关键词: 小麦, 反义TRXs基因, 萌发, 储藏蛋白质

Abstract:

Thioredoxin, some small proteins catalyzing thiol-disulfide interchange, are involved in the cell regulation of redox environment. Both the TRX s (thioredoxin s) gene from Phalaris coerulescens and the TRX h (thioredoxin h) gene from wheat belong to the thioredoxin gene family. The sequences of their cDNA are highly homologous, and their expression products have similar biological functions. Transgenic wheat (Triticum aestivum L.) was obtained by introducing antisense trxs into wheat with particle bombardment. It has been shown that the thioredoxin h content in the transgenic wheat seeds is reduced, and that the transgenic wheat is highly resistant to pre-harvest sprouting. The leading function of the wheat storage proteins is to offer N for the germination of wheat seeds and the growth of the seedlings. Thiocalsin (a 14-kD serine protease which is specific for gliadins and glutenins), malate dehyrogenase(MDH) and glutamic-pyruvic transaminase (GPT) have the close relation to the hydrolysis of the storage proteins in the germination of wheat seeds. To clarify the mechanism of pre-harvest sprouting resistance of transgenic wheat seeds, the activities of thiocalsin, MDH and GPT and the content of amino acids in wheat endosperm were determined during the germination of wheat seeds. The inhibitory effects to thiocalsin and MDH were enhanced so that their activities were reduced in transgenic wheat. During the synthesis of the wheat storage-proteins, protein disulfide isomerase (PDI) can catalyze the disulfide-bond-forming reaction, helping the protein to fold into protein polymer. PDI is one of the target proteins of thioredoxin h, and its activity can be depressed by TRX h. The TRX h content of seeds was lower and the PDI activity was higher in transgenic wheat than in wild-type wheat. Compared with wild-type wheat seeds, the results indicated that it was easier for cysteines to form inter-molecular disulfide bond at N-end and C-end of HMW-GS, and that bigger molecular weight protein was formed in transgenic wheat ones. The PAGE analysis of glutenin showed that a low molecular weight protein was absent and more glutenin macropolymer occurred in transgenic wheat seeds, implying it was more difficult for storage proteins to be degraded during the initial stage of wheat seed germination. Amino acids are the products of proteins degradation and the materials for the synthesis of new proteins, and Glu is the primary amino acid providing N for the germination of wheat seeds. The activity of GPT and the content of amino acids in seeds were lower in transgenic wheat than in wild-type wheat, which indicated the metabolism of amino acid and the hydrolysis of the storage proteins were weakened in transgenic wheat seeds. These results made clear that the hydrolysis rate of the storage proteins in seeds was slower in transgenic wheat than in wild-type wheat, which was a primary reason for the transgenic wheat more resistant to pre-harvest sprouting.

Key words: Wheat, Antisense TRX s, Germination, Storage protein