Research On The Isolation Of Mutated Major Gene Controlling Wheat Grain Hardness From Aegilops

Grain hardness is one of the most important characters in wheat quality improvement. Puroindoline a (PinA) and puroindoline b (PinB) play critical roles in determining wheat grain texture, and mutation or absence of either genes results in different grain hardness. To further the knowledge of grain texture determination, two aspects have been studied: (1) Puroindoline proteins are special TritonX-114 soluble proteins which play critical roles in determining wheat grain hardness. Based on extracted puroindoline proteins from diploid wheat, hexaploid wheat, and Aegilops tauschii, an optimized SDS-PAGE condition was built up with stacking gel (T=4, C=2.6) voltage 128V, separating gel (T=13.5, C=2.6) voltage 240V to reach more steady and repetitive electrophoresis results. At the same time, coomassie brilliant blue (CBB) staining and silver staining were tested to meet different experimental purpose. The series tests can be used for the investigation of puroindolines in wheat and the related species. (2) Based on the reported homologues of PinA, one pair of degenerate primers was designed, and PinA was PCR-amplified from 34 accessions belonging to 4 sections of Aegilops genus. 25 positive PCR products were sequenced and aligned with those already submitted in GenBank. The results revealed that 7 PinA sequences were identical with those in GenBank but from different species, while 14 PinA sequences were mutated. The 14 mutated PinA, 447bp in full length, shared a highest identity, varying from 97.8% to 100%, and they were deduced to encode 149 amino acid residues with the tryptophan-rich domain (WRWWKWWK) except RM0070 and RM0072, who mutated from "W"to a terminator at position 69. The isolation of PinA mutants made it possible to further enlarge the variety of wheat grain hardness by genetic engineering.