Phenotypic Identification And Gene Mapping Of A High-temperature-sausing-death Mutant Htcd1 In Rice(Oryza Sativa L.)

Temperature-sensitive mutant is a kind of common,phenotypic mutant,and it usually present characters from seedling stage in rice（Oryza sativa L.）.In recent years,extensive and in-depth studies have been carried out on temperature-sensitive mutants in rice,but few studies have been conducted on the high-temperature-causing-death phenotype,and its physiological mechanism and regulatory pathways remain need to be further explored.Mutant htcd1 is a stable genetic high-temperature-casing-death mutant,which was sensitive to high temperature,and it was chemically induced from the indica rice maintainer line Xinong 1B by ethyl methyl sulfonate（EMS）.This paper compared mutant htcd1 wih wild type rice Xinong 1B,doing some experements such as sowing at different times,and then investigate the morphological identification,agronomic traits,temperature gradient treatment and observation analysis,chemical analysis,physiological and biochemical index determination,determination of related parameters of photosynthetic and analysis,transmission electron microscope,the chloroplast fluorescence microscopy and researchs,and also fine mapping and locking the gene that controlling the mutant traits by using molecular markers and mutation sequencing.The main research results are as follows:1.Observation of phenotypic characteristicsThe mutant htcd1 seeded in early stage could complete the growth,development,seed bearing and other growth processes due to more suitable temperature conditions.The late seeding mutant htcd1 was affected to different degrees due to different growth periods when exposed to high temperature.The later the sowing was,the more severely affected by high temperature and the earlier the phenotype appeared,even unable to grow and develop normally.In particular,the mutant htcd1 could germinate into seedlings as well as the wild type in the late high temperature,but it could not grow and develop normally after the second-leaf stage.And the growth stagnated under continuous high temperature,and quickly yellowed to death.The heat-sensitive phenotype characteristics of the mutant htcd1 under continuous high temperature condition were as follows:Plant follow the upward leaf sequence successive yellowing of the far leaves,most of the leaves yellowing with death,until the whole plant died.2.Investigation of main agronomic traitsThe mutant htcd1 seeded in the early stage was able to grow without being seriously affected by continuous high temperature.There was no significant difference between the number of tillers per plant and the number of effective panicles per plant and the wild type.but its plant height,panicle length,number of grains per panicle,seed setting rate,1000-grain weight and yield per plant were all significantly or highly significantly lower than that of wild type.The mutant htcd1 seeded in the late stage was seriously affected by the continuous high temperature.And the later the sowing date was,the more serious the decline in plant height and tillering power per plant was,and it was unable to produce normal fruit or grouting,even unable to booting.3.Temperature treatment seedling stageThe results of temperature gradient processing experiment during seedling stage showed that the mutant htcd1 grown slowly than wild type in low temperature（18℃）.Under the suitable temperature（20～30℃）,it grown and developed normally like the wild type.But at high temperatures（32～42℃）,it grown slowly compared with wild type and even died,and that’s closely related to the high temperature and processing time.Those results indicated that the high-temperature-tolerence value of htcd1 was about 32℃.4.Histochemical analysisTrypan blue staining showed that wild-type rice leaves were not stained with dark blue color,while the yellowing and dead parts of mutant htcd1 leaves were stained with dark blue color.DAB staining results showed that there was no brown residue in wild-type rice leaves,while the yellowing and dead parts of mutant htcd1 leaves all retained tan deposits.These results demonstrated that the cells of yellowing and dead htcd1 leaves was dead and the accumulation of H₂O₂ were more than wide type under high temperature stress.5.Determination and analysis of physiological and biochemical indicatorsThe activity of antioxidant enzymes such as peroxidase,catalase and total superoxide dismutase in the leaves of the mutant htcd1 was highly significantly lower than that of the wild type,and the content of reactive oxygen species such as hydrogen peroxide,hydroxyl radical and superoxide anion in the leaves was highly significantly higher than that of the wild type.Therefore,it is speculated that the decrease of antioxidant enzyme activity in the leaves of mutant htcd1 leads to the decrease of its ability to remove reactive oxygen species under high temperature,which leads to the accumulation of reactive oxygen species.Malondialdehyde content and free proline content in htcd1 were significantly and highly significantly higher than that in wild type under high temperature stress respectively,indicating that high temperature cause membrane lipid peroxidation and related protein degradation of mutant htcd1.6.Determination of photosynthetic pigment content and comparative analysis of photosynthetic efficiency parametersThe contents of chlorophyll a,chlorophyll b,carotenoids and the ratio of chlorophyll a to chlorophyll b of the mutant htcd1 at each growth stage（tillering stage,heading stage,grain-filling stage,and maturation stage）were all significantly or highly significantly lower than those of the wild type under the same high temperature stress.Through the analysis of photosynthetic characteristics at different growth stages（tillering stage,heading stage and grouting stage）,it was found that the net photosynthetic rate,stomatal conductance and transpiration rate of leaves of the mutant htcd1 were all highly significently lower than those of the wild-type under the same high temperature stress.7.Observation of chloroplast spontaneous fluorescenceObservation of frozen section showed that in natural light,the wild-type cross section was uniformly dark green,while the mutant htcd1 showed a lighter green color.Fluorescence microscopy showed that the red fluorescence of leave transsection mutant htcd1 was obviously dimmer than that of the wild-type,which indicated that chlorophyll degradation or chlorophyll destruction occurred in the mesophyll part of mutant htcd1under high temperature stress.8.Ultrastructure observation of mesophyll cellsUltrastructure observation of mesophyll cells of wild-type and mutant htcd1 under high temperature stress by transmission electron microscopy,the results shows that the thylakoid lamellae in the chloroplast were arranged neatly and closely,and there were a few small osmiophilic and starch granules.However,the chloroplast structure in mutant htcd1 mesophyll cells was seriously abnormal,and the intact thylakoid lamella and starch granules could not be observed.Also,the number of osmiophilic granules was large and the size was large.That directly indicated that the high-temperature environment destroyed the chloroplast structure in mutant htcd1 mesophyll cells.9.Genetic analysis and gene mappingGenetic analysis result indicated that the phenotype of htcd1 was controlled by a pair of recessive nuclear genes.The mutated gene htcd1 was located in an interval of about 359Kb at the end of the long arm of chromosome 9 by molecular localization.10.Mutation sequencing and gene lockingThe candidate gene analysis and mutation sequencing results showed that the mutant gene was LOC＿Os09g29630,which was a new gene that had not been cloned.That mutation was the replacement mutation from T to A of the second base of the 6th intron in the gene coding region.Due to the mutation of that site,the splicing process of the post-transcriptional m RNA of this gene may be affected,which in turn affected the expression of related proteins and made them lose their resistance to high temperature and adversity.