【作者】 李辉；

【导师】 孙焕良； 李德芳；

【作者基本信息】 湖南农业大学 ， 种子科学与工程， 2013， 博士

【摘要】 烟叶是卷烟工业的原材料,是行业发展的物质基础。烟叶的质量和风格直接影响卷烟产品的品质和风格。浓香型烟叶在中式卷烟原料中占有重要地位。浓香型风格烤烟形成机制的研究对浓香型风格烤烟的育种、生产具有重要意义。本研究以湖南省浓香型特色优质烟叶为研究对象,通过对烟草叶片解剖图谱的构建、叶片质体色素含量和碳氮代谢相关酶活性的测定、叶片基因组DNA的MSAP分析和叶片叶绿体差异蛋白质组分析,探求湖南浓香型风格烤烟的形态发育和物质代谢规律,以及浓香型烤烟风格形成的分子机制,具体的研究结果如下：(1)通过对桂阳不同烤烟品种和K326在不同烟区生长过程中烟叶解剖结构显微观察测量表明：浓香型风格烤烟,在烤烟生长前期,气候条件适宜,叶片碳氮代谢旺盛,各组织结构生长迅速,叶片厚度平均增长幅度为67.4%、栅栏组织厚度平均增长幅度为68.6%、海绵组织厚度平均增长幅度为68.1%,在烤烟成熟期,高温、少雨,叶片各组织结构快速降解,叶片迅速变黄成熟。这是浓香型风格烤烟形成过程中烟叶组织结构变化的一个特点。(2)通过对桂阳不同烤烟品种和K326在不同烟区生长过程中质体色素含量变化的分析结果表明：在烤烟大田的生长前期,叶绿素含量的变化幅度比较小,下降幅度为19.6%,在烤烟大田的成熟期,叶绿素含量下降幅度大,下降幅度为62%,在烤烟大田生长期内,类胡萝卜素含量的变化不大。这是浓香型风格烤烟形成的质体色素代谢的特点。(3)通过对桂阳不同烤烟品种和K326在不同烟区生长过程中碳氮代谢相关酶活性的分析结果表明：浓香型风格的烤烟,碳代谢相关酶转化酶是下降的曲线,淀粉酶是双峰曲线,在成熟期转化酶的平均活是3.395mg/g·h,淀粉酶的平均活是6.33u/mg,显著低于中间香型烟区的酶活性；氮代谢相关酶硝酸还原酶是下降的曲线,谷氨酰胺合成酶是双峰曲线,在成熟期酶硝酸还原酶的平均活是0.865u/mg,谷氨酰胺合成酶的平均活是0.291u/mgport,显著高于中间香型烟区的酶活性,有利于烟碱的合成。这是浓香型风格烤烟形成的碳氮代谢的一个重要特点。(4)同一烤烟品种在不同的生态烟区具有不同的香型风格,为了研究揭示香型风格形成的机制,以烤烟品种K326为材料,对其在典型清香型烟区玉溪和浓香型烟区永州烟叶基因组DNA进行MSAP分析,结果表明：永州烟草叶片的甲基化水平明显高于玉溪的甲基化水平,其中半甲基化比率要高出3.97%,全甲基化比率高出1.74%,MSAP比率高出5.78%。BLAST比对表明,永州K326的基因组中发生了甲基化的MSAP差异片段与编码tRNA-Asn、tRNA-Arg的基因和参与编码叶绿体核糖体蛋白的基因高度同源。DNA甲基化水平和模式的不同影响了永州K326基因组的调控和表达,从而形成了浓香型风格的烤烟。(5)同一烤烟品种在不同的生态烟区具有不同的香型风格,为了研究揭示香型风格形成的机制,以烤烟品种K326为材料,利用双向电泳联合质谱技术对其在典型清香型烟区玉溪和浓香型烟区桂阳烟叶叶绿体差异蛋白质组进行分析,结果表明：桂阳叶绿体蛋白质在分子量为14.3KD-97.2KD之间可以检测到的蛋白质点数分别为1270,上升表达的蛋白质点为41个,其中特异表达的蛋白质点为10个。对质谱鉴定结果,用Mascot软件对烟草数据库和NCBInr数据库进行检索得到4个同源匹配蛋白质,经过生物信息学的综合比对确定特异表达的蛋白为ATP合酶的β亚基和核酮糖-1,5-二磷酸羧化酶／加氧酶。更多还原

【Abstract】 Tobacco is the raw material of cigarette industry, is the material basis for the development of the cigarette industry. The quality and style of tobacco directly influence the quality and style of cigarette product. Luzhou-flavor Flue-cured tobacco leaf production occupies an important position in the Chinese style cigarette materials. It is important to study the mechanism of the formation of strong aroma style flue-cured tobacco on the breeding and production of strong aroma style flue-cured tobacco.This research took Luzhou-flavor high-quality tobacco leaves in Hunan province as the object of study, through the developing anatomical mapping of tobacco leaf, studying the changes of leaf pigment content and the activities of carbon and nitrogen metabolism related enzyme, the MSAP analysis of leaf genomic DNA,the analysis of different proteom of chloroplast, coformed the developmental foundation of flue-cured tobacco varieties, metabolism rules of flue-cured tobacco style formation, explored the molecular mechanism of the formation of flue-cured tobacco style, the specific research results were as follows:(1) Through the observation and measurement of leaf anatomical structure which was different flue-cured tobacco varieties in Guiyang and K326in different tobacco-growing areas,the results showed:Strong aroma style flue-cured tobacco, at the early growth stage of flue-cured tobacco, climatic conditions was suitable, carbon and nitrogen metabolism was exuberant, the organizational structure growed rapidly, the average growth rate of leaf thickness was67.4%, average growth rate of palisade tissue thickness was68.6%, the average growth rate of sponge tissue thickness was68.1%, during the flue-cured tobacco mature period, high temperature, little rain, the organizational structure of leaves degradated rapidly, leaf yellowing mature quickly.This is a feature of the change of leaf tissue structure during the formation of strong aroma style flue-cured tobacco.(2) Through analysing the change of plastid pigment content which were different flue-cured tobacco varieties in Guiyang and K326in different tobacco-growing areas,the results showed:at the early growth stage of flue-cured tobacco in the field, changes of chlorophyll content was relatively small, a decline of19.6%, during the flue-cured tobacco mature period, the chlorophyll content decreased greatly, decreased by62%, during the field growth period, the change of carotenoid content was little. This is a characteristic of plastid pigment metabolism during the formation of strong aroma style flue-cured tobacco(3) Through the analysis enzyme activities associated carbon and nitrogen metabolism of different flue-cured tobacco varieties in Guiyang and of K326in different areas in the growth process,the results showedrduring the tobacco growth process in the field, carbon metabolism related enzyme invertase was a downward curve, amylase was a Shuangfeng curve, the average activity of invertase in mature period was3.395mg/g· h, the average activity of amylase was6.33u/mg, which was significantly lower than the activity of middle flavor tobacco area; Nitrogen metabolism related enzyme nitrate reductase was a downward curve, glutamine synthetase was a Shuangfeng curve, the average maturity of enzyme activity of nitrate reductase was0.865u/mg, the average activity of glutamine synthetase was0.29lu/mgport,which was significantly higher than the enzyme activity of middle flavor tobacco area, which was suitable for synthesis of nicotine. This is an important feature of the carbon and nitrogen metabolism of the formation of Luzhou style Flue-cured tobacco.(4) The same flue-cured tobacco variety in different ecological tobacco area has a different style of flavor, to reveal the mechanism of the flavor style formation, Flue-cured tobacco K326as the material, We used MSAP to analysis tabacco genomic DNA wich was in typical faint scent tobacco area Yuxi and thick aroma tobacco area Yongzhou. The results showed that The methylation level of Yongzhou tobacco leaf is significantly higher than Yuxi,Of which the hemimethylated ratio is3.97%higher, full methylation ratio is1.74%higher, MSAP ratio is5.78%higher.BLAST comparison showed that The methylated MSAP fragments of Yongzhou K326genome and coding tRNA-Asn, tRNA-Arg genes,encoding chloroplast ribosomal protein genes are highly homologous. The difference of DNA methylation level and pattern impacted the regulation and expression of Yongzhou K326genome, thus forming a strong aroma style flue-cured tobacco.(5) The same flue-cured tobacco variety in different ecological tobacco area has a different style of flavor, to reveal the mechanism of the flavor style formation, Flue-cured tobacco K326as the material, Using two-dimensional electrophoresis combined with mass spectrometry to analysis Chloroplast proteome of tobacco leaves in typical Fen-flavor tobacc area Yuxi and Luzhou-flavor tobacco area Guiyan.The results showedGuiyang chloroplast protein spots were1270wich can be detected in the molecular weight of14.3KD-97.2KD of wich the increased expressed protein was41points, the specific expressed protein was10points.According Mass spectrometry identification results,using Mascot softwaren to search the tobacco database and the NCBInr database,we found4homologous matching protein, after a comprehensive bioinformatic comparison, the specific expressed protein were ATP synthase beta subunit and ribulose-1,5-two phosphate carboxylase/oxygenase.更多还原