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首页> 外文期刊>Cereal Chemistry >Methods for Determining Relative Average Number of Channels per Maize Starch Granule and Digestion of Raw Granules of Mutant Maize Cultivars by Amyloglucosidase
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Methods for Determining Relative Average Number of Channels per Maize Starch Granule and Digestion of Raw Granules of Mutant Maize Cultivars by Amyloglucosidase

机译：淀粉葡糖苷酶测定每个玉米淀粉颗粒相对平均通道数和突变玉米品种粗颗粒消化率的方法

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Six methods were investigated for determining the relative average numbers of channels per granule in populations of starch granules. The first method involved digestion of raw starch granules with amyloglucosidase. There were differences in the rates of digestion of starch of the same mutation in two different inbred-line backgrounds, but we concluded that a better method was needed. Three methods involved measuring either all channel proteins or one specific protein. Extraction of granules using 70% 2-propanol containing 1% 2-mercaptoethanol (ME) was successful in removing the majority of external surface proteins (<31 kDa). Further extraction of starch granules using 0.1% SDS containing 0.2% ME, pH 8, removed channel proteins without extracting matrix proteins. By measuring SDS-PAGE band intensities of protein clusters at 31-97.4 kDa or a marker protein at approximate to 42 kDa, methods to quantify relative average degrees of channelization (RADC) in different maize backgrounds were developed. Using either method. Oh43 had the highest RADC and B73 had the lowest RADC among the inbred lines examined. Another method involves extraction of total proteins using 2% SDS containing 0.2% ME from granules with the majority of surface proteins removed. The extract is subjected to SDS-PAGE and Western blotting, followed by location and quantification of the actin band using antibodies to maize pollen actin. These four methods give somewhat different results for six inbred lines of corn with a relatively narrow range of RADC. In an attempt to relate them to the actual number of observed pores (channel openings to the surface), granules were treated with a-amylase to enlarge the pores, with and without prior treatment with a protease. We concluded that this treatment revealed something about the nature of the granules and the action of a-amylase on them, but it was not an indication of the number of channels present. The outcome of this research provides evidence that the number of channels in corn starch granules varies with the genetic makeup of the parent plant. We concluded that measuring granule actin is the preferred method.
机译：研究了六种确定淀粉颗粒种群中每个颗粒相对平均通道数的方法。第一种方法涉及用淀粉葡糖苷酶消化未加工的淀粉颗粒。在两个不同的近交系背景中，相同突变的淀粉的消化率存在差异，但是我们得出结论，需要一种更好的方法。三种方法涉及测量所有通道蛋白或一种特定蛋白。使用含有1％2-巯基乙醇（ME）的70％2-丙醇提取颗粒可成功去除大部分外部表面蛋白（<31 kDa）。使用含0.2％ME，pH 8的0.1％SDS进一步提取淀粉颗粒，可去除通道蛋白，而无需提取基质蛋白。通过测量31-97.4 kDa的蛋白质簇或大约42 kDa的标记蛋白的SDS-PAGE带强度，开发了量化不同玉米背景下相对平均通道化度（RADC）的方法。使用任何一种方法。在所检查的近交系中，Oh43的RADC最高，而B73的RADC最低。另一种方法涉及使用2％含0.2％ME的SDS从颗粒中去除总表面蛋白的方法提取总蛋白。对提取物进行SDS-PAGE和Western blotting，然后使用玉米花粉肌动蛋白抗体对肌动蛋白条带进行定位和定量。对于RADC范围相对较小的六个玉米自交系，这四种方法给出的结果略有不同。为了使它们与观察到的孔的实际数量（通向表面的通道开口）相关联，对颗粒进行了α-淀粉酶处理以扩大孔，有或没有事先用蛋白酶处理过。我们得出的结论是，这种处理揭示了有关颗粒性质和α-淀粉酶对它们的作用的某些信息，但这并不表示存在的通道数。这项研究的结果提供了证据，表明玉米淀粉颗粒中通道的数量随亲本植物的遗传组成而变化。我们得出结论，测量颗粒肌动蛋白是首选方法。

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