Cloning And Functional Analysis Of A Photosynthesis-related Gene C6635 In Rice

Photosynthesis is one of the most important chemical reactions on Earth and can be divided into light reaction and dark reaction?carbon fixation?.The Calvin cycle is the only biochemical pathway used for CO₂ fixation in plants.In C₃ plants,it is completed by 13enzymatic reactions catalyzed by 11 enzymes in the chloroplast.Since the Calvin cycle plays an irreplaceable role in plant metabolism and crop yield,it is important to study the relevant genes in the Calvin cycle pathway and play an important role in crop breeding.In this study,chemically mutagenized EMS was used to treat japonica rice japonica cultivar Zhonghua 11,and a stably inherited rice less tiller mutant c6635 was obtained.Based on the previous gene mapping,genetic analysis,gene cloning and functional analysis of the mutant were performed.The main results are as follows.?1?The phenotype observation and analysis of agronomic traits:Mutant c6635showed a pale green leaves four weeks before growth,and then gradually turned green.The leaves at booting stage returned to normal green.In the whole growth period,the growth was delayed compared to the wild type,and the heading date was delayed by about15 days.The Plant height,number of tillers,grain number of main stems and seed setting rate are significantly reduced,which were reduced by 27.4%,72.7%,41.8%,and 35.7%,respectively.main panicle length and 1000-grain weight also decreased,but there was no significant difference.It was suggested that the c6635 mutation had a great influence on the main agronomic traits of plant growth,especially the number of tillers.?2?Determination of photosynthetic pigment cosntent:compared to its wild type,the c6635 total chlorophyll content,Ch1a,Ch1b,and carotenoids in the seedling stage decreased by 36.45%,35.54%,3.24%,and 17.14%,respectively.With the growth of the plant to the heading stage,c6635 mutant leaf was observed.The color gradually deepened,and the photosynthetic pigments also increased compared with the seedling stage,but the content of the pigments in the wild type was still relatively decreased compared to the wild type pigments.This indicates that the leaves of the c6635 mutant appear pale green at the seedling stage,which is caused by the reduction of chloroplast pigment synthesis.?3?Ultrastructural Observation of Chloroplasts:Compared to wild-type chloroplasts,the c6635 mutant has fewer lamellistone stacks in the chloroplast than the wild type,indicating that the c6635 gene mutation affects chloroplast development.?4?Candidate gene selection:The mutated gene was located on the rice chromosome4 in the early stage of the laboratory,with a physical distance of 5198 kb.Subsequently,high-throughput sequencing and Mut Map methods were used for gene mapping and selection of candidate genes.As a result,it was found that the base sequence of position2143?corresponding to c DNA sequence 1091?of a candidate gene was changed from G to A,resulting in the 364th amino acid of the encoded gene.Glycine?G?is mutated to aspartic acid?D?.The gene is located in the H4 and H5 mapping regions.The encoded protein participates in the Calvin cycle,and the sequence is confirmed by re-sequencing.It is hypothesized that the mutation may be related to a less productive mutant,temporarily named c6635?5?Verification of functional complementation of the transgene:construction of the transgenic expression vector p C2300-Actin-C6635.The wild-type gene C6635 was transformed into the mutant by Agrobacterium-mediated transformation.The number of tillers obtained from the positive transgenic plants was restored to the wild-type level,suggesting that the transgenic mice had fewer tiller mutations.The trait of the body is caused by a C6635 gene mutation.?6?Measuring the Photosynthetic parameters:Mutant c6635 showed a 46%reduction in net photosynthetic rate?P_n?compared to wild-type ZH11,reaching extremely significant levels,with stomatal conductance?G_s?and transpiration rate?T_r?decreasing by 19.23%and 13.67%,respectively.Significant levels were reached,and intercellular CO₂concentration?C_i?was significantly higher,increasing by 14.35%.The photosynthetic parameters of the transgenic plants basically recovered to wild-type levels,indicating that c6635 had a greater impact on plant photosynthesis.?7?Starch potassium iodide staining:The seedling plants were stained with 2%potassium iodide solution.The results showed that wild-type ZH11 staining was deeper than the mutant c6635,indicating that the synthesis of starch in the c6635 mutant was inhibited.?8?Subcellular localization:The p C2300-35S-e GFP expression vector was constructed and transiently expressed in rice protoplasts.Confocal laser scanning microscopy revealed that the protein encoded by the C6635 gene was located on the chloroplast.?9?Tissue expression analysis:RT-PCR analysis showed that C6635 genes were highly expressed in leaves at the seedling and booting stages,but only in trace amounts in stems,leaf sheaths,and panicles at the booting stage,but in the roots at seedling and booting stages.No expression was found,indicating that the C6635 gene was only expressed in plant green tissues.?10?Real-time fluorescence quantitative PCR analysis:Analysis of the expression of six genes in the Calvin cycle pathway showed that the expression levels of rbc L and SBPASE were higher in the mutant than in the wild type,and the expression levels of rbc S,FBP,FBA,and PRK were lower than in the wild.Type,this may be due to their different roles in the Calvin cycle,rbc L and SBPASE are rate-limiting enzyme genes,although rbc S is small subunit gene of Rubisco,but the main catalytic role is the large subunit rbc L,The decline in the expression of several other genes may be due to the downstream or upstream of the C6635 gene,so C6635 directly or indirectly suppresses their expression.