Effects Of Anti-sense Thioredoxin S On Synthesis Of Starch And Protein, Grain Yield And Quality Properties In Wheat Seed

In order to clarify the function mechanism of antisense - thioredoxin s(anti-Trxs ) gene in transgenic wheat with pre-harvest sprouting resistant characteristic, two transgenic wheat materials TY18, TY34 and their corresponding non-transgenic acceptors including weak-gluten cultivar Yumai 18 and strong-gluten cultivar Yumai 34 were used. The effects of anti-sense thioredoxin s(Trx s)gene on the anabolism characteristic of starch and protein, hormone content and grain yield and quality properties in transgenic lines and their non-transgenic cultivars were systematically studied by pot-culture and field experiments during seed filling stage. The main result was as follows:1. Effects of anti-sense Trx s on starch biosynthesis and the activities of the starch key enzymes and expression patterns of the relevant regulatory geneDuring the seed filling stage, the seed starch accumulation was positively affected in two transgenic lines. Compared with non-transgenic control, the total starch content, amylopectin content,in two transgenic lines were significantly higher than those of controls. Although the amylose content in transgenic lines was also higher than that of their controls, the differences were no significant. The activities of ADPG、SSS、GBSS、SBE enzymes in wheat grain varied with the varieties. The activity of ADPG in TY34 increased significantly by 81.1% at the earlier grain filling stage, while, the activity of ADPG in TY18 grain significantly increased by 84.4% at the later grain filling stage. The activities of SSS and SBE in two transgenic lines were all higher than this of their control treatments during all seed filling stage. The activities of SSS in transgenic lines TY18 and TY34 increased significantly by 7.0% and 16.2%. The activity of SBE in TY18 increased significantly by 92.2%, but the significant difference in TY34 was not observed. Although the activities of GBSS in TY34 and TY18 were undulate, the differences were no significant. The reasult of Real time RT-PCR indicated that anti-sense Trx s promoted the expressing contents of SSS enzyme genes(SSSⅠ、SSSⅡ、SSSⅢ) and SBE enzyme-gene (SBEⅠ), restrained transcribe amounts of GBSSⅠ.Based on the above result, we could concluded that the increase of ADPG、SSS、SBE enzyme-genes expression may be the main reason for increasing of starch synthesis in wheat grain.2. Effects of anti-sense Trx s on protein biosynthesis and the activities of the protein key enzymes and expression patterns of the relevant regulatory genesCompared with non-transgenic control, the contents of total protein, albumin and gliadin in transgenic lines TY18 reduced by 2.1%, 14.9% and 10.8% respectively, while the globulin content increased by 11.6% during the grain filling stage. The glutelin content increased by 20.9% at 10～20 day after anthesis(DAA) and decreased by 15.9% at 20～30 DAA. To TY34, the contents of total protein, albumin and glutelin increased by 5.1%, 5.1% and 11.1% respectively and the contents of globulin and gliadin decreased by 7.5% and 26.7% respectively. The activity of NADH-GOGAT enzyme in transgenic lines TY18 decreased and the activity of GS enzyme diminished at before 20 DAA and increased after 20 DAA. The activity changes of NADH-GOGAT and GS enzyme in TY34 were contrary with those in TY18. Simultaneously, the expression patterns of NADH-GOGAT gene in transgenic lines TY18 increased significantly during 10-20 DAA and diminished after the 20 DAA, but the expression of NADH-GOGAT gene in TY34 decreased significantly during 15-25 DAA.3. Effects of anti-sense Trx s on wheat grain hormoneCompared with non-transgenic control, the ABA contents in transgenic lines TY18 and TY34 increased respectively by 18.39% and 16.8% during the seed filling stage. The changes of GA content were different in two cultivars. The GAs content in TY18 were obviously higher during 10-15 DAA(averagely increased by 29.6%) and few lower than the control line(averagely decreased by 9.1%) after the 15 DAA. The IAA contents in two transgenic lines were significantly higher than their controls during 10-15 DAA, averagely increased by 15.2% in TY18 and 21.1 % in TY34. The change trends of ZR contents in two transgenic lines were same, however no significant differences were observed between transgenic lines and their controls.4. Effects of anti-sense Trx s on wheat grain yield and quality propertiesThe grain yield and quality properties of transgenic wheat harboring the foreign gene were greatly affected. In both cultivars, transferring gene treatments increased grain number per ear and grain yield and the differences reached 0.05 significant levels. Compared with non-transgenic control, the total starch content, amylopectin content, peak viscosity, through viscosity and final viscosity were significantly increased in transgenic lines TY18, TY34 and this reduced the ratio of amylase / amylopectin. The effects of transferring gene on protein quality were different in two cultivars. The contents of total protein, albumin, globulin, glutelin were significantly decreased and contents of gliadin were significantly increased in TY18 two transgenic lines. The development time and stability time of flour were significantly decreased. While the changes of above protein quality indexes had no significantly difference except contents of albumin, gliadin and glutelin were decreased in transgenic lines TY34. The contents of -SH in transgenic lines TY18 and TY34 increased significantly by 30.3% and 48.3%. However, the contents of–S-S– in transgenic lines TY18 and TY34 decreased significantly by 38.2% and 54.9%. Contents of total glutenin polymer, glutenin macropolymer and relative size distribution(GMP/TGP) in TY18 were significantly lower than those in control line and reduced respectively by 93.2%, 13.4% and 17.1%. While, contents of total glutenin polymer and glutenin macropolymer in transgenic lines TY34 were significantly higher than those in control and respectively increased by14.8 % and 9.2%. Based on the above results, it can be concluded that transferring anti-trxs gene into wheat will help to improve the starch quality of two wheat cultivars and brake quality of weak-gluten cultivar. These results provide a basis for wheat quality improvement and further use of transgenic materials.