Effects Of Regulating The Key Genes Involved In Leaf Transitory Starch Metabolism On Seed Related Traits In Rice

The source-sink theory of rice summarized the basic pathway of the formation of rice yield.Starch is the major photosynthetic products produced by source organs,and the coordination of source-sink-flow related organs determines starch metabolism rate and transport efficiency.There are already many reports on sink-related genes.In this study,three genes closely related to transitory starch metabolism in source organs,including OsGWD1,OsBMY4 and OsISA3,were selected the following analysis.The generated overexpression and gene editing transgenic rice of these genes were used for systematic analysis of the effects of regulating these genes on rice yield,rice quality,seed germination and stress tolerance,thus providing rich gene resource and genetic materials for breeding elite rice with high quality and yield.The detailed results of this study are as following:1.Generation and identification of positive transgenic rice.We identified an Osl2 promoter with leaf specific expression pattern,whose expression increased gradually with leaf senescence process.Then we fused this promoter with the target genes OsBMY4 and OsISA3 respectively,and cloned them into the same expression vector,thus creating the pOsl2::OsBMY4/pOsl2::OsISA3 transgenic rice with both genes overexpressed.The results of molecular identification and gene expression analysis confirmed that the vector had been successfully introduced and expressed in the recipient variety 9983.In addition,CRISPR/cas9 method was used to edit gene OsBMY4 and OsISA3 in Zhonghua 11,and the transgenic rice with the target site successfully edited were got.Next,homozygous mutant lines were selected for the subsequent analysis.2.Systematic analysis of the OsGWD1 overexpression lines and mutants revealed that overexpression of OsGWD1 increased plant height and 1000 grain weight of rice,and made the grain slender.While knock-down of OsGWD1 promoted the over-accumulation of transitory starch in the leaves,and decreased the chalkiness of the seeds.Overexpression of OsGWD1 significantly increased the initial gelatinization temperature,termination gelatinization temperature,and maximum viscosity of rice flour,whereas the highest viscosity of mutant rice flour was significantly decreased.The other physicochemical characteristics of mutant rice were not changed.Furthermore,OsGWD1 is a positive regulator of seed germination and stress resistance.Overexpression of OsGWD1 remarkably promoted the germination rate of rice seeds under normal and stress germination conditions,whereas the germination of the mutant seeds was significantly slower than that of the wild-type control.The changes in their germination characteristics were mainly mediated by its regulation of the amylase activity of the seeds.3.Simultaneous overexpression of OsBMY4 and OsISA3 increased rice 1000 grain weight and made the grains slender.Analysis of the physicochemical characteristics of rice revealed that the amylose content of the overexpression transgenic rice was increased significantly,and the initial and final gelatinization temperatures were increased.Also,its viscosity properties were changed,such as the increased highest viscosity and decreased the lowest viscosity.Moreover,OsBMY4 and OsISA3 overexpression transgenic rice exhibited faster germination rates than the control,largely due to the enhanced amylase activity.Similarly,the transgenic rice germinated faster than that of the wild-type control under abiotic stress conditions,such as salt and drought stress.Unlike GWD1,knock-out of either OsBMY4 or OsISA3 resulted in no excessive transient starch accumulation in the leaves,but their seed setting rates were significantly reduced.The isa3 mutant did not show any other phenotypic or physicochemical changes.Although the grain shape of the bmy4 mutant was unchanged,its chalkiness degree and the percentage of chalky grain were significantly decreased.And its amylose content was also decreased.In addition,the seed germination rate of bmy4 mutant was lower than that of the wild-type control under both normal and stress germination conditions.