| 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. |
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