Identification And Candidate Gene Analysis Of Yellow-green Leaf Mutant Ygl15 In Rice(Oryza Sativa)

Oryza sativa L.leaf color mutation is a common,easy-to-discover and early-onset mutant trait.In recent years,extensive researches have been carried out on rice leaf color mutations,especially on photosynthesis mechanisms,chlorophyll biosynthetic pathways,development of chloroplasts,and genetic control mechanisms.In this study,we obtained a stable genetic yellow leaf mutant ygl15 from the mutagene sis library produced by the excellent retention of rice at Southwest University by using EMS?Ethyl sulfate sulfonate?.In this study,the wild-type Xinong 1B was used as a control to explore the phenotypic characteristics,major agronomic traits,photosynthetic pigment content,photosynthetic characteristic parameters and physiological and biochemical indicators of the ygl15 mutant,fluorescence microscope observation of chloroplasts,and quantitative expression of related leaf color genes.Using the mutant ygl15 and Zhonghua 11 hybrid populations for genetic analysis and molecular markers,as well as gene map cloning.The main results are as follows:1.Phenotypic identification and agronomic traits.The mutant ygl15 began to appear phenotype at the two leaf stage after sowing,and the leaves showed yellowish green,and the phenotype continued until maturity.For the main agronomic traits,compared with the wild type,the plant height,total grain number per panicle,number of grains per panicle and 1000-grain weight of the mutant were significantly reduced,the panicle length was significantly reduced,but the number of effective panicles between the wide-tipe and the mutants were not far apart and did not reach significant level.2.Determination of Pigment Content in Photosynthesis.The contents of related pigments in the wild-type and mutant ygl15 leaves at the seedling and heading stages were determined.Compared with the wild type,the content of chlorophyll a,chlorophyll b,carotenoids and total chlorophyll in the mutant ygl15was significantly reduced at the seedling stage,while the content of chlorophyll a,carotenoids and total chlorophyll in the heading stage was at the inverted leaf pole.Significantly decreased,but the difference of chlorophyll b was not significant;the content of chlorophyll in the second leaf declined significantly;in the inverted trifoliate,the content of chlorophyll b was significantly reduced except that the chlorophyll b was significantly reduced.Although the chlorophyll content increased after the tillering period,the phenotype did not change significantly.3.Analysis of photosynthetic parametersThe net photosynthetic rate,stomatal conductance,intercellular CO₂ concentration and transpiration rate of the wild type and mutant ygl15 were measured in the heading period.Consequents display that contrasted with wild type,the stomatal conductance,net photosynthetic rate,intercellular CO₂ concentration of ygl15 had no obvious difference with the wide type.but stomatal conductance,intercellular CO₂concentration increased,and the transpiration rate increased significantly;Because of the increase of stomatal conductance,the transpiration rate is accelerated,and the CO₂in the air is more easily absorbed,thus increasing the concentration of intercellular CO₂.4.Frozen section observation.The leaf of wild-type and the leaf of mutant ygl15 were prepared in the childbirth period to make frozen slices.Under the fluorescence microscope,the observation showed that the chloroplast of mutant ygl15 is weaker than wild-type in natural light,and the resulting red fluorescence was weaker than wild-type,which indicated that the chlorophyll was degraded in the mutant.5.Genetic analysis.The F₁ phenotype of Zhonghua11 and mutant ygl15 was the same as that of wild-type west nong 1B.The phenotypic separation occurred in F₁ from the F₂ generation,the normal single plant number was 2202,and the number of single plants with yellow green leaf phenotype was 518.After the chi-square test,it was consistent with Mendelian separation ratio 3:1(c²=1.20<c²_?0.05,1?=3.84),this shows that a pair of recessive genes control the phenotype of the ygl15 mutant.6.Determination of physiological and biochemical indexes.The physiological indexes of wild-type and mutant ygl15 were detected during heading stage.The results showed that in the determination of hydroxyl radical?·OH?,superoxide anion?O₂^-?and hydrogen peroxide?H₂O₂?content,the content of·OH in the ygl15 mutant was inverted,and that of inverted leaves was significantly lower than that of wild type.However,the difference of the inverted trifoliate leaf was not significant;the content of O₂^-was significantly higher than that of the wild type in the first,second and last three leaves;the content of H₂O₂ was higher than that of the wild-type one-,two-,and three-leaf clover.The two leaves reached a significant difference level,while the inverted one and the inverted three leaves had no significant difference.In the determination of total superoxide-oxidase?T-SOD?,peroxidase?POD?,and catalase?CAT?activity,the T-SOD of the ygl15 mutant was inverted,inverted,and inverted.The enzyme activity was significantly lower than that of the wild type;the activity of POD enzyme was lower than that of the wild type,and the difference between the inverted and inverted trefoil was extremely significant,but the difference between the two was not significant;the CAT enzyme activity was also lower than that of the wild type,and the difference between the three leaves was significant,while the difference between the first and the second leaves was not significant.7.Localization and analysis of genes.By molecular markers of mutants.The mutant ygl15 was located in the region between RM25654 and r3 of the 10th chromosome of rice.The genetic distance between the two molecular markers were 0.38cM and 0.57cM,physical distance is about 56 KB.Ten annotated genes were found in the region,and sequencing analysis revealed that the candidate gene in the region:the 121st base of the first intron?total length of 138bp?in the coding region of LOC_Os10g35370 was mutated from A to T.Because the mutation of this site may affect the splicing process of mRNA after transcription of this gene,we preliminarily speculated that ygl15 might be the candidate target gene?8.Gene expression analysis.Quantitative Real-time PCR was used to analyze the photosynthetic pathway related genes?petA,petB,petD,psaA,psbA,Cab1R?and chlorophyll metabolism pathways of wild-type Xinong 1B and mutant ygl15.Genes?HEMD,HEMA1,CHLD,HEME,CAO1?and genes that regulate carotenoid metabolic pathway genes?PSY1 and PSY3?are expressed at the time of delivery.The results showed that the expression level of the mutant ygl15 was extremely significantly lower than that of the wild type.