【作者】 邬珺超；

【导师】 蒋滢； 汪成富；

【作者基本信息】 苏州大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 本研究主要分为三部分：第一部分，将采用随机引物K(5’-CGGCCCCTGT-3’)寻找到的高产蜜西蜂的特异DNA分子标记K700bp片段克隆进载体，然后进行测序。第二部分，以3种随机引物(K、W、P)对黑环蜂和黄环蜂的各品系蜂进行RAPD-PCR扩增，将扩增产物进行DNA多态性的差异比较，以作品系鉴定。第三部分，对已经用随机引物w(5’-CGGCCCCGGT-3’)寻找到的高产浆西蜂的特异DNA分子标记W316bp片段进行五个方面的验证。结果：第一部分，测得K700bp片段的全序列，共719bp，与预测结果相符。第二部分，三种引物中，只有P引物扩增得到的多态性图谱能将黑环蜂和黄环蜂及其各品系之间进行逐一区分。第三部分，(1)Southern杂交、(2)斑点杂交表明W316bp分子标记只出现在高产浆西蜂的PCR扩增产物及其基因组DNA中：(3)在雄、工蜂中扩增结果显示只有工蜂具有W316bp分子标记，这与事实一致：(4)根据已有的W316bp片段序列和SCARs方法设计一对特异引物来扩增高、低产浆西蜂，结果仅在高产浆西蜂中得到316bp大小的单一条带，证明W316bp片段确为高产浆的特异DNA分子标记：(5)以W引物对5个蜂场的25群蜂样进行扩增，凡是扩增到W316bp片段的蜂群均是与蜂农的经验结论相一致，属于高产浆西蜂。以上结果可以说明：RAPD-PCR方法是 蜜蜂优良性状相关的特异DNA分子标记的研究摘要 一种非常有效的寻找和鉴定蜜蜂优良性状特异DNA分子标记的方法。用 此法获得的特异DNA分子片段经由验证均可证实该片段是与蜜蜂优良胜 状紧密连锁的DNA片段。为此，通过RAPD一PCR分析方法获得的优良哇 状的分子标记不仅可以广泛应用于优质蜂种的选择，同时还可以作为蜜蜂 种、亚种以及品系之间差别的遗传标记，这为蜂农在分子水平上选育蜜蜂 提供了科学依据，并为将来寻找与蜜蜂优良性状相连锁的基因奠定了坚实 的基础。 作者:乌区裙超 指导教师:蒋澄汪成富更多还原

【Abstract】 There are three parts in this report. The first part is to sequence the special molecular DNA marker - K700bp fragment, which was detected by using the random primer K (5’-CGGCCCCTGT-3’) in A. M. Lindauer of high honey quantity in former experiments. The second part is using three random primers (K> W, P) and the method of RAPD-PCR (Random Amplified Polymorphic DNA-PCR) to differentiate every assession of the Black-ring bees and the Yellow-ring bees respectively. The third part is to identify the W316bp fragment, produced by random primer W (5"-CGGCCCCGGT-3’), as a special molecular DNA marker in A. M. Lindauer of high royal jelly quantity in five ways.The results is as follows: (1) Study got the full sequence of the K700bp fragment, exactly 719bp, and it was accordant with our surmise. (2) Among three primers, only primer P can distinguish every assession of the Black-ring bees and the Yellow-ring bees respectively. (3) Identification of W316bp fragment: (1)Southern blotting and (2)Dot blotting had been tested before and the positive results appeared only in the PCR products or genetic DNA of high royal jelly quantity bees; (3)The result of the amplification in male and worker bees of high royal jelly quantity showed that the W316bp fragment only consisted in worker bees; (4)According to the sequence of the W316bp fragment, designing a pair of primers on the basis of the method of SCARs (Sequence-characterized Amplified Regions), and then amplifying the A. M. Lindauer which were different in royal jelly quantity, and study got an unique fragment only in the high royal jelly quantity bees; (5)Identification of practice. To use primer W to amplify 25 samples gathered from 5 apiaries, and those samples that had the W316bp fragments were the high royal jelly quantity bees that agreed with the conclusion of the apiarists.All these results explained that RAPD-PCR method is an efficient means of searching and confirming the special molecular DNA markers of the good traits and genetic difference of bees. These DNA markers found by the analysis of RAPD-PCR can not only be widely used in selecting of good breed of bees and also be the genetic markers of the differences in the breeds, sub-breeds and assessions of bees. It offers scientific foundation for the apiarists to select and breed bees on the molecular level and establishes a strong base of searching the gene related to the good traits of bees in the future.Written by: Wu Jun-chao Supervised by: Jiang YingWang Cheng-fu更多还原