【作者】 潘舒群；

【导师】 叶庆生；

【作者基本信息】 华南师范大学 ， 植物学， 2007， 硕士

【摘要】 本文以春石斛(Dedrobium.Pink BEAUTY’Queen’×Dendrobium.Pink GEM’ELEGANCE’)为实验材料，进行春化处理：23±1℃／10±1℃[12h／12h(白天／晚上)]，研究低温对春石斛花芽诱导和分化的效应，在不同时间取样，分析测定了叶片中的碳水化合物，植物各内源激素，EST，POD，SOD同工酶活性及酶谱变化，克隆了与植物开花相关的MADS-box家族的基因，D-MADS，并进行了同源比对及进行叶片、叶芽、不同阶段花芽中的半定量RT-PCR分析。主要实验结果如下：(1)春石斛在40d左右完成春化，芽诱导率达98％，且100％的芽为花芽，2～3朵／节的比例高达80％，每枝有14朵以上的花；未完成春化的植株，芽诱导率低，且大部分发育成叶芽。(2)在低温处理过程中，碳水化合物含量发生变化。在处理的前20d，可溶性糖含量显著增加，随后降低，30d后，糖含量继续增加，40d时达到高峰，179.03mg／g。在处理早期(20d内)淀粉含量逐渐降低，处理20d后又显著上升，40d出现高峰，达到632.96mg／g。处理期间，对照淀粉含量几乎保持不变。淀粉含量与可溶性糖含量变化趋势相反，表明糖含量的增加可能来自淀粉的分解。(3)低温处理过程中，玉米素变化呈双峰曲线。分别在20d和35d达到峰值；IAA含量逐渐增加，20d达到高峰(约为60pmol／gFW)，并维持到30d，随后降低；赤霉素呈递增趋势，在35d达到最高水平(约为48pmol／gFW)，并保持较高水平到45d，随后含量逐渐降低：ABA含量在处理前期(10d内)显著增加，第10d达到最高水平(约为51pmol／gFW)，随后下降，并保持较低水平。IAA／Z在5～35d之间有显著的增加，在35d后又大大降低，几乎与对照保持一致。表明植物内源激素与植物开花有着密切的关联。(4)在处理期间，SOD，POD，EST活性随花芽发育有着明显波动。从EST酶谱上看，春化期间有酶带的消失和重现。春化完成时，出现新的酶带(R_f=0.609)。SOD酶带发生较大的变化，春化完成后，R_f=0.400酶带消失。以上表明同工酶与春化存在一定的关系。(5)根据植物cDNA-3’末端序列的特殊性设计引物(引物2：5’-GACTCGAGTC GATCG ATTTT TTTTT TTTTT TTT-3’)进行cDNA一链的合成，并根据MADS-box基因的保守区域设计上游引物(即引物1：5’-GATCAAG(A／C)G(G／C)ATCGAGAA-3’)，克隆得到895 bp的片段，序列分析表明该MADS-box基因的cDNA中包含一个738 bp读码框(ORF)，是编码246个氨基酸的MADS-box基因，命名为D-MADS。序列多重比较表明D-MADS与球花石斛(Dendrobium thyrsiflorum)的DthyrFL1具有97％的相似性，对应的蛋白序列与拟南芥的AP1蛋白具67％的同源性，与球花石斛蛋白DthyrFL1且具有95％的相似性。通过半定量RT-PCR测定D-MADS在春石斛不同组织和花芽发育不同时期的表达情况，结果显示D-MADS在叶和叶芽中均不表达，但随着花的发育，它的表达不断增强。以上结果进一步表明本实验所克隆的D-MADS基因与春石斛花芽发育密切相关。更多还原

【Abstract】 Dendrobium （Dedrobium. Pink BEAUTY ’Queen’×Dedrobium. Pink GEM ’ELEGANCE’） were treated under low temperature of 10±1℃/23±1℃for 12h/12h （night/day）. The effect of chilling to flower buds inducement and differentiation was studied; The content of carbohydrate, phytohormones and activities of EST, POD, SOD were measured; An MADS-box gene that may related to flowering was cloned, and some analyses about this gene were done. The results were as follows:（1） The Dendrobium finished vernalization in about 40 days. The induction rate of buds was 98%, and all of them were floral bud, the rate of single node with 2-3 buds was high, attaining 80%, each one contained more than 14 flowers. The induction rate of buds for 10 days, 20 days and 30days were lower, and most of them were leaf buds.（2） The content of carbohydrate was variety in low temperature process. In the former 20 days, the dissoluble sugar content increased notably, then decreased. and 30 days later, it increased again, attained the peak of 179.03 mg/g on 40th day. In the former 20 days, the starch content decreased gradually, 30 days later, it increased notably, attained the peak 632.96mg/g on the 40th day. The CK almost kept constant. The variety trend of starch and dissoluble sugar was just opposite, which showed that the increasement of sugar may result from the decomposition of the starch.（3） The Z variety presented as a pair of curves. It got two peaks on 20th day and 35th day; IAA increased gradually, got the peak（about 60 p mol/g FW） on 20th day, and maintained to 35th day, then it declined; GA₃ showed an increaseing trend, got the highest on 30th day, it declined gradually after 45th day; ABA increased rapidly at the beginning, got the peak （51 pmol/g FW） on 10th day, declined later, and kept the lower level. The IAA/Z increased rapidly between 5th day and 35th day. Declined quickly after 35th day. All of these showed that phytohormone had some relationship with flowering.（4） There were some changes in EST, SOD, POD activities during the process. Some special EST isozyme bands disappeared or appeared. On 30th day, a band, R_f=0.609, appeared. There were some obvious changes in SOD. A band, R_f=0.40, disappeared on 30th day. The above showed that isozyme had some relationship with vernalization.（5） Many studies showed that most of genes related to floral differentiation and development belonged to MADS box gene family. According to the special 3’ end sequence of plant cDNA, design a peculiar primer （primer2: 5’-GACTC GAGTC GATCG ATTTT TTTTT TTTTT TTT-3’）; Basing on reported conservative sequence of MADS, design a degenerate primer （primer1:5’-GATCA AG（A/C）G（G/C） ATCGA GAA-3’）, a MADS box cDNA fragment of 895 bps was cloned, named D-MADS. It contains a ORF, coding for 246 amino acids. Multiple alignment about this sequence showed that D-MADS had 97% similarity with DthyrFL1 cloned from Dendrobium thyrsiflorum. Semi-quantitative RT-PCR analysis of D-MADS showed that it was expressed only in flower bud, and its expression increased with the development of flower bud. All of these showed that D-MADS had some relationship with flower bud differentiation of Dendrobium.更多还原