【作者】 曲益涛；

【导师】 董玲；

【作者基本信息】 安徽农业大学 ， 果树学， 2010， 硕士

【摘要】 栝楼(Trichosanthes kirilowii Maxim)是葫芦科栝楼属的一种药食两用植物,多年生草质藤本。栝楼种植历史悠久,栝楼根、果皮、果实均可入药。随着近年栝楼籽休闲食品的开发,栝楼的根与果实的主要用途发生很大变化,市场价格相差悬殊,从综合效益考虑,有效控制雌、雄株比例在生产上是非常必要的。栝楼雌、雄异株,在栝楼未开花前从形态上很难辨别雌雄株,所以,所以在苗期利用分子生物学的方法鉴别未成熟栝楼的性别,可为栝楼生产上合理的雌、雄性植株田间配置以及育种工作中的早期选择等提供科学依据,对栝楼产业持续、快速、稳定发展奠定了基础。本文利用RAPD和SCAR分子标记技术对同一来源栝楼块根经组织培养后分化成的不同性别的二年生栝楼进行研究,发现了在雄性栝楼中,可以稳定的得到一条569bp的条带,而雌性栝楼中没有。该结果为栝楼苗期性别鉴定筛选提供了新的途径,为进一步的栝楼性别分化的机理研究提供了参考和依据。主要试验研究内容与结果如下所述：1.栝楼叶片含有多糖和多酚,通过改良的CTAB法,所提取的栝楼基因组DNA效果较好,其OD260/OD280的值均在1.7～1.9之间,通过1.5%琼脂糖凝胶电泳后,谱带明亮清晰,没有拖尾现象,能够满足PCR扩增需要。2.采用L16 (44) RAPD反应体系正交实验设计,对RAPD-PCR影响反应较大的Mg2+浓度、TaqDNA聚合酶、dNTPs、引物浓度进行4因素4水平正交实验设计。优化后的反应体系为：在25μL反应体系中,含10×buffer 2.5μL,Mg2+2.0mmol·L-1, TaqDNA聚合酶1U,引物0.8μmol·L-1,dNTPs 0.1mmol·L-1。反应程序为94℃预变性2mim；94℃变性0.5mim,37℃退火40s,72℃延伸1.5mim,36次循环；72℃延伸10mim,4℃保存。3.利用92个随机引物对雌、雄栝楼DNA分别进行PCR扩增,共产生大约600多条带,经过多次重复,发现在所选择雌、雄性栝楼样品DNA之间出现的差异性的引物只有1条。4.采取上述优化体系,找到1条引物S1200(序列5’-GTGAACGCTC-3’)能在雄性栝楼DNA中产生1条600bp左右的特异性的扩增带,将这一特异条带命名为S1200-600。经过多次重复试验,结果表明其重复性较好。5.对S1200-600进行回收、克隆和测序,得到569bp的序列,根据测得的序列,利用Primer Premier5.0软件设计了一对特异性引物,通过退火温度的筛选,最终选择55℃为最适合的退火温度,得到适合于SCAR标记的体系为：在25μL反应体系中,含10×buffer 2.5μL,Mg2+2μL,TaqDNA聚合酶0.2μL,引物Ⅰ2μL,引物Ⅱ2μL,dNTPs0.25μL。反应程序为94℃预变性2mim；94℃变性50s,55℃退火温度40s,72℃延伸1.5mim,36次循环；72℃延伸10mim,10℃保存。经过多次验证,最终成功实现RAPD转化为SCAR标记。可作为栝楼品种早期性别鉴定的遗传标记。更多还原

【Abstract】 Snakegourd, or Trichosanthes (Trichosanthes kirilowii Maxim) is Perennial Herb liana of medicinal plant in family Cucurbitaceae. Trichosanthes have a long history of cultivation in china. With the development of Trichosanthes as a leisure food, great changes had been taken place in the main Purpose of the root and fruit and there is also have great difference in their price. From the consideration of Comprehensive Benefit, it is very necessary to control the rate of male and female plant economically. Nowadays it is very difficult to distinguish the male and female plants from their Morphology prior to flowering. This article is to determinate the sex of premature Trichosanthes seedlings by the way of molecular biology, which will give scientific basis to the earlier selection of Trichosanthes seedlings. This research will not only lay a foundation for reasonable configuration of Trichosanthes seedlings of different sex in the production but also promote the industry development of Trichosanthes.In this article the DNA of male and female Trichosanthes seedlings that originate from same clone by the way of tissue culture is amplified by RAPD primers. A primer named S1200 can amplify a specific band of 600bp in DNA of the male, but the DNA of female did not. This result will not only provide a reference for earlier sex selection of Trichosanthes seedlings but also give a basis for Trichosanthes breeding as well as the study of mechanism of Sexual Development.The main contents and results studied are as follows:1.In order to get high quality DNA from Trichosanthes’s leaves enriching polysaccharides and polyphenols, the genomic DNA obtained by improvement method of CTAB extraction showed to be more integral and pure. The value of OD260/OD280 was between 1.7 and 1.9. The extracted DNA by this method showed a clear band when electrophoresed in 1.5% agrarose gel, and produced clear polymorphic patterns when amplified by PCR.2. Using L16(44) orthogonal experiment to get the optimized RAPD-PCR reaction system of Trichosanthes. The result shows that the optimal concentration was 10×buffer 2.5μL,Mg2+ 2.0mmol·L-1,Taq polymerase 1U,primer 0.8μmol·L-1,dNTPs 0.1mmol·L-1 per 25μL and the most suitable program was one cycle at 94℃pre-denaturation for 2 min,36cycles of 94℃for 30s,37℃for 40s,72℃for 1.5 min, and 10 min 72℃extension. Store at 4℃.3. The DNA of male and female Trichosanthes seedlings by using 92 random RAPD primers, more than 600 bands was produced. After repeated experiments, the result revealed that only one primers can amplified polymorphism between the DNA of male and female. The level of polymorphism is very low, about 1%.4. Through the mentioned optimized system, a RAPD primer named S1200 (5’-GTGAACGCTC-3’) can amplified a specific band of 600bp in the male after many repeated experiments. We called this band S1200-600.5. The fragment S1200-600 was recycled, cloned and sequenced. According to the sequence, a specific primer was designed by the Primer Premier 5.0 software. Through the screening of annealing temperature,55℃was proved to be the suitable annealing temperature. The optimized reaction system for specific primer is as follow:10×buffer 2.5μL, Mg2+2μL, Taq polymerase 0.2μL, primerⅠ2μL, primerⅡ2μL, dNTPs 0.25μL per 25μL reaction system and the most suitable program was one cycle at 94℃pre-denaturation for 2min,36cycles of 94℃for 50s,55℃for 40s,72℃for 1.5 min, and 72℃10 min for extension, Store at 10℃. Through many repeated experiments, RAPD marker was proved to be successfully converted into SCAR marker. This will provide a new way to determinate the sex of Trichosanthes seedlings.更多还原