转反义硫氧还蛋白基因小麦萌发种子中蛋白质的变化

硫氧还蛋白h（thioredoxin h，Trx h）是一类广泛存在于生物体内的多功能活性蛋白，分子量约为12kD，它通过还原靶蛋白中的二硫键参与酶活性调节、抗胁迫、信号传导等许多重要的生命活动。硫氧还蛋白h能促进谷物类种子萌发过程，主要表现在以下2个方面：（1）在籽粒萌发期间，硫氧还蛋白可通过还原储存蛋白的分子内二硫键使其更易于被降解；（2）硫氧还蛋白也可以直接地通过将酶还原或者间接地通过使酶抑制蛋白失活而激活酶。源于Phalaris coerulescens的trxs基因（thioredoxin s，trxs）与小麦硫氧还蛋白h基因（thioredoxin h，trx h）同属于硫氧还蛋白基因家族，它们的cDNA有94％的同源性，表达产物也有相似的生物功能。我们采用基因枪法将反义trxs基因导入小麦，获得了可稳定遗传的小麦，并检测出转基因种子中硫氧还蛋白h表达量、水溶蛋白和醇溶蛋白的还原状态以及α-淀粉酶活性均低于对照小麦；另外，通过模拟降雨抗穗发芽试验证实转基因株系具有很强的抗穗发芽能力。以转反义trxs基因抗穗发芽小麦为材料，检测反义trxs基因小麦籽粒萌发过程中蛋白质的变化，探讨转反义trxs基因小麦的抗穗发芽机理。研究表明反义trxs基因能够减缓KCl可溶性蛋白中Chloroform-methanol（CM）蛋白向代谢类蛋白的转化进程，在萌发初期降低籽粒代谢类蛋白的含量，使籽粒代谢速度下降，而CM蛋白主要包含一些分子量小于20kD的蛋白质。在籽粒成熟过程中，硫氧还蛋白能够阻止麦谷蛋白亚基形成谷蛋白聚合体的过程，在转基因小麦中麦谷蛋白更易于形成大分子量的谷蛋白大聚合体，使得转基因小麦中的谷蛋白在萌发初期更难于被水解，因此转基因小麦籽粒会因谷蛋白难于降解而萌发较慢。另外，反义trxs基因减慢了麦胚中10kD蛋白的降解过程。

Changes in Proteins within Germinating Seeds of Transgenic Wheat with an Antisense Construct Directed Gainst the Thioredoxin

Thioredoxin h is closely related to germina-tion of cereal seeds. The mechanism of transgenic wheat seeds with antisense trxs gene,which is responsible for low germination rate was studied through analyzing the changes in proteins of wheat seeds during germination. The antisense trxs could weaken the metabolism of wheat seeds by decreasing the quantity of proteins involved in metabolism,while chloroform-methanol (CM) protein fraction consisted mostly of some low molecular weight proteins (<20 kD). Compared with wild-type wheat seeds,the folding of glutenin in transgenic wheat ones was af-fected during the wheat maturating. Big glutenin macropolymers could be formed more easily in transgenic wheat seeds than in wild-type wheat ones. Therefore,the degradation speed of glutenin in transgenic wheat seeds was slower than that in wild-type wheat ones during seed germination. In addition,the degradation of some pro-teins in transgenic wheat embryos was also delayed dur-ing germination.