Transgenic Wheat Seeds Protemics And Regulation Effects Of Anti-Trx S Gene On Wheat Seeds Physiology

The study on transgenic wheat with antisense Trx s gene and wild-type wheat seeds('Yumai 18')was undertaken to clarify the effect of antisense Trx s gene on physiology metabolism in wheat seeds.Changes in seeds proteomics and composition, key enzymes catalyzing the hydrolysis of protein and carbohydrate,antioxidant enzyme were investigated by the technology in proteomics,bioinformatics and biochemistry.The work will broaden the research on biology function of Trx s,laying the foundation for the use of Trx s in crop breeding.The following are the main results of the study.1.The differential proteomics maps of transgenic wheat with antisense Trx s gene and wild-type wheat seeds were obtained.27 kinds of proteins are over-expression in mature wild-type wheat seeds,and 20 kinds of proteins are over-expression in mature transgenic wheat seeds.15 kinds of proteins are over-expression in wild-type wheat seeds imbibited for 2d,and 10 kinds of proteins are over-expression in transgenic wheat seeds imbibited for 2d.2.Analyzing the differential proteins between transgenic wheat seeds and wild-type wheat ones,it is demonstrated that the seeds metabolism is regulated by antisense Trx s through the following ways.(1)The activities of xylanase inhibitor proteinⅠ(XIP-Ⅰ)and serpin are affected by antisense Trx s.It has been proved that XIP-Ⅰcan inhibit barley amylase.Serpin is the inhibitor of thiocalsin,a serine protease.(2)The activities of enzymes in starch hydrolysis,protein and amino acid metabolism are affected by antisense Trx s.The enzyme participating starch hydrolysis include glyceraldehyde-3-phosphate dehydrogenase(GAPDH),glucose-6-phosphate isomerase, phosphoenolpyruvate carboxylase(PEPC).Glutamate dehydrogenase(GDH),aspartate transaminase(AST)and adenylylsulfate kinase(APK)participate protein and amino acid metabolism.S3-RNase catalyzes the hydrolysis of RNA,and ATP synthase take part in energy metabolism.(3)Some transcription factor and metabolism regulating protein is affected by antisense Trx s.WRKY transcription factor 6 can bind W-box of some gene promoters(such as storage protein,α-amylase andβ- amylase).14-3-3 proteins can regulate gene transcription,primary metabolism,the activities of plasmalemma H~+-ATPase,enzymes in chlorophyll and mitochondria,amino acid synthetase, carbohydrate synthetase,sucrose hydrolyase,nitrate reductase.Maturase K-like protein participates the process after gene is transcripted.(4)Puroindoline b,a protein related seeds hardness,Glutathione transferase and disease resistance protein OB5,the protein related resistant are overexpressed in transgenic wheat.3.The transformation from CM(chloroform-methanol)protein to metablic protein is slowed in transgenic seeds.After imbibited,the metabolism in transgenic seeds is slowed, because the content of metablic protein is reduced.4.The activity of protein disulfide isomerase(PDI)is enhanced in transgenic seeds so that the glutenin is promoted to fold into protein polymer.On the other hand,the reduction status of storage protein is decreased in transgenic seeds.Therefore the solubility of storage protein in transgenic wheat seeds is lower than that in wild-type wheat ones.Serpin is a kind of serine proteinase inhibitors,and can specially inhibit thiocalsin.The content of active serpin in transgenic wheat seeds is more than that in wild-type wheat ones,so that the activity of thiocalsin in transgenic wheat seeds is lower than that in wild-type wheat ones.Glutamate dehydrogenase(GDH)and malate dehydrogenase(MHD)are the enzymes whose activities depend on mercaptal.Since the content of thioredoxin is low in transgenic wheat seeds,the activities of the two enzymes is lower than that in wild-type wheat ones.In a word,the low solubility of storage protein and activity of enzymes in protein hydrolysis make the hydrolysis metabolism of storage proteins slow.5.α-amylase,β-amylase and amylopectase in wheat endosperm is the three key enzymes in starch hydrolysis.During germination,the activity of the activated enzyme of the three enzymes in transgenic wheat seeds was lower than that in wild-type wheat ones, and the activity of the inhibited enzyme of the three enzymes in transgenic wheat seeds was higher than that in wild-type wheat ones.These results indicate that the higher inhibition of the enzymes is one of the important reasons that the activity of the enzymes in transgenic wheat seeds is lower.In addition,thioredoxin can also regulate some enzymes,such as aldolase,whose activity depend on mercaptal during carbohydrate hydrolysis.6.Among the three key enzymes in starch hydrolysis,there are the biggest difference in the activity ofα-amylase between transgenic wheat seeds and wild-type wheat ones on 1d after imbibited.Therefore,α-amylase has more important role in the onset of wheat seeds germination.The activity ofα-amylase is affected by thioredoxin with the following way,(1)regulating the synthesis and degradation ofα-amylase protein, (2)regulating the activity ofα-amylase inhibitor.7.The activity of some isozyme of antioxidant enzyme is inhibited in transgenic wheat seeds,so the activity of peroxidase(POD)and catalase(CAT)in transgenic wheat seeds is lower than that in wild-type ones.The water uptake in transgenic wheat seeds is slower than that wild-type ones.