| 1.Gene Cloning of Apical Panicle Extension Mutant 8358Mutant is one of the fastest means of gene discovery.We used indica rice variety188 R as the experimental mutagenesis parent and treated with chemical mutagen(ethyl methylsulfonate).At the heading stage of M2 generation,we found a mutant8358 with panicle elongation.After many generations of self-pollination,we found that this panicle elongation trait could be inherited steadily.Therefore,The panicle elongation mutant was used as the experimental material to study the panicle type development and differentiation of rice.The genetic mode,physical location and gene function of 8358 mutant were studied by genetic analysis,gene map cloning,candidate gene screening and functional complementary verification of the mutant.The main results were as follows:(1)Observation of mutant phenotype and analysis of Agronomic traits:Compared with wild type 188 R,the phenotype at heading stage was branch elongation at the top of panicle.The results of field and indoor agronomic traits showed that the effective panicle,seed setting rate,1000-grain weight and plant height of the mutant decreased by 10.5%,1.4%,13.4% and 11.2% respectively,while the panicle length and the number of primary branches increased by 7.0% and 4.1%respectively,and the number of secondary branches did not change significantly in statistics.According to the results of agronomic traits investigation,we believe that8358 gene is a Rice Panicle Development related gene,and related to the primary branch development of rice.(2)Observation and electron microscopic scanning of young panicles of wild type and mutant: Compared with young panicles of mutant,the spike tip of mutant8358 was significantly elongated.Electron microscopic scanning of young panicles at the same developmental stage showed that the primary branch of the barley tip of the mutant was obviously elongated.(3)Genetic analysis and gene mapping of mutants: By constructing genetic analysis population and statistical investigation,genetic analysis and chi-square test showed that the 8358 mutant trait was controlled by a pair of recessive nuclear genes.The candidate genes were initially located between X1 and RM303 on chromosome 4by SSR primers and In Del primers,with genetic distances of 7.4 c M and 10.6 c M,respectively.Finely located between In Del primers X2 and X3,the physical distance between the two primers was 280 kb,and the genetic distance between the two primers and candidate genes was 2.2c M and 0.6c M,respectively.(4)Screening and validation of candidate genes: Through high-throughput sequencing results and prediction screening in rice gene annotation database,and then through PCR sequencing verification,the full length of DNA sequence of candidate genes was 1869 bp,the base 104 of the whole genome was mutated from G to A,resulting in 35 amino acids from C mutation Y,amino acids from cysteine mutation to tyrosine,the gene had no intron,and the full length of CDS was 1869 bp.It encodes a protein containing 623 amino acid sequences.(5)Complementary verification of transgenic function and gene knockout verification: In order to study the function of genes,we constructed a superexpression vector p C2300-8358-actin,and transferred it into mutant plants by Agrobacterium-mediated method.The obtained positive transgenic plants were observed and counted after heading stage of rice,and all the positive plants showed normal panicles.At the same time,Nipponare was used as an experiment.Material,CRISPR / Cas9 technology was used to knock out candidate genes.It was found that the positive plants in knockout plants showed barley tip elongation.(6)Tissue expression analysis: Roots,leaf sheaths,stems,leaves and young spikes at booting stage of wild type were selected as semi-quantitative analysis objects.The results showed that 8358 gene was mainly expressed in young panicles of rice,but hardly expressed in roots.Real-time fluorescence quantitative analysis of rice tissues was carried out at seedling stage,tillering stage and booting stage.The results showed that the expression of 8358 gene was very high at booting stage,very low in roots at seedling stage and booting stage,and only a small amount in leaves and sheaths at booting stage.2.Genetic analysis and gene mapping of rice narrow-leaf mutant8316As an important organ of plant photosynthesis,rice leaf is the production site of photosynthate of green plants.It plays a "source" role in plant "source,flow,sink" and plays a decisive role in the final yield of food crops.The restorer line 188 R of indica rice was treated with chemical mutagen Ethyl Methylsulfonate(EMS).A mutant material with narrower leaf shape was found in its M2 generation.The mutant material was self-bred for many generations.It was found that this narrow leaf trait could be inherited stably.We named it 8316 temporarily.The agronomic traits of 8316 mutant were observed,analyzed and the candidate base was analyzed.Because of preliminary positioning.The main results are as follows:(1)Phenotypic observation and Agronomic Traits Analysis of 8316 mutant:Compared with wild type,we can find that the leaves of 8316 mutant obviously narrowed and straightened from tillering stage.Compared with the wild type,the plant height became shorter,the grain became thinner and longer,the seed setting rate and 1000-grain weight decreased significantly,but the number of effective panicles increased.(2)Genetic analysis and gene mapping of mutants: Using 188 R cross between mutants and wild-type parents,F1 represents normal plants.The number of wild-type and mutant plants in F2 generation was investigated and analyzed.The results showed that the ratio of normal and mutant plants in F2 generation was close to 3:1.According to Mendel’s law of gene segregation,8316 mutant trait is controlled by nuclear single gene of recessive trait.The F2 population was constructed by crossing 8316 with Nippon Sunshine.The candidate genes were eventually located between the primers H2 and H3 with genetic distance of 1.74 c M and 2.32 c M and physical distance of738 kb. |
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