| Starch is the main storage material in rice seeds.The ratio of amylopectin and amylose content in rice,as well as the structure of starch,largely determines the taste quality of rice.The formation of starch in rice is a complex and precise process,which requires the participation of a series of enzymes and regulatory factors.At present,we do not have a clear understanding of the molecular regulation mechanism of powder development and starch synthesis in rice endosperm,so we explore and develop The new impact factor of starch synthesis is a very interesting research work.In the early stage of our laboratory,the conventional indica rice variety Xiushui 134 was induced by EMS,and the mutant of the mutant which was stable and phenotypically identified was selected from the mutant library,and it was named flo16.The main research results are as follows:1.The mutant flo16 showed a distinct white phenotype,the white part was white,the starch granules in the middle part of the endosperm were loosely arranged,the white area was large,and the periphery was translucent;compared with the wild type,the flo16 seed Slender,low seed setting rate;total starch and amylose content of mature seeds decreased significantly;ratio of short chain(DP6-8)and long chain(DP>34)of amylopectin decreased,medium and long chain(DP9-19 The ratio increases;the gel consistency and the viscosity of the rice flour are significantly reduced;the swelling volume of the flo16 mutant rice powder becomes smaller.These results show that the FLO16 gene plays an important role in the starch synthesis process in rice endosperm.2.The results of semi-thin section of endosperm showed that most of the starch granules in the wild type were in the form of compound starch granules,and the starch granules polymerized to form a complete amyloplast,while the morphology of starch granules and the development of amyloplasts in the flo 16 mutant endosperm existed.Abnormally,there are a large number of small and scattered starch granules in the mutant body,and the shape is irregular;and the starch single particles are arranged loosely,and the gap gap is large,resulting in the inability to form composite particles.These results indicate that the FLO16 gene plays a key role in the formation of endosperm composite particle morphology.3.In this study,we used the F2 genetic population formed by the hybrid of the flo16 mutant and the conventional indica rice variety Guanglu Dwarf 4.We located the mutant gene between the two markers Indel4 and RM7 on chromosome 3;The flo16 mutant was sequenced by transcriptome.RNA-seq results showed that 6 genes were mutated in the localization interval.For further screening validation,we designed upstream and downstream primers near the mutation sites of these six genes,and selected Twelve phenotypic mutants and three wild-type extracted genomic DNA were amplified by PCR with 6 pairs of primers designed,and the PCR products were sequenced.The sequencing results showed that only the sixth gene was in the 12 mutants.A single base substitution occurred,and the first base of the 5’ cleavage site of the fourth intron was mutated from G to A,resulting in the fourth intron not being normally cleaved,and the protein translation was advanced.Termination;the results of functional complementation,overexpression and other results confirmed that the gene is the target gene.4.Phylogenetic tree analysis revealed that this gene has homologous genes in eukaryotes such as animals,plants and yeast,and proteins expressed in orthologous genes in Arabidopsis,yeast and animals are localized to the mitochondrial inner membrane.In the Arabidopsis study,it was found that the FLO16 orthologous gene deletion mutation is lethal,indicating that this gene plays an important role in the growth and development of plants.Thus,we anticipate that the flo16 mutant may not be a completely deleted mutant,and that a pair of primers spanning the fourth intron revealed that FLO16 was weakly expressed in the flo16 mutant. |
