利用反义trxs基因特异引物，对扬麦5号转反义trxs基因株系00TY5的T4和T5代进行了PCR分子鉴定，并对其纯合株系籽粒蛋白质组分的巯基含量、α-淀粉酶活性及二者的相关性进行了测试。结果表明，00TY5的T4和T5代PCR分子检测均呈阳性。与非转基因植株相比，在籽粒成熟和后熟过程中，籽粒清蛋白巯基含量在开花后30 d至成熟后5 d平均降低33.2%，球蛋白巯基含量在开花后20 d至成熟后5 d平均降低42.5%，均达极显著水平(P<0.01)；醇溶蛋白和谷蛋白巯基含量有下降的趋势，但降低幅度较小；α-淀粉酶活性低，变异小，尤其在开花后35 d至成熟后10 d，平均比非转基因植株下降36.5%。各种蛋白质组分的巯基含量与α-淀粉酶的活性呈正相关关系，清蛋白巯基含量在成熟前与α-淀粉酶活性相关性达极显著水平(P<0.01)；球蛋白巯基含量在成熟前10 d至成熟后10 d达显著水平(P<0.05)；贮藏蛋白巯基含量与α-淀粉酶活性的相关性在各时段均较高。
Thioredoxin (Trx) is an important protein in enzymes of deoxidizing S-S to -SH. Wheat pre-harvest sprouting is caused by a series of proteases, in which α-amylase plays a key role. We have transferred the anti-trxs gene into wheat (Triticum aestivum L.) cultivar Yangmai 5 since 2000 for the sake of decreasing Trx h reductive capability and α-amylase activity in wheat seed, and obtained the transgenic generations of T0 – T3. A pair of specific anti-trxs primer was used in PCR test to validate the transgenic plants in the present study. To investigate the biochemical mechanism of pre-harvest sprouting resistance, the -SH content of protein ingredients, α-amylase activity and their relationship in the seeds of pure transgenic line 00TY5 were studied. The PCR analysis showed a positive result indicating exogenous anti-trxs gene has been successfully transferred and steadily inherited in the T4 and T5 generations of 00TY5 (Fig.1, Fig.2). The -SH contents changes of albumin and globulin showed the parabola-like and wave-like trends in the period of grain maturing and after ripening, respectively, with the peak value at 40 d after anthesis. Compared with non-transgenic plant (Yangmai 5, CK), the -SH content of albumin in 00TY5 decreased significantly (P<0.01) by 33.2% at the period of 30 d after anthesis to 5 d after ripeness, while that of globulin decreased significantly (P<0.01) by 42.5% at the period of 20 d after anthesis to 5 d after ripeness (Fig.3). The -SH contents of stored proteins (gliadin and glutenin) increased throughout in 00TY5, but were lower than those of CK at less extent (Fig.4). The order of -SH content of protein ingredients was glutenin>gliadin>albumin>globulin. The α-amylase activity in transgenic grains decreased averagely by 36.5% from 35 d after anthesis to 10 d after ripeness (Fig.5). Based on further correlative analysis, the -SH contents of protein ingredients were positively correlated with the α-amylase activity, and the closer to mature the bigger correlative coefficient. The correlation between the -SH content of albumin and the α-amylase activity reached a significant level (P<0.05) before mature. So was that of globulin from 10 d before mature to 10 d after mature. The correlation between the -SH contents of stored proteins and the α-amylase activity was bigger in seed maturing period and after ripeness (Table 1). Thus, it can be concluded that the redox state of protein ingredients is adjusted, and the α-amylase activity is reduced by the transformation with anti-trxs gene. Therefore, the wheat pre-harvest sprouting can be controlled effectively as a result of the expression of anti-trxs gene.
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