| As an important plateau cereal crops, Hulless Barley, the main source of food for the Tibetan people in China, it’s also an important industrial raw materials. Starch is the major component in plant seeds and is the most important carbon reserve. It provides more than80%calories in the world, and has become an essential commodity that is widely used in food, feed, fuel and industry. About60%of the hulless barley endosperm is starch, which can affect the yield and quality of hulless barley directly. So it is very important to do further research on starch metabolism. AGPase is considered as an key enzyme among starch synthesis process, which has two large subunit (LSU) and small subunit (SSU), encoded by separate genes. The cytoplasmic is main form, which AGPase consists in poaceae endosperm and generating ADPG reactions occur mainly in the cytoplasm. The cytoplasmic AGPase have never benn found in other plant.The research of improve AGPase’s activity and starch content, which by genetic engineering techniques, was focus on the cytoplasmic small subunit SSUI and cytoplasmic large subunit LSUI.In order to investigate the function of SSUI and LSUI gene in the hulless barley, SSUI and LSUI were cloned from86020(high starch content) and Aqing5(low starch content) by RT-PCR, their expression were detected by Real-time PCR. The main results are as follows:1.The length of SSUI cDNA was1438bp with1419bp ORF which encoded a protein with472amino acids. It was a stable and hydrophobic protein with the molecular weight of52.01kD, theoretical pI5.59, coefficient of instability33.54, the average hydrophilic-0.176. The protein was rich in Ile and Ala. Its transmembrane prediction results show that:the protein SSUI gene encoded is non-transmembrane protein belonging to membrane proteins. The sequence alignment showed the highly homologous between SSUI gene and the barley (Hordeum vulgare L.). The lowly homologous between SSUI gene and the I.batatas, S.tuberosum. The express pattern of SSUI gene was similar in two materials, the seeds were the most abundant in the expression of the gene, then the leaves and stems. In relative expression levels of grains was approximately normally distribution, Peaked at about15days after flowering then began to declined, And the relative expression level in86020was significantly higher than the Aqing5. It also similar in the Leaves and stems, and the obvious expression pattern didn’t exist. 2.The length of LSUI cDNA was1786bp with1572bp ORF which encoded a protein with523amino acids. It was a unstable and hydrophobic protein with the molecular weight of58.032kD, theoretical pI6.30, coefficient of instability44.59, the average hydrophilic-0.25. The protein was rich in Ser and Gly. Its transmembrane prediction results show that:the protein LSUI gene encoded without transmembrane protein, with SSUI encoding proteins belong to the same protein in membrane. The homology comparison showed the homologous was high between LSUI gene and the barley(Hordeum vulgare L.), and low between LSUI gene and the I.batatas. The express pattern of SSUI gene was similar in two materials, but had obvious differences in expression levels in the seeds, leaves and stems, seeds had the highest expression of all, and in two materials, the similar trends was seeds> leaves> stems. In relative expression levels of grains was approximately normally distribution, Peaked at about11days after flowering then began to declined, it’s no significant difference in the expression levels of LSUI in Grain Formation of two materials, but the relative expression levels declined slowly in filling and ripening stage of high starch material, and maintained at a relatively high level, the relative expression levelss was similar in leaves and stems of two materials, and the obvious expression pattern didn’t exist.3.The expression trends of SSUI and LSUI are similar with accumulation of starch in hulless barley endosperm, suggesting SSUI and LSUI maybe play an important role in hulless barley starch synthesis. |
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