Cloning And Genetic Transformation Of Gene Encoding Antifreeze Protein In Wheat (Triticum Aestivvm. L)

Wheat (Triticum aestivum L.) is one of the most important cereal crops and is widely cultivated throughout the world. First domesticated in the Middle East, now it has the second largest production among the major world staple foods. Since the introduction of first genetically modified wheat in USA, many transgenic approaches have been applied to improve the wheat cultivars best suitable for China. Many breakthroughs have been achieved concerning biotic as well as abiotic stress resistance to improve quality as well as the quantity of wheat. Improvements in transgenic technology can greatly promote the process of wheat breeding. Frozen injury is one of the serious natural disasters in agricultural and also the limit factor in some crops of regional and seasonal reasons. Isolation of plant antifreeze genes and analysis their expression regulation are of great significance to clarify the mechanism of resistance to low temperature in plant and to enhance wheat cold tolerance breeding by transgenic technology.This study consists of two parts:The first part of this study covers the study carried out in our lab to study the function of antifreeze proteins in wheat. The second part of the study covers research work on exploring the possibility of AtMYB12 as novel novel gene marker and its role of regulating flavonols metabolism in transgenic wheat.First part of this dissertation describes research work on isolation and functional characterization of antifreeze protein gene isolated by screening a wheat cDNA library, after exposure to chilling temperatures. Homology analysis by using bioinformatics tools revealed that it had 93% homology with type III antifreeze proteins in fish. For further study full length of the gene was cloned by using RACE-PCR. The full length of cDNA was 782 bp, the ORF was 504 bp which translate 168 Aa. The protein was 18.19 kD and pI was 8.8. Sequencing analysis revealed that this protein found in wheat have different attributes as compared with chilling stress related proteins found in plants. We named the gene TaAFPⅢ. This is the first time discovery of a plant protein which is homologous with fish antifreeze proteins. The discovery provides a new starting point for the molecular mechanism of plant freezing. We analyzed the expression of TaAFPIII response to low temperature and homone treatments by real time PCR. The results showed that the expression of AFP gene was significantly higher in leaves as compared with roots. After 2 days of cold treatment, the AFP gene expression suddenly increased 266 times in leaves as compared with the control. A small enhancement was observed in the expression levels of this gene with 0.2 mM methyl jasmonate and 15%PEG10000, whereas 0.1 mM ABA inhibited its expression.Discovery of this antifreeze protein gene in wheat and its further study in prokaryotic systems would open new windows in research, for instance, making TaAFPⅢantibodies. We constructed prokaryotic expression vector pET32a-AFP. We also constructed binary vector pBI121-AFP under CaMV 35S promoter and tuansformed into tobacco successfully through agrobacterium. The results showed that the antifreezing capacity of transgenic tobacco has been significantly improved. After treatment with -20℃for 4 h, the transgenic tobaccos can 100% survive when put them to room temperature while the control tobaccos were all died. The results indicated that TaAFPⅢplay an important role in plant to resistance the low temperature.AtMYB12 is a flavonol biosynthesis pathway activator in Arabidopsis. To find the possible that AtMYB12 can regulate the flavonoid and be as a novel reporter gene in transgenic plant. With the construction of recombinant vector pLRPT-MGH and pBI121-MYB12, we successfully observed AtMYB12 expression in Chinese Spring by using gene gun. It is clearly evident from our experimental study that AtMYB12 can be used as a novel marker gene and has obvious advantages over GUS and GFP.The result of this study have important reference value for clarify the molecular mechanism of plant resistance to low temperature and the use of transgenic technology to enhance the capability of antifreeze in crop.