关键词: 小麦, 反义trxs基因, 蛋白组分, 巯基, α-淀粉酶

Abstract:

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.

Key words: Wheat, Anti-trxs gene, Protein ingredient, -SH content, α-amylase activity