论文标题:转基因小麦抗穗发芽特性的分子鉴定、遗传分析与抗性机理研究
Genetic and Biochemical-Physiologic Dissection of the Antisense Trxs Gene Mediated Pre-Harvest Sprouting Resistance in Transgenetic Wheat Lines
论文作者 李志岗
论文导师 尹钧,论文学位 博士,论文专业 作物栽培学与耕作学
论文单位 山西农业大学,点击次数 4,论文页数 122页File Size15047k
2005-06-01论文免费下载 http://paper.dic123.com/lunwen_106419477/ Trx,硫氧还蛋白;trx,硫氧还蛋白基因;SH,巯基;anti-trxs,硫氧还蛋白反义基因;00T89,受体为895004的转基因小麦;00TY5,受体为扬麦5号的转基因小麦;OOTY18,受体为豫麦18-64的转基因小麦
Anti-trxs gene Transgenic wheat Thioredoxin Anti pre-harvest sprouting ; Molecular detection ; Physiologic and biochemical characteristics
小麦是世界上第一大粮食作物,它不但推动了世界人类文明的演进,也极大地影响着世界农业经济和粮食安全。但由于穗发芽影响,从1983到1985年世界小麦的产量损失达20%,每年近1亿吨,相当于我国小麦年总产量;在我国黄淮麦区因成熟期遇雨也导致多次发生大面积小麦穗发芽,而造成断种和面粉不能食用的灾情,在北方麦区,常年虽无梅雨季节,但个别年份也曾出现过麦收期连阴雨小麦穗发芽的情况。因此,小麦穗发芽问题早就引起了国内外植物育种家和生理学家的广泛关注,成为农业生产中的殛待解决的难题。 近年来,国内外有关硫氧还蛋白(thioredoxin,Trx)生物学功能和反义基因作用机理的研究为创育抗穗发芽小麦资源提供了一条新的途径。Trx是一种能催化二硫键氧化还原反应的功能蛋白,通过调节Trx h活性能改变种子中淀粉酶和蛋白质酶等重要酶的活性,增加贮藏蛋白的水溶性和促进营养物质水解的作用,并对作物的品质产生影响。 源于蓝色黑鸭草(Phalaris coerulescens)的trxs基因(thioredoxin s,trxs)与trx基因(thioredoxin,trx)同属于硫氧还蛋白基因家族,其蛋白质表达产物有相似的生物活性。将反义trxs基因导入小麦有望降低种子中TRX的活性,减慢种子发芽时的碳氮代谢和淀粉、蛋白质的水解,抑制淀粉酶的活性,最终导致转基因小麦子粒发芽能力减弱。 本课题组早在2000年就开始进行反义trxs基因导入小麦的研究,到目前,已经得到了转基因第四代小麦。为了明确反义转基因在转基因小麦中的遗传规律和作用机制,本研究以转基因小麦后代株系为材料,追踪反义转基因的传递规律,并筛选纯合系;通过对目的基因的分子检测,确定了目的基因在小麦基因组中的遗传稳定情况;通过对转基因大麦籽粒和胚乳的生化生理特性测定,证明了目的基因在转基因小麦部分株系中能正常表达;通过对转基因稳定遗传和表达的株系进行模拟降雨抗穗发芽试验,证实部分转基因株系与对照相比,具有很强的抗穗发芽能力。研究的主要结果如下: 1、转反义trxs基因小麦株系分子检测和遗传分析 用PCR、Nested PCR、限制性酶切、Southern杂交等技术对转基因小麦T_1、T_2、T_3、T_4代部分株系进行分子鉴定,并连续2年追踪研究反义trxs基因在这些株系中的遗传规律和遗传稳定性。结果表明,反义trxs基因在转基因小麦株系中呈孟德尔单基因遗传,能稳定传递;根据分子检测结果,确定了4个反义trxs基因纯合的株系(00T89和
Wheat is one of most important corps in the world, it not only contribute to the evolvement of human civilization in the world, but also impact international economy and the food safty in most nations. The Pre-Harvest Sprouting, a natural disaster, often take place in the wheat plant area in the worldwide. In China, my hometown, the harvest season often meet the overcast and rainy day. Because of long time imbibition, the maturing wheat seeds germinated in their spikes, in other words, that is pre-harvest sprouting. The sprouting seeds of wheat will lose the main commodity characteristics, following sequel is the huge loss. The disaster of pre-harvest sprouting had been recognized as an urgent task. However, the attention about the wheat pre-harvest sprouting be taken by the scientists who are working in the fields of breeding, physiological, biochemical and molecular biological.Recently, a new approaches to dissolve the pre-harvest sprouting are cleared gradually due to the function of thioredoxin and antisense gene well knowing. Thioredoxins are small ubiquitous proteins, containing a readily reducible disulfide bridge that play a role in a wide variety of physiological processes by acting as general protein disulfide reductases. Plant thioredoxin h have been implicated in a wide variety biological functions, and it is likely that cytoplasmic thioredoxins could participate in seedling development-related processes.Thioredoxin genes are expressed mostly in organs or tissues that contain growing cells. Thioredoxin h are involved in Phalaris coerulescens self-incompatibility systems. Several functions have been hypothesized for a rice thioredoxin h that is a major protein translocating in sieve tubes.The presence of multiple thioredoxin h has been reported in plant seed. In wheat, three different thioredoxins have been characterized, and in wheat seed the NADP-thioredoxin system (NTS) has been credited with physiological activity. It has been shown that thioredo- xin h is able to reduce low-molecular-mass cystine-rich proteins, including chloroform- methanol soluble proteins (CM protein) that are inhibitors of a -amylases. Gliadins and glutenins, the major storage proteins of wheat endosperm, were reduced by the NTS, and during seed germination thioredoxin h functions as a signal to enhance metabolic processes for germination and seedling development. Therefore, the manipulation of the thioredoxin gene offers considerable promise for modifying the seed sprouting properties.Thioredoxin h gene, involving in Phalaris coerulescens self-incompatibility systems (named trxs byXinming LI), isolating from genomic DNA and cDNA library, is compose of five exons. The ORF (open reading frame) has been cloned. And its antisense gene, complementary with the sense strand, has also been cloned into an expression vactor, pBluescript SK+, and a gliadin promotor is inserted the upstream of it.If the antisense trxs gene are transformed into wheat genome, the anticipation trait of wheat should be reasonable supposition. When the antisense trxs gene express in the period of wheat seed maturing, the trx h from wheat genome will be interfered, then the activities of a -amylases will decrease, the free SH content of albumins, gliadins, globulins and glutelins will drop down, and the catabolism speed of endosperm proteins will be slowed, finally, the germination and seedling development of wheat seed would be inhibited.The transgenic T3 and T4 generation wheat plants, created and propagated by the former researchers who are graduate or PhD students supervised by Dr. YIN, are as materials to analyse. The production of transgenic crop plants is an expanding component of agricultural biotechnology. For commercial success and antagonizing hazard, it will be crucial that the introduced traits be transmitted faithfully through successive generations in a predictable manner. Up to now, the elementary identification of transgene antisense trxs transferred into wheat genome has be done, but the genetic mothed or rule in transgenic wheat lines is unclear.
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