Expressions Of Starch Synthase Genes And Starch Synthesis In Wheat Grains

The expression levels of ADP-Glc Pyrophosphorylase gene(AGPase1), Granule-Bound Starch Synthase gene(GBSSI), Soluble Starch Synthase gene(SSSIII), and Starch Branching Enzyme gene(SBEI) and their relationships with the starch synthesis in wheat grains of different gluten types were investigated with non-waxy wheat cultivars and waxy wheat varieties. The relationship between GBSSI gene expression and amylose synthesis in wheat grains with different missing Wx proteins, and the responses of the expression levels of starch synthase genes in weak-gluten wheat grains to nitrogen and high temperature after anthesis were analyzed . These studies are of crucial importance both in exploring the physiological mechanism for starch synthase gene expression regulating starch synthesis and starch quality in wheat grains with different gluten types and in providing theoretical basis and technical guidance for efficient production in wheat grains with high yield and goodquality. The main results were as follows.1. Changes of starch synthase genes expression levels, starch synthases activities and starch accumulation in grains of different wheat cultivars during grain fillingSingle-peak trends were observed in the expression levels of AGPase1, GBSSI, SSSIII and SBEI during grain filling. The expressions of these genes were first detected on DAA 5 (the 5th day after anthesis), reached the maximum on DAA 15, and declined significantly from DAA 15. The lowest expression levels of starch synthase genes were recorded during the period of DAA 30-40, only at around 1.0. The activities of AGPase, SSS, GBSS, and SBE performed in the pattern of a single-peak curve during grain filling. AGPase, SSS and SBE activities reached their peaks on DAA 25 and GBSS activity reached its maximum on DAA 30. The starch content and accumulation in grains rose gradually after anthesis,Among the different gluten types of wheat varieties, the expression levels of starch synthase genes, the activities of starch synthase, and starch accumulation were recorded in a descending order of Zhongyou9507 (a strong-gluten cultivar), Huaimai18 (a medium-gluten cultivar), Ningmai9 (a weak-gluten cultivar), and Wx11 (a Waxy variety). The expression of GBSSI and the activity of GBSS in Wx 11 grains were maintained at very low levels approaching zero, which led to amy-lose content in Wx11 grains were lower than 2%. Between different wheat varieties with the same gluten type, the order of above-mentioned parameters was: Zhongyou9507 > Qinmai11 in strong-gluten cultivars; Yangmai16 > Huaimai18 in medium-gluten cultivars; Yangmai15 > Ningmai9 in weak-gluten cultivars.2 Changes of SS activity, sucrose and total soluble sugar contents in grains of different wheat cultivars during grain fillingThe activities of SS performed in the pattern of a single-peak curve in wheat grains with different gluten types during grain filling. Its peak appeared on DAA 20. The sucrose and total soluble sugar contents in wheat grains dropped constantly respectively after anthesis and at early grain filling stage.Among different gluten types of wheat varieties, the activity of SS in a descending order of Zhongyou9507, Huaimai18, Ningmai9, and Wx11, and the order of sucrose and total soluble sugar contents in wheat grains were expressed as Wx11 > Ningmai9 > Huaimai18 > Zhongyou9507. The results showed that a large amount of sugar in strong-gluten wheat grains had been converted into the substrate for starch synthesis, therefore the starch accumulation in its grains was much higher than that in the grains of the other three gluten types. In waxy wheat grains there were a large amount of sugar which was incapable of being converted into the substrate for starch synthesis. Hence, the starch accumulation in its grains was much lower than that in non-waxy wheat grains. Between different wheat varieties with the same gluten type, the above mentioned parameters were in the following order: Zhongyou9507 > Qinmai11 in strong-gluten cultivars; Yangmai16 > Huaimai18 in medium-gluten cultivars; Yangmai15 > Ningmai9 in weak-gluten cultivars.3. Relationship of the expression levels of starch synthase genes with the activities of starch synthases and starch accumulation in wheat grainsThe relationship between the maximum expression levels of AGPase1, SSSIII and the peak activities of AGPase, GBSS, SSS, SBE, SS was significantly correlated at 0.01 or 0.05 levels. The maximum expression level of GBSSI was significantly correlated with the peak activities of AGPase and SS, while there were no significant correlations between it and the peak activities of GBSS, SSS, and SBE. The maximum expression level of SBEI was highly significantly correlated with the peak activities of SSS and SBE, (p<0.01), and insignificantly correlated with the peak activities of AGPase, GBSS, and SS. The expression levels of AGPase1, GBSSI, SSSIII, and SBEI and the activities of AGPase, GBSS, SSS, and SBE were highly significantly correlated with the accumulatiing rates of amylase, amylopectin, and total starch. These suggested that starch synthase genes and starch synthases played collaborative roles in starch synthesis in wheat grains. AGPase, SSS,and SBE might probably control starch synthesis at transcriptional level, and GBSSI might probably control starch synthesis at post-transcriptional level.4 Relationship between GBSSI gene expression and amylose synthesis in wheat grains with different missing Wx proteinsThe expression level of GBSSI, GBSS activity, and amylase content were recorded in the following order: normal type > single null allels of Wx-A1 > single null allels of Wx-D1 > single null allels of Wx-B1 > double null allels of Wx-A1 and Wx-B1 > waxy type. Similar trends were observed in final viscosity, rebound value, and gelatinization temperature, while opposite trends were measured in peak viscosity, trough viscosity, breakdown, and sedimentation value.5 Responses of starch synthase gene expressions in weak-gluten wheat grains to nitrogenThe expression levels of starch synthase genes (AGPase1, GBSSI, SSSIII, SBEI), the activities of starch synthases (AGPase, GBSS, SSS, SBE), and starch accumulation rose in the grains of Yangmai15, a weak-gluten wheat cultivar, with the increase of nitrogen application amount varying from 150kg hm-2 to 210kg hm-2. When nitrogen application amount was higher than 210kg hm-2, those decreased. The expression levels of starch synthase gene, the activities of starch synthase, and starch accumulation were stimulated when topdressing N percentage decreased reasonably.6 Responses of starch synthase gene expression in weak-gluten wheat grains to high temperature after anthesisThe expression levels of starch synthase genes (AGPase1, GBSSI, SSSIII, SBEI), the activities of starch synthases (AGPase, GBSS, SSS, SBE), and starch accumulation in the grains of Yangmai15 were significantly inhibited when the wheat plants were subjected to high temperatures during grain filling. The decrements of these parameters among different high temperature treating periods were in the following order: 5-7DAA > 10-12DAA > 15-17DAA > 20-22DAA > 25-27DAA > 30-32DAA > 35-37DAA. Compared to 25℃, the inhibitive effects were more significant at 35℃. Similar trends were found in these parameters for the treatment of 25℃and the control. The expression levels of AGPase1, SSSIII, and SBEI, and the activities of AGPase, SSS, SBE reached their peaks on earlier dates when subjected to 35℃on 5-7 DAA. GBSSI and GBSS were insensitive to high temperatures. SSSIII and SSS were sensitive to high temperatures.7 Improvement of real-time quantitative PCR techniqueReal-time quantitative PCR (RT-PCR) is a kind of nucleicacid technology, which can monitor the entire real-time PCR process through the accumulation of fluorescence signal and analysis the unknown sample through the standard curve. Therefore, it has the characteristics of real-time monitoring, simple and cost saving. In this experiment, a series of measures had been taken to further improve RT-PCR technology, which included removing DNA in mRNA with DNA enzyme, strictly designing primers in accordance with the requirements of RT-PCR, mixing different template cDNA to make standard curve, optimizing the reaction system of RT-PCR and the use of two-step RT-PCR reaction. These measures above mentioned can accurately reflect the process of reverse transcription and amplification.