Activity Assay Of Some Key Enzyme In Seed Amylose Synthesis And Cloning Of Waxy Genes Of Yunnan Wild Rice Species

Rice is one of the most important crops.More than half of the world's population takes rice as staple food.The more improvement of the living condition, the higher quality of rice is demanded for. In order to investigate and utilize the traits related to good quality of the low amylose content in wild rice, study the mechanism of amylose synthsis and key starch synthsis enzymes,cloning the related genes,comparing the molecular mechanism and gene between wild rice and cultivated rice have important sense for utilizing good gene from wild rice to improve cultivated rice quality.In the early study, it was found that the amylose content of these wild rice varied from 8.78 to 12.99%. Indeed the amylose content level among 9%-14% is considered as good quality of rice. So we continued to study the key starch synthsis enzymes in the wild rice and some cultivated rice and cloned Waxy gene which relate to amylose content. The results were as followed:1. Four key enzymes (ADPG pyrophosphorylase(ADPGP), soluble starch synthase(SSS), granule bound starch synthase(GBSS), starch branching enzyme(Q- enzyme)) activities were measured in three Yunnan wild rice species(Oryza rufipogon Griff, Oryza officinalis Wall, Oryza granulata Baill). The results indicated that only the activity of GBSS in the wild rice was positively correlated with the amylose content. Therefor it is necessary to study Waxy gene in wild rice and use these genes to improve cultivated rice quality.2. The results indicated that SSS and Q-enzyme showed opposition change trend in terms of activity during the grain filling stage. It may suggest these two enzymes had some feedback suppression effect.3. The Waxy genes had been cloned from 3 Yunnan wild rice species and cultivated rice Dianlong 201# by PCR with the genomic DNA as template. The coding fragments of the Waxy genes had been cloned by RT-PCR with the mRNA of young seeds during filling stage. We got a complete structure Waxy gene-4285bp of Waxy gene DNA sequence and 2054bp coding sequence from O.rufipogon; a complete structure Waxy gene-3733bp of Waxy gene DNA sequence and 2085bp coding sequence from O.officinalis; a complete structure Waxy gene-3342bp of Waxy gene DNA sequence and 2065bp coding sequence from O.granulata; a complete structure Waxy gene-4864bp of Waxy gene DNA sequence from Dianlong 201#.4. Through the DNA sequence and RNA sequence comparative analysis, it was found that the Waxy genes of O. rufipogon, O. officinalis and O. granulata had big difference with cultivars in terms of the gene length, DNA sequence. Also the Waxy genes were quite different from each other among 3 yunnan wild rice species. In these 3 yunnan wild rice species, the Waxy gene of O. rufipogon had the highest similarity to the cultivars. And this was followed by the O. officinalis. The Waxy gene of O. granulata and the cultivated rice had the biggest DNA sequence disparity.5. The intron regions and the exon regions of the Waxy genes were identified in the 3 Yunnan wild rice species. It's showed that O.officinalis and O. rufipogon have 14 exons and 13 introns, they had one more exon and intron than that in cultivated rice species.6. The amino acid sequences deduced from the Waxy genes of the three yunnan wild rice species had significant difference each other and with the cultivars. But the amino acids difference was less compared with the DNA sequence difference of the Waxy genes. There were 7 unique amino acids only existed in the WAXY of the three yunnan wild rice species. The predicted second protein structure of WAXY of the 3 wild rice species had significant difference each other and also with the cultivars. All of these suggested that there might be different regulation mechanism controlling amylose content by Waxy genes in the 3 yunnan wild rice species from that of the cultivars.7. Three pairs of specific primers were designed from the Waxy genes of the wild rice species for MAS in the generations after hybridization between wild rice and cultivars.This is the first report about amylsoe synthesase activity and the Waxy gene from wild rice worldwide. From the results, we could know the enzyme basic and molecular basic of key gene relating with the low amylose content in some extent. These results also made a foundation to completly discover the molecular basic. According to the three pairs of specific primers from wild rice's Waxy gene, these primers can be used as molecular markers in selection of low amylose content in rice breeding.