【作者】 丁茂予；

【导师】 马传喜；

【作者基本信息】 安徽农业大学 ， 作物遗传育种， 2005， 硕士

【摘要】 本研究用 SKCS、特异引物的 PCR 扩增和改良的 SDS-PAGE 三种方法对 85 份中国小麦品种的硬度类型进行了逐类淘汰筛选和分类,在为小麦硬度性状的品种资源筛选、早代鉴定、育种材料的选择、品质性状的分析及应用提供了理论依据的同时,进一步讨论了各种筛选方法的可行性和改进之处,得到了以下结果: 1. 利用单籽粒特性测定仪(SKCS)分析得到:SKCS 硬度值>50 的硬质品种有 37 个,介于 25-65 的混合型品种有 17 个,硬度值<41 的软质品种有 31 个。分别占 43.5%,20%和 36.5%。其中硬质和混合型都为硬度主效基因突变型,而混合型可能为人工混杂或环境造成的部分籽粒硬度测量值降低。 2. 用根据 Pinb-D1b 相对野生型的突变位点设计的特异鉴定引物和根据野生型 Pinb-D1a 对应位点设计的对照引物对 54 份硬质和混合型小麦品种进行 STS 引物扩增鉴定,56℃退火温度时,在设计引物和对照引物间扩增条带能出现差异,且条带清晰,稳定性好,因而根据 Pinb-D1b 突变位点设计的引物是一种可行的突变体筛选引物。在 54 份硬质及混合型小麦品种里,有 42 份经 PCR 检测为 Pinb-D1b 突变型,占 77.8%。 3. 当酶切位点为野生型时,Pinb 全长的扩增产物才能被限制性内切酶 PvuⅡ酶切成 264bp 和 184bp 两个片段;否则为 448bp 单一条带。结果表明,除野生型软麦和 Pinb-D1b 突变型外,其余 12 份小麦品种都能被酶切,即都不是 Pinb-D1c 突变型。在 54 份硬质小麦品种里,不存在 Pinb-D1c 型突变。而针对 Pinb-D1d、Pinb-D1e、Pinb-D1f 和 Pinb-D1g 相对野生型的已知突变位点设计引物,在与根据非突变基因设计的对照引物对除已鉴定出的三种类型外的 6 份硬质和混合型小麦进行的共同扩增中,发现扩增稳定性较差。这几种突变体的筛选上,不能像 Pinb-D1b 突变体一样根据突变位点设计引物使用。 4. 用 PDA 代替双叉丙烯酰胺制作凝胶,经 SDS-PAGE 检测,能清晰分辨Pruoindoline-a 和 Pruoindoline-b 亚基,Pin a 基因表达产物蛋白质的缺失或存在能在凝胶上清晰的反映出来。在 54 份硬质及混合型小麦品种里,有 6 份为 Pin a 基因表达产物蛋白质的缺失型,占 11.1%。 5. 虽然突变类型众多,但已设计出的筛选方法有限。对本实验中未鉴定成更多还原

【Abstract】 In this research, the hardness types of 85 varieties of Chinese wheat were screened and classified by such methods as SKCS、PCR amplification using specific primers and modified SDS-PAGE while the screening was eliminated one kind by one kind. The feasibility and improvement were further discussed at the same time as the theory bases were supplied for the variety resources screening, identification in early generations, breeding materials selecting, quality and character analyzing of the wheat hardness. Then we got the results as follows: 1. Measured by SKCS, there are 37 hard wheat varieties whose hardness indexes were more than 50, 17 mixed varieties whose hardness indexes were between 25-65 and 31 soft varieties whose hardness indexes were less that 41. They occupy 43.5%,20% and 36.5% respectively in whole. Both the hard and mixed varieties have mutations in the major genes controlling the grain hardness. The mixed types may be caused by people’s behavior or environment reasons to make the hardness index of some kernels lower. 2. 54 varieties were amplified and identified by STS specific primers which were designed on the mutated-type site of Pinb-D1b and the corresponding wild-type site of Pinb-D1a. The results show that at 56℃ of the annealing temperature, there are polymorphism between the two amplified clear bands which is good on stability. So the primers designed on the mutated-type site of Pinb-D1b are feasible for screening. 42 varieties of wheat are determined to be the mutated-type of Pinb-D1b and occupy 77.8% in the whole 54 hard and mixed varieties. 3. The amplified whole Pinb can be cleaved to two bands by restriction enzyme PvuⅡwhen the site is wild, or can’t be cleaved. Except the wild and Pinb-D1b two alle types, all the other 12 varieties can be cleaved and determined to be not the mutated-type of Pinb-D1c. The 12 varieties were amplified and identified by STS primers which were designed on the mutated-type site of Pinb-D1d、Pinb-D1e、Pinb-D1f and Pinb-D1g and the corresponding wild-type site of the four. The amplified products are not good on stability. We can’t use these designed primers as primers designed on the mutated-type site of Pinb-D1b. 4. The subunits Pruoindoline-a and Pruoindoline-b can be discerned easily in the gel of SDS-PAGE when the N’-N’ acrylamide was displaced of PDA. And the expressed protein’s absence or presence of pinA can be discerned easily too. Detected by SDS-PAGE, 6 varieties which occupy 11.1% were pinA-null in the whole 54 hard and mixed varieties. 5. Though there are a lot of mutated types, the designed screening methods are limited. Measured to the gene sequences of remained 6 varieties unidentified, we can find all of them are Pinb-D1p which is abundant in Chinese varieties and they occupy 11.1% in the whole 54 hard and mixed varieties too. 6. The grain hardness are affected by different mutation types: the hardness mean of pinA-null is 69.000 and the largest in all the three types which means that pinA-null affect the hardness most. The hardness means of Pina-D1a/Pinb-D1b and Pina-D1a/Pinb-D1p are 58.810 and 55.500. The smaller means mean pinb mutation types affect the hardness comparatively less.更多还原