Study on molecular identification and pre-harvest sprouting characteristic of the transgenic anti-trxs-gene wheat line 00T89 / 生物工程学报

ABSTRACT

Pre-harvest sprouting of wheat is caused by a series of enzymes, in which alpha-amylase plays a key role. Thioredoxin (trx) is an important protein capable of reducing S-S to -SH. The authors transferred the anti-trxs gene into wheat ( Triticum aestivum L.) cultivar Wanmai 48 in 2000 in order to obtain a transgenic line with low activities of trx and alpha-amylase in wheat seeds to enhance their resistance to pre-harvest sprouting. In this work, a pair of specific anti-trxs primers were used in PCR test to validate the transgenic plants in T4 lines. A pair of primers of a wheat actin gene cDNA and a pair of trxs gene primers were used in the reverse transcription PCR test to measure the relative content of trx mRNA in the transgenic positive lines. The experiments of pre-harvest sprouting were carried out to test the resistance to pre-harvest sprouting. The results showed that there were 13 positive lines with the anti-trxs gene among 18 transgenic lines. In the maturity stage, the 8 relative contents of the trx mRNA in the 13 positive lines were reduced evidently (P < 0.01). The trx mRNA was correlated with the sprouting parameters significantly (r = 0.7181) . 6 lines with low trx mRNA showed stronger resistance to pre-harvest sprouting during the period from 30 days after anthesis to 10 days after maturity. Compared with non-transgenic plant (Wanmai 48, CK), the average sprouting time of these lines was postponed by 2.7 days (P < 0.01), the sprouting ratio of seeds on spikes and the spike sprouting degree were reduced by 35.5% (P < 0.01) and 47.5% (P < 0.01), respectively. However, 25 days after maturity, the sprouting capacity of the positive lines recovered gradually, and no significant differences were found between them (P > 0.05) . Thus, it can be concluded that the wheat pre-sprouting was controlled effectively in some 00T89 transgenic lines as a result of the expression of anti-trxs gene.