| The transport of photosynthesis products plays an important role in the growth of plants and the increase of grain yield.It has been reported that sugar transporters,intercellular hyphae and sieve molecular in vascular plants play an important role in the loading and unloading of sugars,.In this paper,we screened two rice mutants with abnormal starch accumulation in the leaves,sem1-1(starch excess mutants 1)and sem1-2.We obtained a gene encoding a callose synthetase by map based cloning.In order to further verify the correctness of the target gene,we let the full-length CDS sequence transfer into sem1-1 mutant plant and drived by actin promoter to produce transgenic complementary plant line,then we select the positive transgenic plants by using the methods of resistance callus screening and PCR identification.Through the observation of the phenotype of the transgenic plants and the statistical analysis of the agronomic characters,we found that the complementary lines can completely restore the wild phenotype.In order to further study the gene function,we carried out potassium iodide staining and transmission electron microscopy experiment at 6:00 a.m.,12:00 p.m.and 18:00 p.m.,respectively,to refine the starch accumulation phenotype of the mutant sem1-1 and sem1-2.It was found that the starch content in the mesophyll cells of the mutant sem1-1 and sem1-2 was significantly higher than that of the wild type The content of soluble glucose,fructose,sucrose and maltose in the wild type and mutant sem1-1 were measured.It was found that the content of soluble sugar in the functional leaves which could carry out photosynthesis was much higher than that in the wild type,but in the new leaves which did not grow out and photosynthesis,the content of soluble sugar in the mutant was lower than that in the wild type,which indicated that the process of soluble sugar transport impedient in the mutant.In order to further confirm whether there are obstacles in sugar transport process,we used CFDA fluorescent dye to treat the tip of three-leaf stage seedlings,and let the plants carry out normal photosynthesis under light,so as to judge whether there are obstacles in the pathway of sugar transport of mutants by the movement of fluorescent dye.After 12 hours of treatment,we found that the fluorescent dye in wild type can be transported to the leaf sheath and leaf primordium normally However,it is difficμlt for the mutant to detect the fluorescence signal in these two tissues,which indicates that the transport of dye in the plant is blocked.Through the sequence analysis of the target gene,we know that the gene SEM1 has ten members in the same family of rice,all of which are transmembrane proteins with multiple transmembrane domains.The protein SEM1 contains 13 transmembrane domains.This gene has high homology with AtGSL7 and AtGSL11 in Arabidopsis.The subcellular localization of SEM1 protein was carried out by injecting tobacco and the protein was further confirmed to be located on the cytoplasmic membrane.The results of quantitative PCR analysis showed that the gene was expressed in many tissues in the two stages of rice seedling and maturity,but the relative expression was high in the leaves of seedling and the leaf sheath of maturity.It was known that the gene SEM1 could be expressed in the leaf primordium of seedling by in situ hybridization.Callose is a kind of polysaccharide,which can protect and support the plant cell structure.As a callose synthetase,the mainly function of SEM1 protein is to control the synthesis of callose.In order to study the change of the content of callose in mutant sem1,we carried out immunofluorescence and immunogold labeling experiments,and found the callose on the cell wall of leaf and stem vascular bundle cells decreased obviously in mutant sem1,which led to the relative decrease of vascular bundle cell volume due to the lack of support.This is one of the reasons for the transportation obstacle of photosynthetic products.Vacuole is a unique organelle in plant cells,which can temporarily store sugar in the process of sugar transport,and in vascular plant phloem parenchyma cells there are larger vacuoles,which can continuously supply sugar as a large container for carbohydrate storage during the long-distance transport of photosynthetic products.Through the observation and analysis of the leaf anatomical structure of wild-type and sem1-1/2 mutants,we found that the number of phloem cells in the leaves of the mutant sem1-1/2 decreased in the mature stage of flag leaves,which made it difficult for photosynthetic products to be transported normally due to the lack of buffering vacuoles in the long-distance transportation process,which was the second reason for the transportation obstacle of photosynthetic products.At the same time,RNA was extracted from the growth site and sequenced by RNA-seq.It was found that the relative expression of cyclin genes involved in cell mitosis in the mutant sem1 was significantly lower than that in the wild type.We further verified the experimental results by qPCR and in situ hybridization,which may be the main reason for the decrease of phloem cell number.In order to further refine the phenotype of phloem structure,we observed the paraffin section and transmission electron microscope section of wild-type and mutant plant leaves and found that the cell walls of the thin-walled phloem cells and sieve element cells of the mutant sem1 / 2 were slightly thickened,and these positions were the key cell structures for the transportation of photosynthate,and the thickened cell walls would further increase the photosynthate between cells The difficulty of transportation,which is the third reason for the transportation obstacle of photosynthetic products.These results indicate that sem1,the gene encoding callose synthetase,plays an important role in the transportation of rice photosynthetic products... |
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