【作者】 刘彩云；

【导师】 刘洪祥；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2011， 博士

【摘要】 本论文选用白肋型烟草品种Burley21和正常绿色型马里兰烟品种Maryland609为供试材料,在分析了白肋型烟草叶色性状遗传特点的基础上,对Burley21和Maryland609生长发育过程中叶片质体色素含量的差异及其可能机理进行了探讨,以期为白肋型烟草叶色性状、香气品质及高等植物叶色突变研究提供参考依据。论文研究的主要内容及所得结果如下:1、通过大田常规遗传群体的构建,分析了白肋型烟草叶色性状的遗传特点。结果发现, Burley21和Maryland609两类烟草品种的正交与反交F1代植株,全部表现为正常绿色型,而在其BC1代和F2代群体中,既有正常绿色型植株,也有白肋型植株。统计分析表明,杂交F2代群体中正常绿色型和白肋型分离比例为12.5:1,BC1代群体的分离比例为4.9:1。卡平方检验分析表明,两类烟草品种间的F2代和BC1代的分离比例符合双隐性性状遗传规律。从而进一步验证了白肋型烟草叶色的黄绿色性状遗传为两对隐性重叠基因控制的细胞核遗传。2、生育期的连续调查发现,Burley21植株主茎与叶片主脉呈乳白色的性状表现,从苗床期就明显显现,且在整个生长发育过程中均稳定保持该性状表现;但叶色黄绿的表型性状,则是从旺长期开始明显显现,而在苗期和旺长期之前,与Maryland609之间并无明显差异。3、采用分光光度法测定比较了Burley21和Maryland609植株生长发育的假植期、团棵期、旺长期、现蕾期和成熟期中部叶片中质体色素的含量。结果表明, Burley21在生长前期,叶脉中的叶绿素含量均显著低于Maryland609,但类胡萝卜素含量差异在上述五个发育阶段均不明显。生长前期,二者叶肉组织中的叶绿素和类胡萝卜素含量在同一发育时期均无明显差异。从旺长期开始,Burley21叶肉组织中的叶绿素含量明显低于Maryland609,二者在同一发育时期的差异性达到极显著水平。成熟期,这种差异达到最大值,Maryland609叶绿素含量为Burley21中的3.35倍。旺长期之后,Burley21叶肉组织中的类胡萝卜素含量也明显低于Maryland609,但差异幅度明显少于叶绿素。4、测定比较了Burley21和Maryland609旺长期植株叶片中影响叶绿素合成的7种主要前体物含量。结果表明,两种烟草品种旺长期植株叶片中叶绿素合成前体物δ-氨基乙酰丙酸（ALA）、胆色素原（PBG）、尿卟啉原Ⅲ（UrogenⅢ）、粪卟啉Ⅲ（CoprogenⅢ）的含量并没有明显差异,但Burley21叶片中原卟啉Ⅸ（ProtoⅨ）、镁原卟啉Ⅸ（Mg-ProtoⅨ）和原叶绿素酸酯（Pchlide）的含量明显低于Maryland609中。这说明Burley21叶片中叶绿素合成在这几步受阻,从而导致叶绿素含量降低,叶片呈现黄绿色。据此推断,白肋型烟草叶色突变的分子机制,可能与叶绿素生物合成途径中的基因突变有关。5、叶绿素酶是植物体叶绿素降解的关键限速酶。实验采用分光光度法,测定了旺长期Burley21和Maryland609植株叶片叶绿素酶活性,并采用实时定量PCR技术对这两类烟草品种旺长期叶片叶绿素酶基因的表达水平进行了检测比较。酶活性测定结果发现,Burley21和Maryland609叶片中叶绿素酶活性分别为525.17nmol g^-1h-1和257.60nmolg^-1h^-1,前者为后者的2.04倍。实时定量PCR检测结果发现,Burley21叶片中叶绿素酶基因表达量为Maryland609中的3.01倍。这一结果表明,旺长期Burley21叶片的黄绿色性状显现与叶片叶绿素酶活性升高及其基因表达增强有关。由此推断,叶绿素酶基因表达增强及酶活性升高的协同作用,可能加速了叶绿素的降解,进而对白肋型烟草叶片的黄绿色表型性状的显现起到了促进作用。6、采用Southern杂交技术,对Burley21和Maryland609叶绿素酶基因的拷贝数进行了检测。杂交结果表明,这两类烟草中叶绿素酶基因均以单拷贝形式存在。7、采用气质联用技术,对Burley21和Maryland609晾制烟叶中叶绿素和类胡萝卜素降解产生的挥发性香气物质进行了检测分析。结果表明,检测到的16种质体色素类降解物中,有5种为叶绿素降解产生,11种为类胡萝卜素降解产生,但是种类上占优势的类胡萝卜素降解物的总含量却远远小于叶绿素降解产物。在这16种香气成分中,新植二烯含量在两种烟叶样品中均最高。由此推测,晾制过程中,白肋型烟草叶片中质体色素类的降解可能比正常绿色型马里兰烟更加充分,进而对白肋型烟草特有的香气品质的形成产生了影响。更多还原

【Abstract】 In this paper, the Burley21 and Maryland609 were chose as the experimental plant materials, and the heredity of yellowing leaf color and the variation of plastid pigment contents and the mechanism of this variation between Burley21 and Maryland609 were researched. The research results of this paper will give the experimental proof for the causes of burley leaf color, the aroma quanlity of white burley as well as green-deficient mutant in other higher plants. The main researches and conclusions of this paper were as follows:1. The leaf color heredity of white burley was studied by way of construction of field breeding groups. The results found that the F1 generation plants of reciprocal cross between Burley21 and Maryland609 showed the normal green type, and the BC1 generation, F2 generation have both normal green and white burley type. Statictical analysis showed that the ratio of normal green type and white burley type in F2 generation and BC1 generation were 12.5:1 and 4.9:1, respectively. Chi square test showed that the segregation ratio of F2 generation and BC1 generation were consistent with the inheritance law of double-recessive trait. These results further estimated that the leaf color in white burley was the double-recessive trait controlled by nucleus.2. Continuous observation found that the white vein trait in Burley21 can be seen during the whole growth and development period. There was no significant difference between Burley21 and Maryland609 in leaf color before the vigorous growing period, but the yellowing leaf color trait of Burley21 can be seen clearly from the vigorous growing period.3. Chlorophyll and carotenoid contents in the leaf mesophyll and vein tissue of Burely21 and Maryland609 were determined using spectrophotometer during the development of temporary planting period, rosette period, vigorous growing period, flower budding period and mature period, respectively. The results indicated that the contents of chlorophyll in the leaf vein of Burley21 was significantly lower than in that of Maryland609 at the earlier growing periods, but there was no obvious difference in the contents of carotenoids during all those five periods. There were no significant differences in chlorophyll and crotenoid contents before vigorous growing periods in the leaf mesophyll, but from vigorous growing period, the difference of chlorophyll content between the two tobacco varieties was significantly at the same growth and development period. The difference of chlorophyll contents in leaf mesophyll ranked the largest at the mature period, and the chlorophyll contents in Burley21 was 3.35 times as high as in that of Maryland609. The contents of crotenoid in the leaf mesophyll of Burley21 also lower than in that of Maryland609 at the same growh and development period from vigorous growing period, but these differences were much smaller than chlorophyll contents.4. Seven kinds of precursors in the biosynthesis of chlorophyll in leaf mesophyll of Burley21 and Maryland609 during viogous growing period were measured. The results showed that there were no significant difference in the contents of AlA, PBG, UrogenIII, Coprogen III in leaf mesophyll between Burley21 and Maryland609, but the contents of ProtoⅨ, Mg-ProtoⅨand pchlide in Burley21 were significantly lower than in that of Maryland609. This indicated that the synthesis of chlorophyll was inhibited during these steps, and this might led to the lower chlorophyll contents and yellowing leaf phenotype of Burley21 from vigorous growing period. It can be supposed that the mechanism of yellwong leaf phenotype might relate with the mutation of gene in the chlorophyll biosynthesis.5. Chlorophyllase is the rate-limiting enzyme in the degradation of chlorophyll（Matile et al.,1999; Gan et al.,2002; Tang, 2002）. The activities of chlorophyllase as well as its gene expression level in the leaf mesophyll of Burley21 and Maryland609 in vigorous growing period were measured using the technologies of spectrophotometer and real-time quantitative PCR, respectively. The results showed that the activities of chlorophyllase in leaf mesophyll of Burley21 and Maryland609 in vigorous growing period were 525.17nmolg^-1h-1 and 257.60nmolg^-1h^-1. The relative activitiy of chlorophyllase in Burley21 leaf was 2.04 times as high as in Maryland609. The gene expression level of chlorophyllase analysized by real-time quantitative PCR showed 2.01 times higher in Burley21 than in that of Maryland609. These results indicated that the increased activities and its gene expression level of chlorophyllase in the leaf mesophyll of Burley21 in vigorous growing period resulted in the speed up chlorophyll degradation and yellowing leaf color of Burley21.6. The gene copies of chlorophyllase in Burley21 and Maryland609 were studied by using the Southern blotting method. The results indicated that the chlorophyllase was encoded by single-copied gene in both Burley21 and Maryland609. 7. Using Gas Chromatography-Mass Spectrometry （GC-MS） technology, the volatile aroma ingredients produced from the degradation of chlorophyll and caroteniod in the tobacco leaf of Burley21 and Maryland609 were analyzed. The results indicated that sixteen kinds of aromatic components detected from the degradation of these plastid pigments in all volatile aroma ingredients, and in which of sixteen kinds of aromatic components, five kinds were get from the degradation of chlorophyll and the other eleven kinds were from that of caroteniod. But the total contents of these eleven kinds degradated from carotenoid were lower than that of five kinds degradated from chlorophyll. The contents of neophytadiene was the highest in all these detected aroma compounds in both tobacco leaf of Burley21 and Maryland609. We might concluded from these results that the plastid pigments were degradated more fully in curing process in white burley than in that of Maryland tobacco, and this affected the the unique arom quality of white burley.更多还原