The factors affecting the binding characteristics of GBSS with starch granule were studied using a wheat (Triticum aestivum) cultivar Chinese Spring with different temperature treatments. After sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, we got GBSS concentration by coomassie brilliant blue G-250(CBB G-250) method. The results showed that the temperature affected the binding of GBSS and starch granule. In 50–80℃, the concentration of GBSS unbound from starch increased with the temperature rising, which the maximal concentration was 11.361 μg mL-1 at 80℃, while that was reduced when the temperature was 85–95℃. Furthermore, Mg2+ could also affect the quantity of GBSS unbound from starch. When Mg2+ concentration was lower than 1.75 mmol L-1, the concentration of GBSS unbound increased. The lower Mg2+ concentration, the higher the concentration of GBSS unbound. However, when Mg2+ concentration was higher than 2.5 mmol L-1, it could restrain GBSS unbinding. These results showed that GBSS bound starch granule by non-covalent bonds. The best GBSS extracting conditions were 55 mmol L-1 Tris-HCl (pH 6.8), 0.75 mmol L-1 MgCl2, 2.3% SDS, 5% 2-ME and 10% glycerol, 15 min in boiling water, or 55 mmol L-1 Tris-HCl (pH 6.8), 0.75 mmol L-1 MgCl2, 2.3% SDS, 5% 2-ME and 10% glycerol, 80℃ 30 min. The results are helpful to investigate 3-D structure of biological activated GBSS and mechanism of GBSS binding with starch granule.
SSR is a good molecule marker with broad application. Bulk sampling is feasible to analyze genetic diversity of maize population by SSR. It is necessary to study influence of genetic distance as using bulk sampling. In present study, the genetic distances between two populations were compared and analyzed by 50 pairs of SSR primers, checked by inbred lines Huangzao 4 and Mo17, and using 60 individuals DNA samples of Golden Queen and Yuzong 5 maize populations with six treatments (the individual DNA samples, the mixed DNA samples with 5 individuals, 10 individuals, 15 individuals, 20 individuals, and 30 individuals, respectively). The results indicated that using the individual DNA samples can detect the alleles with very low frequencies, and make the detected allele number decreased gradually and the frequency of missing alleles increased with the increase of mixed individuals in the treatmen. The results of the genetic distance between two populations calculated by three models suggested that the genetic distances GD, GD(J) and GD(N) showed a gradually increasing trend with the increasing of the mixed individual number, but the increasing extent of GD(J) and GD(N) was wider. There was an obvious deviation between GD(J), GD(N) from mixed samples and the GD from 60 individual DNA samples based on allele frequency. The results and the comparison of the genetic distances based on different calculation methods suggested that the 10 individuals’ DNA mixture is the best sampling strategy when genetic diversity and polymorphism in maize populations are studied by SSR.
If the Nei’s genetic distance GD(N) and Jaccord’s genetic distance GD(J) are calculated according to 0, 1 data, (GD(N)+GD(J))/2 will be calculated close to the genetic distance by allele frequency of individuals. Gene frequency can be studied by using capillary electrophoresis and SSR fluorescence detection. There will be a broad application prospect about bulk sampling strategy with SSR marker.
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