Functional Analysis Of Rice Yellow Leaf Mutation Gene 5941YS And Genetic Identification Of Premature Leaf Senescence Mutant Pls8255

1.Functional analysis of rice yellow green leaf mutation gene5941ysWhen plants are damaged by high light,in order to protect them from the damage caused by high light,plants form three lines of defense:defense Ⅰ--high light defense,defense Ⅱ--high light protection,and defense ⅡⅠ--PSⅡ damage repair.When the first two lines of defense fail to withstand the damage caused by high light,plants will initiate the third line of defense--PSⅡ repair.PSⅡ repair is mainly completed by PSⅡ disassembly and assembly.The study of the components involved in PSⅡ disassembly and assembly was beneficial to the study of photosynthesis.The yellow leaf mutant 5941ys was obtained by EMS mutagenesis.Based on the preliminary positioning in the laboratory,the gene was cloned in this experiment and relevant functional analysis was conducted.The main research results are as follows:（1）Phenotypic and agronomic analysis:the whole growth period of 5941ys was yellow leaves,and the heading stage was 7 days later than the wild type.Plant height,panicle length and each panicle’s grain number,seed setting rate and 1000-grain weight were significantly lower than wild-type G46B,and the effective panicle number was significantly lower.This indicated that 5941ys yellow leaf mutant had a great influence on agronomic traits.（2）Determination of photosynthetic pigment content:the pigment content of5941ys and G46B in the seedling stage and the pregnant stage was determined.The results showed that the pigment content of 5941ys in the two stages was significantly lower than that of G46B.The total chlorophyll,chlorophyll a,chlorophyll b and carotenoid in the seedling stage were significantly lower than that of the wild type,which was reduced by 53.2%,55.4%,43.5%and 51.7%,respectively.At the booting stage,the components decreased by 36.8%,37.5%,33.9%and 19.6%.This indicated that the phenotype of 5941ys was caused by chlorophyll reduction.（3）Temperature-and light-sensitivity of 5941ys mutant:processing temperature,light incubator(80 including mol.m^-2 s^-1),23℃and 30℃under the condition of planting mutant and wild type respectively,pigment content determination of trilobites period.Ⅰn 23℃after processing mutants of chlorophyll,chlorophyll a and chlorophyll and carotenoid wild type b fell 46.0%,47.9%,36.7%and 27.9%respectively,30℃after processing mutant of chlorophyll,chlorophyll a and chlorophyll b and carotenoids fell by 31.8%,34.2%,22.4%and 20.9%respectively.Light treatment,30℃illumination incubator(including 80 mol.m^-2 s^-1（weak light）,and salween in chengdu in August after an average of 32℃outside（light）treatment pigment determination results showed that under low light mutant chlorophyll,chlorophyll a and chlorophyll b and carotenoids decreased by 31.8%,34.2%,22.4%and 20.9%respectively.Compared with the wild-type chlorophyll,chlorophyll a,chlorophyll b and carotenoids,the mutant decreased by 44.4%,44.8%,43.8%and 12.8%.Chlorophyll content determination after5941ys at cryogenic temperatures lower than normal 30℃,under the strong light pigment content is more than the weak light decline.This indicated that low temperature and strong light had different effects on the chlorophyll content of the mutant.（4）determination of photosynthetic rate:during the rice boot stage,the photosynthetic rate of wild-type and mutant 5941ys swordleaf was measured,and the results showed that the net photosynthetic rate of mutant was significantly reduced by65.0%.Compared with the wild type G46,the intercellular CO₂ concentration of the5941ys mutant was significantly increased by 10%.There was no change in stomatal conductance or transpiration rate.This indicated that the yellowing phenotype of leaves caused the decrease of photosynthetic rate of plants.（5）TEM analysis:the full-leafed leaves of the four-leafed mutant 5941ys and wild-type G46B were observed by tem,and it was found that the wild-type chloroplasts had a complete double-layer membrane structure,and the inner grana thylakoids were arranged neatly.The mutant had a bilayer membrane structure,but the thylakoid in the granule was abnormal,and some vesicular structures were similar to those observed in the preplasmid period.（6）Accumulation of reactive oxygen species:DAB and NBT staining was performed on wild type and mutant treated with different temperature and light intensity.DAB the results found that the mutant under strong light brown spots more than the weak light,brown flecks of 23℃more than 30℃.Mutant leaves in bright light and the accumulation of H₂O₂ under 23℃.NBT staining had two mutant under strong light,weak light brown spots more than 23℃blue spots more than 30℃,Mutant under natural conditions（light and low temperature of 23℃）more conducive to the accumulation of H₂O₂ and O^2-.（7）Complementation experiment:because genetic transformation of indica rice was difficult,5941ys was cross-bred with hareback from Japan for multiple generations to obtain the yellow leaf mutant 7861ys with japonica rice background and 5941ys mutant gene and stable inheritance for transgenic functional complementarity experiment.The expression vector pc2300-actin-5941ys was constructed.Through agrobacterium-mediated transformation,the 5941YS gene in wild-type G46B was transferred into the mutant 7861ys to obtain the transgenic vaccine.The transgenic plants showed normal green color,and the pigment content in the seedling stage and the pregnant stage was determined,and the pigment content also recovered to the wild-type level.This indicated that the phenotype of yellowing was caused by the gene mutation of5941ys.（8）Expression pattern analysis:qRT-PCR quantitative analysis showed that5941YS was expressed in roots and leaves at the seedling stage and roots,stems,leaves,ears and leaf sheaths at the booting stage.The expression level of leaves was higher at the seedling stage and booting stage,while the expression level of roots and ears at the seedling stage was the lowest.（9）qRT-PCR analysis:the four main protein complexes involved in photosynthesis light system Ⅰ,Ⅱ light system,cytochrome b6f and 12 of ATP synthase gene analysis results showed a significant increase in PSⅡ related genes,PSⅠ related gene difference and ATP synthase significantly raised and the base of the ATP and cytochrome b6f related gene significantly lowered.This indicated that 5941ys was a gene closely related to photosynthesis.2.Genetic identification of premature leaf senescence mutant pls8255The aging process of plants occurs in the late growth stage,and aging involves plant hormones,proteins,pigments and other substances.Plant early consenescence will break the material balance inside body,affect material metabolization.And leaf aging in advance will directly or indirectly affect the degradation of chlorophyll,results in the decrease of photosynthetic capacity,resulting in decreased the yield of crops,so the study of the molecular mechanism of rice leaf premature aging,genetic improvement of rice and increase crop yield is of great importance.Pls8255 is a premature leaf senescence isolated from normal rice variety 188R treated by EMS.The mutant of pls8255 was green in vegetative growth stage,and began to appear orange-red in reproductive growth stage.Orange-red develops from old to new leaves and from tip to base.The agronomic traits of pls8255 were investigated.The results showed that plant height,ear length and 1000-seed weight of pls8255 were significantly decreased compared with wild-type 188R,and the seed setting rate was significantly decreased.Genetic analysis showed that pls8255 was controlled by a pair of recessive nuclear genes.Using F₂ hybridized with pls8255 and NP as the locational population,the mutation loci were located between the SSR primer RM6483 and Ⅰn/Del labeled ch3-212 on chromosome 3,and the genetic distances were 5.4cm and 4.0cm,respectively.