Construction Of Gapdh Gene Expression Vector Of Wheat Changwu134and Genetic Transformation Of Arabidopsis Thaliana

Wheat (Triticum aestivum L), Gramineae Triticum, its total output is second only tocorn(Zea mays) in food crop, occupies a very important position in agricultural production ofour country. Drought is a serious problem and affecting normal production of agriculture innorth China. To find and breed new crops have drought resistance ability is an important issuewhich need to be solved for agricultural scientists. GAPDH(glyceraldehyde-3-phosphatedehydrogenase) gene is usually considered as a housekeeping gene, can express in high levelsin almost all tissues or cells, so it is widely used in volume research as the standardization ofgene expression in biological tissue. But in recent years the study found that its expressionquantity is not always stable in some organizations and parts. We had found the expressionGAPDH of strong drought resistance of wheat Changwu134had increased under droughtstress, and when compared with weak drought-resistant wheat it was much higher, so weconsidered GAPDH protein involved in the drought resistance of wheat. This paper alsochoose strong drought resistance of wheat Changwu134for the material, extracted total RNAafter48h treated by-1.2MPaPEG6000. Then obtained its cDNA sequence by reversetranscription. By using traditional enzyme-connection method to construct the GAPDH geneexpression vector pTCK303-GAPDH, and genetic transform arabidopsis by agrobacteriumimpregnation method of inflorescence.The experiment results are as follows:This Experiment used Trizol method to extract total RNA of wheat Changwu134after48h treated by-1.2MPaPEG6000, the total RNA integrity is good. After the reversetranscribed into cDNA, with cDNA as a template for PCR amplification, then constructedpTCK303-GAPDH expression vector, the sequencing result compared with known sequenceGAPC (code: EF592180.1),the similarity is99.7%, showed that successfully constructed therecombinant plasmid.Determined the culture conditions of arabidopsis thaliana: light intensity of8800~13200lx,16h/22℃, dark8h/20℃,70%humidity.By agrobacterium impregnation method of inflorescence transgenic arabidopsis，and get the seeds.Found the suitable concentration50mg/L of kanamycin to screen transgenicarabidopsis.