Transgenic Regulation Of The Expression Of OsGBSS Ⅱ And OsPull Genes And Its Effect On Grain Starch Quality In Rice

Starch is the main storage component in rice endosperm, and accounts for about 70~90 percentage of the dry weight of grain. Therefore, it is the key determinant of rice quality. A series of key enzymes, such as GBSS (Granule-Bound Starch Synthase) and PULL (Pullulanase), are involved in the pathway of starch biosynthesis in rice endosperm. However, it was not verified of the functions of some of these enzymes ,especially the effects on rice quality. In present study, the over-expression and RNA interference constructs of OsGBSSâ…¡and the RNA interference constructs of OsPull, encoding rice typeâ…¡GBSS and Pullulanase, respectively, were introduced into three rice genotypes containing distinct grain starch quality by Agrobacterium-mediated transformation. Lots of transgenic rice lines were regenerated and further confirmed by both PCR and Southern blot analyses. Both the expression of target gene and grain quality in those transgenic rice were carefully analyzed, and the results were as followings.1. Over-expression construct of OsGBSSâ…¡gene, in which the OsGBSSâ…¡cDNA is controlled by the promoter of rice endosperm-specific glutelin gene Gt1, was introduced into two japonica rice varieties Suyunuo and Nipponbare. RT-PCR and Northern blot analyes showed that the introduced OsGBSSâ…¡gene was expressed with different levels in the endosperm of most of the transgenic lines, while there was no any OsGBSSâ…¡transcript detected in that of the wild type. The amount of GBSS protein was increased in the endosperm of some transgenic rice plants derived from Nipponbare, while no any GBSS detected in those derived from the waxy rice Suyunuo. Quality analysis indicated that the amylose content was a little decreased in the grain in T2 transgenic rice of Suyunuo, while it was contrary in those from Nipponbare. The gelatinization temperature did not change in T2 transgenic rice of Nipponbare. The gel consistency of T2 transgenic rice of Nipponbare became more stiffen when compared with that of the wild type, while it did not chang in those from Suyunuo.2. The RNA interference construct of OsGBSSâ…¡gene was introduced into Suyunuo, Nipponbare and Longtefu, respectively. Northern blotting and RT-PCR analyses showed that the expression of endogenous OsGBSSâ…¡in leaves of transgenic plants was significantly decreased comparing to that of the wild type control and the decrease level among different transformants was different. The amylose content of single rice grains decreased in T1 transgenic rice from Suyunuo, while the change of AC was not disciplinary in that of the transgenic rice from Nipponbare and Longtefu. The change of AC was not disciplinary in that of transgenic rice plants derived from Suyunuo, Nipponbare and Longtefu in T2 generation. The gelatinization temperature did not change in T2 transgenic rice of Nipponbare and Longtefu, while it increased in T2 transgenic rice of Suyunuo. The gel consistency of T2 transgenic rice of Nipponbare became more stiffen compared with that of the wild type, while it did not chang in those from Suyunuo and Longtefu.3. Transgenic rice plants containing the the RNA interference construct of OsPull gene was produced in three varieties Suyunuo, Nipponbare and Longtefu. The gene expression analysis showed that the OsPull transcript in developing seeds of transgenic plants was significantly decreased comparing to that of the wild type, and the decrease level was different among different transformants. Which was consistent to the results from pullulanase activity assay. The preliminary data of quality analysis implied that there was no significant effect on grain qualtiy after down-regulation of the target gene's expression in transgenic rice.