Genetic And Biochemical-Physiologic Dissection Of The Antisense Trxs Gene Mediated Pre-Harvest Sprouting Resistance In Transgenetic Wheat Lines

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. And the biochemical-physiological characteristics of transgenic wheat need to be clarified.In this dissertation, the mechanism of anti-sprouting in transgenic wheat had been investigated: the inheritance stability of the two transgenic wheat lines by using the molecular data of PCR and the nested PCR, and the PCR products are digested with restriction enzyme Csp6l to verify the integrality and the reliability of the transgene anti-trxs, which has been integrated into the transgenie wheat plants genome verifying by genomic DNA Southern blotting using trxs as probe. The molecular data suggested that the transgene anti-trxs is under the control of a pair of genes in the two transgenic wheat lines, and the transgene anti-trxs is transmitted to the progeny following Mendelian rules.On the other hand, the anti pre-harvest sprouting ability of T3 and T? seed the two wheat lines are tested by employing the semiautomatic disconnected sprinkler in 2003 and 2004, respectively. The pre-harvest sprouting test data indicated that the two transgenic wheat lines plants have possessed the strong anti-sprouting characteristics.In biochemical and physiological experiments, earlier germination experiments with wheat revealed that thioredoxin h first undergoes reduction and then rapid proteolysis after imbibition. In this study with transgenic wheat seed and endosperm, thioredoxin was more highly reduced in the latter maturation grain of both the null segregant and transgene homozygote. Further reduction after earlier imbibition was either sluggish or drop sharply (homozygote). After imbibition 36 hours, the more reduction occurred in seed andendosperm of both the null segregant and transgene homozygote. The total amount of thioredoxin h increased during the earlier maturation, decreased during the mid maturation, and decreased earlier imbibition, after imbibition 36 hours the total amount begin to climb up. All changes similar to those were either observed with deembryonated grain and grain, or both of the null segregant and transgene homozygote. The difference between the null segregant and transgene homozygote is that the total content of thioredoxin in null plant seed and deembryonated grain have more activities than the transgenic wheat, the absolute amount of reduced thioredoxin h was 1.77-fold higher in the null segregant than the homozygote.The above results raise the question whether the low level of reduced thioredoxin in the homozygous endosperm and grain effects change in the redox state of storage proteins of the starchy endosperm. We therefore assessed the sulfhydryl disulfide (SH-SS) redox status of the soluble and insoluble protein fractions of transgenic wheat in different maturation period, and germinating period grain. The results suggest that the low thioredoxin h redox activities in the transgenic seed led to a 41% decrease in the ratio of relative reduction of the albumins, and to respective 77%, 14% and 13% decreases in the globulins, gliadins(l-propanol and 2-mercaptoethanol extractable) and glutelins. In another lines 00TY5, the low thioredoxin h activities led to 6.3%, 15%-. 4.9% and 2.7% decrease as above describe proteins order, respectively. There was little change in the aqueous and propanol fractions of the different maturation and imbibition period grain. However, as wheat seed sprouting (imbibition and spraying), relative to the null plant, there was a decrease in the sulfhydryl groups in the albumins, globulins, the gliadins and glutelins fractions of the homozygote. The change in the glutelins was less consistent.By changing the sulfhydryl status of the appropriate protein inhibitor, thioredoxin may control or effect the activity of a specific amylase within a cell. A notable question: what effect is occured by thioredoxin h in transgenic wheat seed cell. Earlier work showed that amylase activity is increased in vitro by the thioredoxin-linked reductive inactivation of its disulfide inhibitor protein.In the present study with whole seed of the trans antisense trxs gene wheat, we observed that the appearance of amylase is decelerated by about 24 hours imbibition with respect to both activity and amount of isoenzymes. The isoelectric focusing gel electrophoresis showed that the decline was due mainly to a decrease in the amount of the high pi rather than the low pi form isoenzymes. Atypical minor isoenzymes component with two pi of 6.4 in addition to the typical component with a pi of 3.7-5.7.The interesting thing is that two proteins, molecular weight 43 KDa approximately, from null plant are detected by SDS-PAGE, however, only one protein is find in the aquatic soluble extraction of transgenic wheat seed.Based on the molecular information from the two transgenic wheat lines genome, the changes in...