生物炭对烟草类胡萝卜素代谢的影响及LCY-b基因过表达调控研究
发布时间：2018-09-14 11:42
【摘要】：为阐明添加生物炭对烟草类胡萝卜素代谢的影响机制,本试验以烟草品种K326为材料,在大田试验中设置不同的生物炭处理,从烟株发育过程中叶片类胡萝卜素积累动态、类胡萝卜素代谢途径关键基因表达和烤后样中性致香物质三个方面进行了系统研究。在此基础上,以类胡萝卜素合成途径中的关键分支点酶LCY-b为切入点,通过同源克隆的方法获得烟草品种K326 LCY-b基因全长序列,利用in-fusion方法成功构建了LCY-b基因过表达载体,采用农杆菌介导法转化烟草植株,得到LCY-b过表达阳性植株共6株。将T0代阳性植株进行大田种植,进一步探究LCY-b基因对烟草类胡萝卜素代谢及烤后样中性致香物质的影响,为烟草类胡萝卜素代谢的分子调控奠定了基础。主要研究结果如下:1.不同生物炭肥施用量对烟草类胡萝卜素代谢影响的分析结果表明:随着烟叶成熟,类胡萝卜素合成相关基因(PSY、ZDS、LCY-b)表达逐渐减弱,降解相关基因(CCD1)表达逐渐增强,与打顶后类胡萝卜素含量逐渐下降的结果一致。烟叶成熟后期,添加生物炭的处理类胡萝卜素积累量较多,其中T1(300 kg·hm-2)处理的类胡萝卜素各组分含量在打顶后一直保持在较高水平。RT-PCR分析结果显示:T1(300 kg·hm-2)和T2(600 kg·hm-2)处理中类胡萝卜素合成及降解相关基因的表达量相对较强,说明适量添加生物炭肥对烟叶类胡萝卜素的合成和降解均有明显的促进作用,整体上减缓了类胡萝卜素含量下降趋势,提高了成熟期烟叶类胡萝卜素积累量。烟叶调制后,T1处理的类胡萝卜素降解产物含量最高,未添加生物炭肥的处理含量最低。以上结果说明,适量添加生物炭能够增强类胡萝卜素代谢相关酶活性,有利于成熟期烟叶类胡萝卜素的积累及调制后中性致香物质的形成。2.采用同源克隆的方法获得烟草LCY-b基因全长序列1557 bp,并对其进行了详细的生物信息学分析。序列分析表明:K326 LCY-b基因包含一个1503 bp的开放读码框(ORF),编码500个氨基酸。预测蛋白质分子量为56.05 KDa,理论等电点(p I)为6.68。蛋白二级结构预测结果显示以α螺旋和无规则卷曲为主。3.利用in-fusion方法成功构建LCY-b过表达载体,采用农杆菌介导法转化烟草植株,获得LCY-b过表达阳性植株。分子检测证明LCY-b基因在烟草基因组中成功实现了整合和转录。过表达阳性植株的q PCR检测结果表明:部分转基因阳性植株LCY-b基因的表达量显著高于K326对照。相应地,类胡萝卜素组分中的β-胡萝卜素、新黄质、紫黄质所占比例增加,而叶黄素所占比例有所降低。对烤后样类胡萝卜降解产物的分析结果显示:LCY-b过表达株系中的β-胡萝卜素降解产物(β-二氢大马酮、β-环柠檬醛、香叶基丙酮、二氢猕猴桃内酯等)显著高于K326未转基因对照,而叶黄素降解产物(巨豆三烯酮)则与K326相当或低于K326对照。研究结果说明在烟草植株中过表达LCY-b基因可以改变类胡萝卜素组分比例,影响烤后样中性致香物质的含量及组分。
[Abstract]:In order to elucidate the mechanism of the effect of biochar addition on carotenoid metabolism in tobacco, a tobacco variety K326 was used as the material in this experiment. Different biochar treatments were set up in the field to study the dynamics of carotenoid accumulation in tobacco leaves, the expression of key genes involved in carotenoid metabolism pathway and the neutral aroma-inducing substances in tobacco leaves. On this basis, the full-length sequence of LCY-b gene of tobacco variety K326 was obtained by homologous cloning, and the LCY-b gene overexpression vector was successfully constructed by in-fusion method. The tobacco plants were transformed by Agrobacterium tumefaciens mediated transformation. Six plants were over-expressed with LCY-b gene. T0 positive plants were planted in the field to further explore the effects of LCY-b gene on carotenoid metabolism and neutral aroma-causing substances in tobacco, which laid the foundation for the molecular regulation of carotenoid metabolism in tobacco. The main results were as follows: 1. Different application of biochar fertilizer on tobacco carotenoids. The results showed that the expression of PSY, ZDS and LCY-b decreased gradually with the maturation of tobacco leaves, and the expression of CCD1 increased gradually, which was consistent with the decrease of carotenoid content after topping. The results of RT-PCR analysis showed that the expression of genes related to carotenoid synthesis and degradation in T1 (300 kg The synthesis and degradation of carotenoids were promoted obviously, and the decreasing trend of carotenoids content was slowed down, and the accumulation of carotenoids in mature tobacco leaves was increased. After curing, the content of carotenoids degradation products in T1 treatment was the highest, while that in the treatment without biochar fertilizer was the lowest. Carbon can enhance the activity of carotenoid metabolism-related enzymes, which is beneficial to the accumulation of carotenoids in mature tobacco leaves and the formation of neutral aroma-inducing substances. 2. The full-length sequence of LCY-b gene in tobacco was obtained by homologous cloning method and its bioinformatics analysis was carried out in detail. The predicted protein molecular weight was 56.05 KDa and the theoretical isoelectric point (PI) was 6.68. The predicted secondary structure of the protein was mainly alpha helix and irregular curl. 3. LCY-b overexpression vector was successfully constructed by in-fusion method and transformed into tobacco plants by Agrobacterium tumefaciens. LCY-b gene was successfully integrated and transcribed in tobacco genome by molecular detection. The results of q-PCR showed that the expression of LCY-b gene in some transgenic plants was significantly higher than that in K326 control. The proportion of violaxanthin increased, but the proportion of lutein decreased. The analysis of the degradation products of carotenoids after baking showed that the degradation products of beta-carotene (beta-dihydro-damascone, beta-cyclic citral, geranyl acetone, dihydro-kiwifruit lactone, etc.) in LCY-b overexpression lines were significantly higher than those in K326 untransformed control. The results showed that over-expression of LCY-b gene in tobacco plants could alter the proportion of carotenoids and affect the content and components of neutral aroma-causing substances.
【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
本文编号：2242612
[Abstract]:In order to elucidate the mechanism of the effect of biochar addition on carotenoid metabolism in tobacco, a tobacco variety K326 was used as the material in this experiment. Different biochar treatments were set up in the field to study the dynamics of carotenoid accumulation in tobacco leaves, the expression of key genes involved in carotenoid metabolism pathway and the neutral aroma-inducing substances in tobacco leaves. On this basis, the full-length sequence of LCY-b gene of tobacco variety K326 was obtained by homologous cloning, and the LCY-b gene overexpression vector was successfully constructed by in-fusion method. The tobacco plants were transformed by Agrobacterium tumefaciens mediated transformation. Six plants were over-expressed with LCY-b gene. T0 positive plants were planted in the field to further explore the effects of LCY-b gene on carotenoid metabolism and neutral aroma-causing substances in tobacco, which laid the foundation for the molecular regulation of carotenoid metabolism in tobacco. The main results were as follows: 1. Different application of biochar fertilizer on tobacco carotenoids. The results showed that the expression of PSY, ZDS and LCY-b decreased gradually with the maturation of tobacco leaves, and the expression of CCD1 increased gradually, which was consistent with the decrease of carotenoid content after topping. The results of RT-PCR analysis showed that the expression of genes related to carotenoid synthesis and degradation in T1 (300 kg The synthesis and degradation of carotenoids were promoted obviously, and the decreasing trend of carotenoids content was slowed down, and the accumulation of carotenoids in mature tobacco leaves was increased. After curing, the content of carotenoids degradation products in T1 treatment was the highest, while that in the treatment without biochar fertilizer was the lowest. Carbon can enhance the activity of carotenoid metabolism-related enzymes, which is beneficial to the accumulation of carotenoids in mature tobacco leaves and the formation of neutral aroma-inducing substances. 2. The full-length sequence of LCY-b gene in tobacco was obtained by homologous cloning method and its bioinformatics analysis was carried out in detail. The predicted protein molecular weight was 56.05 KDa and the theoretical isoelectric point (PI) was 6.68. The predicted secondary structure of the protein was mainly alpha helix and irregular curl. 3. LCY-b overexpression vector was successfully constructed by in-fusion method and transformed into tobacco plants by Agrobacterium tumefaciens. LCY-b gene was successfully integrated and transcribed in tobacco genome by molecular detection. The results of q-PCR showed that the expression of LCY-b gene in some transgenic plants was significantly higher than that in K326 control. The proportion of violaxanthin increased, but the proportion of lutein decreased. The analysis of the degradation products of carotenoids after baking showed that the degradation products of beta-carotene (beta-dihydro-damascone, beta-cyclic citral, geranyl acetone, dihydro-kiwifruit lactone, etc.) in LCY-b overexpression lines were significantly higher than those in K326 untransformed control. The results showed that over-expression of LCY-b gene in tobacco plants could alter the proportion of carotenoids and affect the content and components of neutral aroma-causing substances.
【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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1 张嘉炜;生物炭对烟草类胡萝卜素代谢的影响及LCY-b基因过表达调控研究[D];河南农业大学;2016年
，本文编号：2242612
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