Studies On Changes Of OXO Activity During Different Biological Processes In Triticeae Crops And Cloning Of OXO-Gene In Wheat

In order to study the effects of oxalate oxidase during wheat dedifferentiation and differentiation processes,Yumai 18 mature embryos was used under dichlorophenoxyacetic acid(2,4-D)or not dichlorophenoxyacetic acid (2,4-D)condition;The effects of different materials and different periods on oxalate oxidase activity during the seeds maturation and the changes of oxalate oxidase activity in seeds,seed capsule,embryos,endosperm were investigated during the seeds germination from the Triticeae crops(Barley,rye,triticale,durum wheat, Chinese Spring,Yumai 18).To study the roles of OXO expression and its regulation further,The gene and the promotor of OXO in yumai 18 were cloned from wheat calli, and sequenced and analysised the nucleotide sequence of OXO.The main results were as follows:1)In 2,4-D culturing condition,A higher the enzymatic activity of oxalate oxidase was detected during wheat mature embryo in vitro.Variance analysis showed: 2,4-D was very important to the enzymatic activity of oxalate oxidase.It is possible that 2,4-D might be essential to the expression of oxalate oxidase,also and formated embryogenic callus during Wheat tissue culture dedifferentiation2)The oxalate oxidase activity was significantly different from the different materials and different periods of the seeds during the seeds maturation,The results were as follows:The enzymatic activity of oxalate oxidase in rye was the highest,and were followed by barley,oxalate oxidase activity in China and spring,Yumai 18,rye and durum wheat was relatively low.7 days after flowering in wheat,oxalate oxidase activity was the highest,and decreasing gradually.3)The oxalate oxidase activity in the seeds and different parts of the seeds(seed capsule,endosperm,embryos)was common during the seed maturation:The OXO activity of seed capsule,endosperm,embryo were higher than the OXO activity of the seeds.Therefore,it is speculated that the seeds may contain inhibitory substances,so that oxalate oxidase activity of the seeds was decreased.The seeds were further analysed that the inhibitory substances was likely to exist in the soluble protein of the endosperm.4)The promoter of oxalate oxidase gene was amplified by Tail-PCR,cloned,and sequenced,the results were showed:Fragment obtained included a 1713bp upstream region and a 95bp fragment overlapping the 5'UTR and partial coding sequence(CDS) of the oxalate oxidase cDNA sequence,The TATA box(TATATA)was present at position -33 and a CAAT box at position -157,indeed These evidence showed that it was the promoter sequences of wheat.Analyses of the OXO promoter region with the related software indicated:the presence of a number of regulatory sequences.Some being involves in hormone response:8 pyrimedine box core elements(Y-TTTT-Y) named GARE(Gibberllic Acid Responsive Element)located at positions -195,-305, -541,-1032,-1106,-1159,-1249,-1532;1(CTAACA/TA)ARE element(ABA Responsive Element)locate at positions -1346;In addition,Some regulatory sequences were included:7 DOF(One Finger DNA Binding)motifs(A/TAAAG) which had previously been associated with auxin induction were also present.And located between -1283-969 and -6747-556;13 SEF4(CabMV Embryo Factor 4) sequence(R-TTTTT-R)located between -1383-910 and -775--466.These cis elements was provided basis for research the expression and regulation of the oxalate oxidase gene further5)The cloning of OXO gene was provided a theoretical basis for the species transformation and the improved the species resistant further.