):16-20.GONG Changrong,YUAN Hongtao,CHEN Jianghua.Studies on activity of amylase and degradation of starch and pigment in tobacco leaf during the process of curing[J]. Acta Tabacaria Sinica,2002,8(2):16-20.