Cloning And Functional Analysis Of An Precocious Emerald Gene PE-1 In Rice

Rice（Oryza sativa L.）is one of the world’s major food crops.Heading date is the most important agronomic traits of rice adaptation to environmental changes.It refers to the period of rice that has been experienced from sowing to heading,and it has a decisive influence on the geographical adaptability and seasonality of a variety.Rice has a short sunshine time and is warm and humid.As a typical model crop,its heading stage is affected by genetic factors and environmental factors.In this study,physicalγ-ray mutagenesis was used to treat an indica rice variety shuangkezao,and a mutant pe-1 with precocious emerald phenotype was obtained.Through morphological,physiological and cytological experiments,we explored how the gene PE-1 mutation causes a series of phenotypic changes,such as precocious emerald and so on.Using the map-based cloning technology to locate the PE-1 gene and the mutation site of PE-1 was found by sequencing comparison.On this basis,related experiments such as functional complementarity,subcellular localization and tissue expression analysis were carried out.These lay the foundation for functional analysis of PE-1.PE-1 is a high efficiency gene with short growth period.It has certain significance for guiding breeding practice,improvement of growth period characteristics and variety promotion.The detailed research results are shown as follows:（1）Compared with the wild-type,pe-1 showed obvious phenotypic differences in leaf color at the seedling stage about one week after germination,and the leaf color showed a bright yellow.The number of tillers is roughly the same as the wild-type.However,the leaf color of pe-1 gradually changed from bright yellow to bright green,and the plant height was slightly shorter.It was worth noting that pe-1 heading about18d earlier than the wild-type,and the growth and development of the mutant was significantly faster than the wild-type,and the plant height was significantly lower.At the maturity stage,the plant height,panicle length,primary and secondary branch stem number,grain number per panicle and seed setting rate of pe-1 decreased significantly.（2）During different periods of growth and development of pe-1,the chlorophyll content of the flag leaf of mature stage was determined,and it was found that the chlorophyll a,chlorophyll b and total chlorophyll content of pe-1 showed a significant decrease compared with the wild-type.It was also found that the net photosynthetic rate,stomatal conductance and transpiration rate of mutant pe-1 were significantly higher than wild-type,but there was no significant difference in intercellular CO₂concentration.（3）Transmission electron microscopy（TEM）of the leaves showed that the chloroplast development of the mutant pe-1 was partially defective.The chloroplast morphology of pe-1 was smaller than that of the wild-type,and there was no obvious difference in thylakoid arrangement.The starch granules accumulated abnormally,the volume increased,and the number of osmiophilic granules decreased sharply.（4）The pollen was stained with FDA-PI fluorescence to explore whether pe-1fertility was affected.The results showed that the pollen activity and fertility of pe-1were decreased.Leaf histochemical analysis showed that more hydrogen peroxide and oxygen anions were found in pe-1 leaves compared to wild-type leaves.（5）The results of exogenous hormone treatment of seedlings showed that SA,ABA and MeJA treatments were not sensitive to the plant height and root length of mutant pe-1.The expression patterns of genes related to heading date and chlorophyll metabolism pathway in flag leaves at tillering stage were detected by qRT-PCR quantitative analysis.Compared with the wild-type,the expression of FCA and DTH2in the leaves of pe-1 decreased.The expression of chlorophyll metabolism related gene CAO1 was higher than that of wild-type.In particular,the pale-green leaf gene FGL and the gene lvp1,FT which promote early heading were significantly higher in the leaves of pe-1 than in the wild-type.（6）Genetic analysis indicated that the precocious emerald mutant pe-1 was controlled by a single recessive nuclear gene,and PE-1 was located between the Indel marker M2 and M5 on rice chromosome 6 with a physical distance of 37.9 kb,using the method of map-based cloning.Bioinformatics was used to predict the candidate genes in this region.The results showed that the region contained 5 open reading frame（ORF）.Through sequencing analysis,it was found that only one gene（LOC_Os06g40080）was mutated,and the 580-bit nucleotide C deletion in the coding region resulted in the change of the encoded amino acid,and the termination codon was advanced.This gene encodes a heme oxygenase gene（Se5）.The gene is predicted to be a candidate gene.It is indicated that pe-1 is a allele of cloned Se5.（7）Genetic complementation experiment to confirm that the PE-1 mutation is the cause of the mutant pe-1 phenotype.Using the PE-1 knock-out CRISPR/Cas9vector,it was found that the heading date of T₀ plant was earlier than that of the empty vector.The endogenous expression level of PE-1 was detected using a transgenic plant carrying a PE-1 promoter-GUS fusion construct.The results showed that GUS signals were detected in roots,culms,leaves and young panicles.In addition,by observing the localization of green fluorescence in the infected tobacco leaves,the protein of PE-1 was localized to the chloroplast.