【作者】 马小锋；

【导师】 胡景江；

【作者基本信息】 西北农林科技大学 ， 植物学， 2008， 硕士

【摘要】 糯玉米起源于我国,是玉米属玉米种的一个亚种。糯玉米经数百年的变异,在株高、粒色、产量、抗性、品质等方面都形成了较大差异,是我国宝贵的玉米种质资源。糯玉米具有甜、粘、香的特点,风味优于普通玉米,其可溶性糖高于普通玉米而低于甜玉米,更适合当前发达国家人们的口味要求。用糯玉米作为原料加工的产品,一定会以独特的风味丰富人们的食谱。因此,深入研究糯玉米的遗传多样性,对糯玉米的新品种选育具有重要的现实和理论价值。本研究主要以1997年由R.Waugh等创立的特异序列扩增多态性(sequence-specific amplification polymorphisms,SSAP)为依据,在糯玉米上建立了此方法,为糯玉米种质资源及遗传多样性研究奠定基础。主要结果如下:1、在综合现有DNA提取方法和试验材料本身性质的基础上,对糯玉米基因组DNA提取进行了了改进:采用高盐的2% CTAB裂解细胞;2%的可溶性PVP以及2%β-巯基乙醇除去酚;先用苯酚:氯仿:异戊醇抽提一次,再用氯仿:异戊醇抽提一次除去蛋白质等杂质;最后用RNase消化,去除RNA,得到纯的DNA。此法提取的DNA其A260/ A280的值介于1.8和2.0之间,A260/ A230的比值大于2.0,且能被MseⅠ酶解完全,符合后续试验对DNA纯度的要求。此DNA提取方法快速、高效、经济,提取的DNA质量好,特别对样品数量多、工作量大和时间紧迫的情况下尤为适用。2、通过单酶切(Mse I)和双酶切(EcoR I、Mse I)对比实验,说明了单酶切不适合于糯玉米SSAP分析;在双酶切试验中发现用EcoR I的引物进行特异性扩增所得图谱清晰、条带质量好、多态性高;同时证明现在人们多采用的高频剪切酶的引物不适合于糯玉米SSAP分析,这与前人普遍采用的高频剪切酶引物做SSAP分析不同。3、初步建立了糯玉米的SSAP分析方法为:(1)酶切:EcoRI、MseI双酶切。先用EcoRI在37℃下酶切3h(Buffer:3μl,EcoRI:0.5μl,DNA:500ng,ddH2O补至15μl)。再加入MseI在65℃下酶切1h(Buffer:1μl,MseI:0.2μl,ddH2O:3.8μl)。取10μl用于下步。(2)连接:用EcoRⅠ和MseⅠ的接头进行连接,22℃下连接2h(Buffer:2.0μl,Ead:0.5μl,Mad:0.5μl,T4 DNA连接酶:0.15μl,ddH2O补至10μl)。(3)预扩:反应体系:10XBuffer:2.5μl,连接后DNA:2.4μl,dNTPs(2.5mM):1.6μl,EcoRI引物(25ng/μl):4.0μl,MseⅠ引物(25ng/μl):4.0μl,Taq DNA聚合酶:1U,ddH2O:补至25μl。反应条件:94℃:5min,94℃:30s,56℃:1min,72℃:1min,30个循环,最后72℃:5min。预扩产物用TE稀释4倍后,完成下面的步骤。(4)选择性扩增:反应体系:10XBuffer:1.0μl,dNTPs(2.5mM):0.8μl,预扩后DNA:0.75μl,EcoRI引物(25ng/μl):0.6μl,特异性引物(25ng/μl):1.2μl,Taq DNA聚合酶:0.5U,ddH2O:补至10μl。反应条件:94℃:5min,94℃:30s,65℃:30s(此步采用touchdown,每个循环降0.7℃),72℃1min,此程序进行13个循环,然后在94℃:30s,56℃:30s,72℃:1min下进行26个循环,最后72℃延伸7min。4、SSAP技术是基于反转录转座子的一种新的分子标记技术。在扩增时需要一个在反转录转座子上的特异性引物,所以知道反转录转座子的序列或者其上的保守序列是必须的。我们的试验发现不同的反转录转座子和相同的限制性内切酶的引物相组合得到条带的数目和多态性有着明显的差别,这就要求必须要有足够多的特异性引物来选择。同时由于不同特异性引物的GC含量不同,所以对于不同的特异性引物需要探究它们合适的退火温度。SSAP技术似乎在重复性方面不如AFLP,但其多的条带数目和高的多态性及相对少的多的引物组合,能节约很多时间,因此该方法是可以肯定,同时继续对其进行更加深入研究也是很有必要的。更多还原

【Abstract】 Waxy corn originated in China, it is one subspecies of corn. After several centuries’variation, Waxy corns have large differences in height, color grain, yield, resistance, quality and other aspects. They are our country’s precious maize resources. Waxy corn possesses characters of sweet taste, wax and fragrance beyond of normal maize. Its soluble sugar higher than ordinary corn and lower than sweet corn, more suitable for developed countries’tasting. The products made of waxy corn will join in recipes with the distinctive taste. So, it is important to do further research in genetic diversity of waxy corns. The research based on sequence-specific amplification polymorphisms(SSAP)created by R.Waugh in 1997,and our research made a foundation of germplasm resources and genetic diversity for waxy corn.the results as follows :1. Combine with existing DNA extraction methods and the nature of the waxy corn, waxy corn genome DNA extraction was improved: high salt (1.4mol/lNaCl) in 2% CTAB to break cells;2% of the soluble PVP and 2%β-Mercaptoethanol to remove phenols;first phenol: chloroform: isoamyl alcohol and then chloroform: isoamyl alcohol to remove proteins; at last digest RNA with RNase,the high quality DNA will be get.The value of the DNA in A260 / A280 between 1.8 and 2.0, A260 / A230 greater than 2.0 and genome DNA can be restriction by MseⅠc ompletely, and the DNA can meet the follow-up test. The DNA extraction method is rapid, efficient, economic, the quality of DNA is high, especially suitable for the large number of samples extraction.2. Base on the single enzyme (MseI) and dual-enzyme (EcoRI, MseI) comparative experiments, we confirmed that a single frequency enzyme restriction is not suitable for SSAP analysis of waxy corn; In experiment of EcoR I and Mse I restrction, we found that EcoR I primers combine with specific primers can got more bands and polymorphisms are also high. At the same time we found that many people now use the high frequency enzyme primers do not apply to waxy corn SSAP analysis.3. Initially established of waxy corn SSAP analysis method as follows:(1)Restriction: EcoRI and MseI. First EcoRI restrction at 37℃for 3h(Buffer:3μl,EcoRI:0.5μl,template DNA: 500ng,ddH2O up to 15μl).Then MseI restrction at 65℃for 1h(Buffer:1μl,MseI:0.2μl,ddH2O: 3.8μl).10μl for next step. (2)ligation: EcoRⅠand MseⅠadaptor ligation at 22℃for 2h(Buffer:2.0μl Ead: 0.5μl, Mad:0.5μl, T4 Ligase:0.15μl,ddH2O up to 10μl).(3)Pre-amplified:10XBuffer 2.5μl, ligation DNA 2.4μl, dNTPs(2.5mM) 1.6μl, EcoRI primer (25ng/μl) 4.0μl, MseⅠprimer (25ng/μl) 4.0μl,Taq DNA polymerase 1U,ddH2O: up to 25μl at 94℃:5min, 94℃:30s, 56℃: 1min,72℃:1min, 30cycles, 72℃:5min. then,diluted 4 times by TE.(4)Selective amplification:10XBuffer:1.0μl,dNTPs(2.5mM):0.8μl, pre-amplified DNA: 0.75μl, EcoRI primer(25ng/μl): 0.6μl, specific primer (25ng/μl):1.2μl, Taq DNA polymerase: 0.5U, ddH2O:up to 10μl at 94℃: 5min, 94℃:30s, 65℃:30s(touchdown, -0.7℃for every step), 72℃1min, 13 cycles, then at 94℃:30s, 56℃:30s, 72℃: 1min for 26cycles, 72℃for 7min.4. SSAP is a new molecular marker technology based on retrotransposons. We need to known a specific sequence which is on conservation region of retrotransposons. At the same time we found that it is serious difference in number of bands and polymorphisms when different specific primers combine with same restriction enzyme primer. So we require sufficient specific primers to choose. Because of different GC content in different specific primers, we need to explore suitable annealing temperature respectively. SSAP seems to be lower repetition than AFLP, but more bands, higher polymorphisms and relatively less number of primers, no doubt we can to save a lot of time. At this point, we can confirm it and continue to do more study.更多还原