| The grain hardness of wheat, reflecting character of endosperm texture, is one of the most important determinants of milling, baking and end-use quality. The trait is controlled by puroindolines a (pina) and puroindolines b (pinb) genes located 5DS in wheat. The hard texture of kernel is common associated with mutation in pina or pinb. Polyphenoloxidase in kernel is a major factor causing darkness and discoloration of Asian noodle and steam bread. Here, the characters and molecular mechanism of the two traits were studied. The results are as follows:1. The allelic variations were rich in common wheat, especially in local cultivars of China. In this research, one new variation of pina and three new variations of pinb were identified. Sequencing results showed that they were attributed to single nucleotide mutations. These allelic variations were tightly associated with grain hardness. But no allelic variation was found in Tibet semi-wild wheat, Spelt wheat, Compact wheat and Aegilops. Puroindolines genes were absent in wild Emmer and Thinopyrum.The improved Urea-SDS-PAGE had higher resolving power than TBE-PAGE, so that single nucleotide mutation could be identified. Pina and pinb variations could be discovered using the PAGE in DNA level more efficiently than in protein level. It's unnecessary to design different allelic specific primers in studying puroindolines genes, so it's a good technique for identify single nucleotide polymorphisms (SNP) too.2. The total puroindolines protein in endosperm showed no difference between hard and soft wheat in development of grain. But significant differences of puroindolines protein associated with starch granule could be found among hard and soft wheat. That was, there were more PinA and PinB on the surface of starch granule in soft wheat than in hard wheat. Some wheat cultivars with wild type of puroindolines genes had hard endosperm and the direct reason maybe very faint puroindolines protein "sticking" to the starch granule.3. SDS could improve extraction of PPO in wheat kernel and enhance difference between cultivars. PPO activity behaved varied characters of time and temperature in different common wheats. In wheat and its related species, the variation of PPO activity was very significant, but it lied in the range of middle to low activity generally. Some varieties with very low PPO activity had high value in breeding and relative study. Generally speaking, hexaploid wheat (AABBDD) showed higher PPO activity than Aegilops (DD), and PPO activity of wild Emmer (AABB) was most low. There was certain activity in Thinopyrum grain.4. PPO-1 and PPO-2 genes were located in 2A and 2D chromosomes through nullisomic-tetrasomic lines of Chinese Spring cultivars. The two genes, especially the latter exhibited rich variations. The a-type allelic gene was predominant in PPO-1 allelic genes. As for PPO-2 gene, the D-type gene was the most. In these allelic genes of PPO-1 and PPO-2, the activity was distributed widely in common wheat and it's related. The cultivars with very high or very low activity were very few. The PPO activity of varieties with A, S, E and F type allelic genes was higher than that of H, I, D* and K type. The null of PPO-1 and PPO-2 was close relationship with extremely low activity.5. An intron was identified between CuA-CuB of PPO-2 gene, its variation could be found among different cultivars of wheat. The DNA sequence of functional region (CuA-CuB) in PPO-2 had very high homology among wheat and it's related, but its variation could also affect PPO activity of kernel. With the development of seeds, the isoform enzyme of PPO would generallybecome weak except A and C region on the gel. The weakening degree of the isoform in low PPO activity wheat was more significant than high activity wheat. As for wheat cultivars with high PPO activity, the isoform enzyme in C region of gel became strong gradually, consistent with the change of its PPO activity, during wheat kernel maturation, but low activity cultivars differed from this change. In the transcr...
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