Study Of Using Expression Vector With Two Copies Of Anti-Waxy Gene To Improve The Edible Quality Of Rice

Eating quality is a very important character for cooked rice, and largely depends on the rice starch, as main component of rice. The rice starch consists of amylase and amylopectin, and is synthesized by a series of enzymes, including ADP glucose pyrophosphorylase(AGP), starch branching enzymes(SBE), starch debranching Enzyme(DBE), granule-bound starch synthase (GBSS) and soluble starch synthase (SSS). The ratio of amylase to amylopectin (i.e., the amylase content) is one of the most important factors that determine the eating quality. GBSS is the key enzyme in amylase synthesis, which is encoded by waxy gene. Consequently, the waxy gene plays a major role on the rice eating quality. In recent years, improvement of rice eating quality through biological technology is widely investigated, mainly by using molecular makers and introducing antisense gene.In this study, the anti-waxy gene was introduced into the male sterile line 261S by Agrobacterium tumefaciens-mediated co-transformation. A total of 66 resistant calli differentiated into plantlets.9 co-transformants were obtained by PCR analysis at the co-transformational rate of 12%.With the trait observation of the progeny of 8 surviving co-transformants, T1 seeds of 5 transgenic plants B2, B3, B17, C8 and D2, exhibited the waxy mutant phenotype, and the other three plants D19, D8 and C6, generated normal seeds. The result of genetic analysis showed in the plants B3, C8 and D2, the ratio of the T1 seeds with waxy mutant phenotype to the normal ones agreed with expectation 3:1. The marker-free transgenic plants containing anti-waxy gene were seek out by GUS staining assay and PCR analysis, meanwhile the homozygous transgenic plants with waxy mutant phenotype and normal transgenic plants were identified from T1 generation of the plants B2, B3, B17, C8 and D2, and the plants D19, D8 and C6, respectively. Expression analysis of the homozygous progeny of the 8 co-transformants was carried out. The results indicated that the expression of waxy gene in pollen and endosperm decreased to a great degree in the progeny of the plants B2, B3, B17, C8 and D2, while slightly in D19, D8 and C6 by comparison with the 261S control. It was also found that the structure of the starch granule had changed in the homozygous progeny with the waxy mutant phenotype, which were generated from the transgenic plants B2 and B17.The transgenic anti-Waxy plants, B3 and B17, with good characters were crossed with W99075 respectively,261S×W99075 taken as control. The genotypes of F1 hybrid plants were identified by PCR analysis. Genetic analysis of the traits of brown rice in F2 generation resulting from hybrid cross, B3×W 99075 and B17×W 99075, showed that the ratio of the waxy mutant seeds to the normal is 3:1, which confirmed to Mendel's law.Main agronomic characters, yield traits and the size of the brown rice of the homozygous hybrid plants and their female parents were investigated, and the results revealed that the net weight of transgenic individual decreased significantly, compared with those of the control group, while the other characters had no distinct difference. Study on the physicochemical properties of the hybrid rice indicated that compared with "Minyouxiangjing", "new Minyouxiangjing" had hardly no change in the gelatinization temperature, however, the amylose content and the gel consistency decreased by 210% and 37.8% respectively.Compare to the study introducing the anti-Waxy gene to rice by previous presons,this study is innovative in three aspects as follows:(1)firstly confirmed the expression vector,P13AWY-2,could effectively reduce the amylose content of rice.(2)firstly found while thee anti-Waxy gene significally decreased the the amylose content of brown rice,the starch structure of rice endosperm changed.(3) firstly explained while thee anti-Waxy gene significally decreased the the amylose content of brown rice,the rice weight changed.