| The puroindoline gene is located at Ha (Hardness) locus on the short arm of chromosome 5D which contains puroindoline a (pina) and puroindoline b (pinb), and plays an important role in wheat seed hardness. Puroindoline A (PinA) and Puroindoline B (PinB) proteins encoded by the pina and pinb, respectively, are on the surface of starch granules and the matrix proteins, which is regarded as biochemical marker of wheat grain hardness. Particularly, Pina and PinB are unique because of their tryptophan-rich, hydrophobic domain, which contributes to wheat grain hardness and antimicrobial activities for G+, G- and fungus.In this study, biochemistry and molecular biology methods were used to investigate function of puroindoline gene. Puroindoline was extracted from wheat flour with the 100mM Tris-HCl pH 7.8 buffer, containing 4%Triton X-114. Then Sephadex G-75 and Carboxymethyl-cellulose (CM-23) successively purified the protein mixture including Puroindoline. At last the puroindoline was obtained by freeze-dried. Puroindoline a gene was chosen to construct the mutant pina gene which was expected to have stronger antimicrobial activities.The Puroindoline protein was extracted and purified from wheat flour successfully. During the study, we modified the conditions of protein chromatography, with which the extraction efficiency was greatly improved. The aim of my research is to construct the mutant pina, which contains several tryptophan-rich, hydrophobic domains. After the modification of many aspects such as the designation of experiment, the conditions of PCR, we obtained the mutant pina with two tryptophan-rich, hydrophobic domains, of which the open reading frame was changed as a pity. Further modification could be obtained using the established methods. Result obtained in present provided the basis for the further study of pina functions.
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