Cloning And Expression Of Three Genes Potentially Involved In The Starch Metabolism In Wheat Grains

The development of grain is a critical period that affects the finlal yield and quality of wheat, and metabolic regulation of starch accumulation in the endosperm directly determines the grain weight and flour processing quality. In this study, based on the analysis of proteomic profileing in development wheat seed, three genes potentially involved in the starch metabolism were cloned from wheat grains, and their expression patterns during the seed development were characterized. Our results provide important information for further exploration of molecular mechanism involved in the starch metabolism in endosperm of wheat grains.The main results were showed as follows:Some evidences have suggested that trehalose 6-phosphate is an important regulatory factor that potentially involved in the regulation of starch metabolism. In this study, three c DNAs encoding trehalose 6-phosphate synthase were cloned by RT-PCR and named Ta TPS-1.1, Ta TPS-1.2 and Ta TPS-1.3, which was 2 422, 2 379 and 1 889 bp in length, respectively. Genomic characterization indicated that 14 introns werer included in the cloned region of this gene. Expression analysis showed that the three transcripts of Ta TPS-1 were up-regulated during the grain development in Luohan2, and the expression levels were relatively higher at the stages of 15-25 DAP. Compararively, the expression level of Ta TPS-1.1 was much higher than the other two transcripts, which suggested that Ta TPS-1.1 plays important role during the seed development. However, in Chinese spring, the expression patterns of the three transcripts were quite different from that in Luohan2. The Ta TPS-1.1 was highly expressed at the early stage of seed development, while it was rapidly down-regulated after 25 days;The expression of Ta TPS-1.2 was the highest in the 20 DAP seeds. In the seed of Aikang58, the expression of Ta TPS-1.1 and Ta TPS-1.2 was the highest at 15 DAP, while Ta TPS-1.3 was down-regulated. Generally, our results suggested that Ta TPS-1.1 should play important roles in grain filling process, and the effects of Ta TPS-1.2 was relatively weak, while the expression pattern of Ta TPS-1.3 was uncertain depend on the trait of different wheat cultivars.Amylase inhibitors are a sort of storage proteins accumulated during seed development and their biological function during the seed development is still not clear. Here, three c DNA versions of an α-amylase inhibitor, Ta AAI-1.1, Ta AAI-1.2 and Ta AAI-1.3, were cloned by RT-PCR. Ta AAI-1.1 and Ta AAI-1.2 contains 152 amino acids, and there was a deletion including seven amino acids in the coding region for Ta AAI-1.3, because of this deletion, the protein missed an α-amylase enzyme binding site. Expression patterns analysis showed similar expression patterns of the three transcripts of Ta AAI-1, and their expression levels positively correlated with grain filling rate, which suggesting that the inhibitors play crucial roles during starch accumulation in wheat grains. However, the expression of Ta AAI-1.3 was very weak.H-type thioredoxin is a class of redox regulatory molecules that widely exist in plant. In our previous studies, it was found that the repression of Ta Trx-h9 affected the accumulation of starch and the flour processing quality. Here, we cloned two transcripts of Ta Trx-h9 which containing the complete coding region and named Ta Trx-h9.1 and Ta Trx-h9.2, respectively. There was a deletion including 18 bp at the 5’UTR region in Ta Trx-h9.1 compared to Ta Trx-h9.2. Expression analysis showed that the Ta Trx-h9 was highly expressed at the early stages of seed development(5DAP)in three wheat varieties detected, and slightly decreased during the development. However, the expression level is generally higher and the abundance of it are almost same as that of actin, which is an endogenous house-keeping gene. Its extremely higher expression suggests that Ta Trx-h9 play key regulatory roles in developing wheat seeds.