| Rice is one of the most important crops in the world,and how to enhance the rice yield is the issue that the crop scientists pay close attention to.Meanwhile,as a special branch form of gramineous plants,the technique of tillering plays a crucial role in determining the plant type and rice yield during the growth process.So regulating rice tillering can effectively change rice yield.There are many methods to produce tiller diversity in rice.In this study,ethyl methyl sulfonate(EMS)induction and multiploidy induction techniques were used to screen rich tiller diversity materials.Ethyl methyl sulfonate(EMS)is a common reagent for the construction of plant mutant libraries,which plays an important role in enriching biological gene banks and diversifying plant phenotypes.In this study,9311 wild type seeds were used as materials to construct a rice tiller diversity mutant library by EMS.The results showed that rice tillers increased generally after EMS mutagenization,and some individuals with very many tillers and few tillers were selected.Preliminary studies showed that the comparison of 9311-2x,9311-4x,BLL-2x and BLL-4x showed that tillering significantly decreased after multiploidation of rice.Previous studies have shown that MND1 can repair chromosomes and stabilize homologous recombination during meiosis.However,this study shows that overexpression of MND1 can lead to a significant increase in tillering number of rice,and absence of MND1 can lead to a decrease in tillering number of rice,but the decrease range is smaller than that of overexpressed plants.Further,using BLL and its OsMND1 overexpression mutant OsMND1:OsMND1 and OsMND1 gene cas-9 mutant cas-9-mnd1 as materials,hybrid rice 620 was used as doubling material to obtain 620-4x plants to explore the method and mechanism of controlling tillering in rice.The main research contents are as follows:(1)Create germplasm resources with different tillering characteristics by multiploidy technique.Polyploidization can significantly affect the development of tillering in rice.After multiploidy,tillering decreased by 16.7%-59.8%.Variety 620 with the largest number of tiller declines was selected for subsequent cytological analysis.The results showed that 620-2x began to form tiller buds at 2WAG(2 weeks after germination)and tiller buds gradually increased over time.Three tiller buds were formed at 5WAG(5 weeks after germination)stage.620-4x began to form tiller buds at the WAG stage,the tiller formation rate lagged behind 620-2x,and finally formed two tiller buds at 5WAG.The spatio-temporal expression analysis of tiller related genes indicated that polyploidy may affect the number of tiller buds produced and the degree of development at the base of rice stem through gene expression differentiation,and ultimately regulate the number of tillers in rice.(2)Expression analysis of OsMND1 gene in germplasm resources with different tillering characteristics.The expression levels of diploid and tetraploid rice,which were screened by EMS,were detected by quantitative PCR to further clarify the relationship between the expression of OsMND1 and the number of tillers.Overexpression of MND1 resulted in the significant increase of tillers in rice,and the increase of tillers was 230.1%.After MND1 deletion,tillering decreased by 33.1%.(3)On the basis of confirming that OsMND1 is indeed involved in the regulation of tillering number in rice,further studies on the mechanism of OsMND1 regulating division were carried out.Overexpression of OsMND1 significantly improved tillering in rice,and OsMND1 affected the formation of tillering buds and slowed down rice development.Through dynamic observation of the generation rule of rice tillers,the tiller ability of overexpression mutant OsMND1:OsMND1 gradually increased with the development of rice,and the tiller ability of wild type BLL was significantly higher than that of wild type BLL at the fourth week after germination,and the continuous tiller ability of wild type BLL was significantly weaker than that of overexpression mutant OsMND1:OsMND1.The deletion mutant cas-9-mnd1 lagged behind the wild type BLL at 4WAT(4 weeks after transplanting).By histocytological analysis,it was found that from the third week after germination,the growth number of tiller buds of mutant OsMND1:OsMND1 was significantly higher than that of BLL,and the growth rate was faster.On the contrary,the interfering mutant cas-9-mnd1 lagged behind that of wild type BLL,and OsMND1 helped the tiller stage start faster.Molecular docking predicted that MND1 protein might interact with clarolactone.(4)The germplasm resources of different tiller characteristics were screened by ethyl methyl sulfonate(EMS)mutagenesis.EMS treatment resulted in the increase of tillering of rice,and the increase of tillering was 83.85%.Compared with wild type 9311,the number of plants with increased tillering was much greater than that with decreased tillering.The maximum tillering value was 56 and the minimum tillering value was 3.The number of tillers was about24 after EMS mutagenesis.EMS mutagenesis may disrupt the expression of tiller related genes,leading to the diversity of rice tillers.Further analysis and verification of EMS mutants are expected for many years and generations. |
