施氮量对烟叶成熟期重要物质代谢及基因表达的影响研究

发布时间：2018-01-28 00:09

本文关键词： 施氮量烤烟细胞超微结构常规化学成分质体色素多酚基因表达　出处：《河南农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：为了阐明不同的氮肥施用量量对烟叶风格特色形成的影响,本研究以烤烟品种豫烟10号为试验材料,分别设置低氮处理(T1,22.5 kg/hm2)、中氮处理(T2,26.25 kg/hm2)和高氮处理(T3,30 kg/hm2)三个处理在河南省许昌市襄县进行种植,并根据当地最优的烟叶生产管理方法进行管理;在烟叶生长发育过程中,对打顶后(即移栽后60天)的不同时期的中部叶的细胞超微结构、常规化学成分和质体色素成分以及多酚类化合物成分及其相关的基因表达规律进行比较分析,探索烤烟烟叶在不同氮肥施用量条件下的生长发育规律及物质代谢特点对烟叶质量特色的影响。所获得的结果如下:1.细胞超微结构比较:采用透射电镜的方法对不同施氮量条件下豫烟10号不同发育时期中部叶组织结构特点、叶绿体和淀粉粒的发育、降解规律以及其它的一些细胞器和内含物的变化规律进行了比较研究。结果表明:豫烟10号烤烟品种中部叶在不同氮肥施用量条件下叶片细胞组织结构变化规律差异明显,T1处理下的烟叶淀粉粒体积小且含量少,嗜锇颗粒体积和含量增大增多趋势明显;T2处理下烟叶淀粉粒和嗜锇颗粒的体积及含量增大增多较为明显;T3处理下烟叶淀粉粒和嗜锇颗粒体积及含量增大增多不明显。不同施氮量下的烟叶细胞结构发育比较,正常施氮量下的烟叶淀粉粒降解更为充分,发育更快。2.碳氮代谢相关物质以及相关基因表达行比较分析:(1)采用流动分析仪技术对不同施氮量下烤烟品种豫烟10号中部叶的常规化学成分(淀粉、总糖、还原糖、总氮)进行检测分析:从整体来看,成熟期烤烟中部叶的淀粉、总糖和还原糖含量均表现出逐渐增加的趋势,总氮含量表现出平稳递降的趋势;对氮处理的研究发现,淀粉和总氮含量随着土壤氮肥施用量的增加表现出逐渐增加的趋势,而总糖和还原糖含量则相反,表现出递降的趋势。(2)采用RT-PCR技术对中部叶碳氮代谢的相关基因进行表达分析,发现糖代谢的相关基因INV的表达量较弱,而在移栽后90天T3处理下的表达有所增强;而AGPase基因在T1和T2处理下的表达量增加的较为明显。与淀粉合成的相关基因GBSSI和SBE表达量在T1处理下表现先升高后下降的趋势,而在T2和T3处理下则表现为持续上升趋势,说明随着施氮量的增加,淀粉的积累较为明显。与氮代谢相关的基因NR表达量在三个处理中均较高,尤其在T1处理下,NR基因表达量显著高于T3处理;而Ni R基因在T1处理下基因表达量有高于T3处理的趋势。3.质体色素类物质及其相关基因表达分析:(1)采用LC/MS技术对不同氮肥施用量下豫烟10号中部叶的质体色素含量进行检测分析:从整体来看,成熟期烤烟中部叶的叶绿素a、叶绿素b、新黄质、紫黄质、叶黄素和β-胡萝卜素的含量随着成熟期的推进均表现出逐渐降低的趋势;随着氮肥施用量的增加烤烟烟叶中的质体色素的含量表现出逐渐增加的趋势。(2)采用Q-PCR技术对烤烟中部叶质体色素代谢相关基因进行表达检测分析,发现叶绿素合成的关键基因CAB表达差异较为明显,随着烟叶的成熟CAB基因的表达量增强;同时CAB基因随着氮肥施用量的增加而增强。叶绿素降解的基因CLH在T3处理下的表达较弱,在TI和T2处理下的表达较强,这与细胞超微结构的观察结果较为一致。而与类胡萝素合成的相关基因PSY、PDS、ZDS、LCYB和LCYE,在移栽后62天和76天其表达量表现出逐渐减弱的趋势,而在移栽后90天,类胡萝素合成的相关基因的表达量表现出增强的趋势;而在不同施氮量处理下,类胡萝素相关基因的表达量随着氮肥施用量的增加呈逐渐增强的趋势。4.多酚类物质及其相关基因表达分析:(1)采用LC/MS技术对不同施氮量下豫烟10号中部叶的多酚类物质含量检测分析:整体来看,绿原酸含量随着烟叶的成熟而升高,随着氮肥施用量的增加而降低。随着成熟期生育期的推进芸香苷含量表现出逐渐升高的趋势,达到生理成熟后开始下降,而随着氮肥施用量的增加芸香苷含量表现出逐渐降低的趋势。(2)采用RT-PCR技术对烤烟中部叶黄酮类物质代谢的相关基因进行表达分析,研究发现随着烟叶的成熟PAL、4CL、CHS和CHI基因的表达量表现为先升高再降低的趋势,而随着施氮量的增加该途径相关基因表达量减弱。总之,从不同处理下的叶片超微结构、物质代谢以及相关基因表达分析的研究结果来看,T1处理下的烟叶提早进入衰老期,表现出早衰现象;T2处理下的烟叶适时成熟,代谢产物分配更为合理;T3处理下的烟叶表现为延迟成熟。
[Abstract]:In order to clarify the effects of different amount of nitrogen fertilizer on the formation of the style and characteristics of tobacco leaves, the flue-cured tobacco varieties Yuyan No. 10 as the test material, are respectively provided with low nitrogen treatment (T1,22.5 kg/hm2), nitrogen (T2,26.25 kg/hm2) and high nitrogen (T3,30 kg/hm2) three treatment were planted in Xuchang city of Henan Xiang county and managed according to province, tobacco production management method of local optimum; in the process of growth and development in tobacco leaves after topping on (60 days after transplanting) cell ultrastructure of middle leaves in different periods, the law of comparative analysis of expression of conventional chemical composition and pigment composition and composition of polyphenolic compounds and related genes exploration, flue-cured tobacco in different amount of nitrogen fertilizer under the condition of growth and metabolism characteristics influence on the quality of tobacco leaves. The results obtained are as follows: 1. cell ultrastructure comparison: Methods by using transmission electron microscopy to different nitrogen Yuyan 10 different developmental stages of central leaf tissue structure characteristics of chloroplast and starch granule development, degradation and other organelles and inclusions changes were studied. The results showed that the difference of Yuyan 10 varieties of flue-cured tobacco leaves in the middle in different amount of nitrogen fertilizer under the condition of the cell structure changes significantly, T1 treatment of tobacco starch grains with small volume and content of less osmiophilic particles size and the increase of the content of T2 increased significantly; the treatment volume and content of osmiophilic granules of tobacco starch and increase obviously; T3 treatment of tobacco starch and the osmiophilic granules content and volume increase is not obvious. The development of cell structure of tobacco leaf under different nitrogen levels, the normal application of tobacco starch degradation of nitrogen is more fully developed faster,.2. For the comparative analysis of expression of carbon and nitrogen metabolism related genes and related substances: (1) using the technology of flow analyzer under different nitrogen in flue-cured tobacco varieties Yuyan 10 middle leaves of routine chemical components (starch, total sugar, reducing sugar, total nitrogen) were detected and analyzed: on the whole, the starch maturity of flue-cured tobacco in central leaf the content of total sugar and reducing sugar showed a trend of increase gradually, the total nitrogen content showed a stable trend of degradation; nitrogen, starch and total nitrogen content in soil increased with nitrogen fertilizer showed a gradual increase trend, while the content of total sugar and reducing sugar in performance a descending trend. (2) the genes related to the metabolism of central Ye Tandan expression analysis using RT-PCR technology, found the expression of weak INV gene related to glucose metabolism, and expression in 90 days after transplanting under T3 treatment increased; AGPase gene The expression in T1 and T2 treatment increased significantly. And starch synthesis related genes GBSSI and SBE expression decreased in the treatment of T1, and in T2 and T3 treatment showed a rising trend, with the increase of nitrogen, starch accumulated more obviously. NR gene expression correlated with nitrogen metabolism in the three treatments were higher, especially in the treatment of T1, NR gene expression was significantly higher than that of T3 treatment; and Ni R gene in gene expression under T1 treatment is higher than the trend.3. plastid pigment material T3 processing and related gene expression analysis: (1) were detected and analyzed the content of plastid pigment of different amount of nitrogen fertilizer under the Yuyan No. 10 middle leaves using LC/MS Technology: on the whole, mature period of flue-cured tobacco in central leaf chlorophyll a, chlorophyll b, neoxanthin, violaxanthin, content of lutein and beta carotene as The advance of ripe stage were shown to decrease gradually; with the increase of nitrogen content in flue cured tobacco plastid pigment showed a gradually increasing trend. (2) of the central leaves of flue-cured tobacco plastid pigment metabolism related gene expression analysis using Q-PCR technology, the key gene synthesis of chlorophyll CAB expression difference is obviously, with the expression of mature CAB gene tobacco was enhanced; while CAB gene increased with the increasing of nitrogen fertilizer. Weak expression of gene CLH of chlorophyll degradation in the treatment of T3, TI and T2 in the treatment of strong, the results and observation of cell ultrastructure and related gene PSY is more consistent. Synthesis and type of carotene, PDS, ZDS, LCYB and LCYE, at 62 days after transplanting 76 days and its expression showed a decreasing trend, and in 90 days after transplanting, the expression of genes related to class carotene synthesis. The amount shown increasing trend; and in different nitrogen treatments, the expression of type carotene related genes with nitrogen fertilizer increased the trend was gradually increased.4. of polyphenols and related gene expression analysis: (1) the detection and analysis of polyphenol content under different nitrogen Yuyan 10 middle leaves by LC/MS: Overall, chlorogenic acid content increased with the maturity of tobacco leaves, decreased with the increasing of nitrogen fertilizer. With the maturity of the development stages of rutin content showed a gradually increasing trend, began to decline after physiological maturity reached, and with the increasing of nitrogen fertilizer rutin content exhibited a gradual decrease trend. (2) related genes on Flue-cured Tobacco in central leaf flavonoids metabolism expression analysis using RT-PCR technology, the study found that with the mature PAL, leaf 4CL, expression of CHS and CHI gene The performance of the first increased and then decreased, with the increase of the expression of genes involved in this pathway of nitrogen content decreased. In short, from the ultrastructure of leaves under different treatments, the expression of genes related to metabolism and analysis of the research results, T1 treatment of tobacco leaves early entry into the aging period, showing the phenomenon of premature T2; under the treatment of tobacco timely mature, metabolite distribution more reasonable; leaves under T3 treatment for delayed maturity.

【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S572

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