Analysis Of Gene Expression Differences In Transgenic Wheat Antisence-TrxS Seed Development

The transgenic wheat of "Yumai 70" was taken as materials. By DDRT-PCR technology, the gene expression difference between transgenic and non-transgenic (wild type:WT) wheat seeds were analyzed in access to the specific expression fragment (cDNA) of wheat in development. With differentially expressed cDNA fragment sequence analysis, gene and protein database homology comparison database analysis and function prediction,the role of h-type thioredoxin regulating gene expression of seeds development was found. Two differentially expressed genes, wheat amylase inhibitors, were cloned by electronic-PCR and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Their genome structure and expression patterns were studied. The main results are as follows:1. In order to investigate specific expression during seeds development, the samples was extracted from embryo and endosperm of transgenic and non-transgenic wheat in 5、10、15、20、25 and 30d after anthesis and amplified by reverse transcription polymerase chain reaction among the total 81 cDNA fragments, only 28 posltive cDNA fragments have been cloned and sequeneed after being testified by Reverse Northern bloting.The cDNA fragment sequences were compared with data in GenBank using basic local alignment search tool (Blast). The results showed that sequence similarity is 86% between 1023 and acetyl-CoA carboxylase gene,91% between 0513 and HMW glutenin 85% between 0724 and acetohydroxyacid synthase gene.42% between 0451 and trypsin inhibitor gene. So h-type thioredoxin during wheat seeds development may lead to the expression quantitative changes of functional genes, such as acetyl-CoA carboxylase, HMW glutenin、aquaporin Aqyl、pyruvate dehydrogenase、trypsin inhibitor.2. Two amylase inhibitor genes were cloned by electronic-PCR and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The cDNA of TaAAT3 includes a 621bp sequence and a coding region 462bp、which contains 28bp in 5’UTR and 131bp in 3’UTR. The deduced amino acids sequence (153 aa) of TaAAT3 is about 16.51KD and its pI is 7.86, and contained an AAI_SS conserved domain. The TaWASI2 includes a 608bp sequence and a 513bp coding region, and the deduced amino acids sequence (170aa) of TaWASI2 is about 18.59KD and its pI is 7.86,and contained an STI conserved domain. Expression patterns of cloned genes between transgenic and non-transgenic wheat were studied via SQ-RT-PCR,the results showed that TaAAT3 expression level of transgenic wheat was gradually increased with the time after anthesis, and significantly higher than the expression of non-transgenic wheat.The TaWASI2 expression was gradually increased with the developing grains, and reached the highest level at the 25 days after anthesis and higher in endosperm than other organs.Therefore, antisence-Trxs gene transgenic wheat might promote amylase inhibitor gene expression.