Exogenous Dehydration Response Transcription Factor Cbf4 Gene Into Corn

Maize,one of the three important cereal crops in the world,plays a very important role in food and animal feed production.As its whole production surpassed wheat,maize has become the second largest cereal crops in china.More than fifty percent of maize planted in arid and semiarid region every year.And through the whole growth stage of maize,different degrees of damage to its growth might be caused by drought and less of water,which are the main factors restricting maize production.So,it is very important to breed drought resistant maize.Traditional breeding way using heterosis as a main method has made a great success on improvement of maize production.As quantitative character of drought resistance, traditional breeding has too many limitations to satisfy the needing of breeders in the future. In recent years,to improve drought resistance of maize by transgenic method has got great attention in many countries.There are several kinds of gene we can use in breeding stress resistant plant,Genes for osmotic regulation substance synthesis,for example,genes coding for enzymes for active oxygen clearing,genes coding for proteins induced by abiotics or genes acts in the stress signal transduction pathway.In order to gain high drought resistance maize and speed up the process of drought resistance breeding,the dehydration responsive transcriptional factor CBF4 gene from Arabidopsis thaliana was cloned and transformed into maize with biolistic bombardment.The main results are as follows:1) The CBF4 gene and the promoter of rd29A gene,a preceding drought responsive gene, were amplified from Arabidopsis thaliana by PCR.Using the EPSP gene as the selective marker,pBAC146,the expression vector of CBF4 gene drived by the promoter of rd29A gene was constructed.2) Biolistic bombardment and pollen-tube pathway were used to transform the pBAC146 vector into excellent maize inbred lines.Thirty six transformation plants transformed by Biolistic bombardment were obtained and 1270 seeds were harvested through the pollen-tube pathway.3) PCR analysis suggested that the transgene exist in most of the 36 transformation plants, among which four plants have successfully gotten seeds.PCR,PCR-Southern and Southern analysis proved that the alien CBF4 gene had been integrated into the genome of 3 transgenic lines.4) To test the drought resistence ability of the transgenic lines we got,seeds of T1 generation (T₂ generation) were planted into garden pot in greenhouse and treated by no watering. The relative water content,proline content and chlorophyll content analysis showed that transgenic lines had higher drought resistant ability than non-transgenic plants.5) To select those seeds obtained by pollen tube pathway quickly and effectively,normal maize seeds were treated by different content of glyphosate.The suitable glyphosate content for distinguishing transgenic and non-transgenic plants was found.Thisr study suggested that induced expression of the CBF4 gene in transgenic maize could improve the drought resistance of transgenic maize,and which will pave a way for using CBF4 gene in maize drought resistant breeding.