The Function Of Rice Bsl Gene And Its Application In Mixed-planting Seed Production Of Hybrid Rice

Bentazon is used as a benzothiadiazole herbicide with high selectivity by an inhibition of photophythesis for killing sensitive plant.In general,common rice varieties(wild types) are all tolerant to bentazon,which can metabolize bentazon into 6-OH or 8-OH non-toxic molecules.Bentazon sensitive lethal(bsl) mutants were obtained byγ-ray radiation from wild rice varieties('N8' and 'W6154s').Previous genetic studies have confirmed that the bsl phenotype of is controlled by a single recessive gene.The bsl gene,as selective chemical marker,has a critical usage in seed production of hybrid rice(Oryza sativa.L).In hybrid breeding system,tagging the bsl gene to the male sterile line can ensure the hybrid seeds free of false hybrids(self-pollination of sterile lines) by spraying bentazon at the seedling stage.On the other hand,if the bsl gene is transferred into a restorer line,the restorer line is easily killed by spraying bentazon immediately after pollination,while the sterile line maintains normal growth and development.In order to clarify the function of bsl gene,and provide sufficient evidance for its application in rice breeding,we researched the physiological basis on the difference of bentazon tolerance and rice microsomal proteomics in wild type rice and their bsl mutants.In addition,the bentazon influence on the pollen viability of sensitive restorer line,the relation between bentazon lethal dosage and environmental factor,and the bentazon residues in mixed-planting seed production were studied to further perfect the technology of hybrid rice mixed-planting seed production and accelerate its application.Firstly,this study was to elucidate the physiological mechanism of bentazon tolerance in rice.Tolerant rice varieties(W6154S and Norin 8) as well as their corresponding mutants,sensitive to bentazon,were employed in this study.Plant net photosynthetic rate (P_n),chlorophyll fluorescence,and the level of superoxide radical(O₂^·) as well as the contents of chlorophyll(Chl) and malondialdehyde(MDA),were analyzed for both tolerant and sensitive rice plants treated with bentazon.After treatment,the two sensitive mutants showed a significant reduction in P_n at 0.5 h.A continuous decrease of Chl contents was found over the first 3 d whereas a significant increase of MDA contents was noticed on the 3rd day and thereafter.Analysis of chlorophyll fluorescence revealed a bentazon-induced increase in the proportion of the reduced state of Q_A.In the early stage after bentazon treatment,wild types and their mutants showed no significant difference in the alteration of P_n and chlorophyll fluorescence.While these two parameters then increased progressively in both wild types and remained low in the mutants.A significant generation of O₂^·was found over the 5 d period in the mutants.Both wild types and mutants contained the same level of bentazon after 2 h of treatment.Bentazon content dropped to barely detectable amount in the wild type varieties at 1 d.However,the mutants retained a substantial amount of the herbicide after 5 d.It is proposed that the herbicide might inhibit rice photophythesis and accumulation of oxidative stress with the treatment of bentazon in both lethal mutants.The damaging effect on PSâ…¡system can be significantly alleviated in the wild type varieties due to a higher rate of catabolism of the herbicide.Secondly,we established a modified two-dimensional gel electrophoresis(2-DE) protocol suitable for rice microsomal proteins by using differential centrifugation in sample preparation,optimizing length and pH range of immobilized pH gradient(IPG) strips,and modifying the electrophoresis program.In order to search bentazon tolerance-related proteins,we used the 2-DE technique to detect the change of rice microsomal proteins expression induced by bentazon in N8 as well as its bentazon sensitive lethal mutant N8m. Results showed that 15 differentially expressed protein spots were found after bentazon treatment between or within the two lines.The proteins in these spots were classified into three types.Typeâ… proteins showed differential expression between the two lines while typeâ…¡proteins differed between the treated and untreated samples,and typeâ…¢proteins were only induced in N8 after the treatment.Typeâ… proteins arise from rice DNA damage byγ-ray radiation,based on the fact that N8m is a new strain from N8 mutated byγ-ray. Our previous study indicated that N8 and N8m showed no significant difference in the alteration of photosynthesis in the early stage after bentazon treatment,which induced accumulation of oxidative stress.It can be confirmed that typeâ…¡proteins are related to the reactive oxygen scavenging system or systemic acquired resistance.N8 could metabolize the absorbed bentazon thoroughly while residual bentazon was present in the treated leaves of N8m.Therefore,we predict the typeâ…¢proteins to be related to bentazon metabolism.Thirdly,A culture solution for rice's pollen germination in vitro was established through Studying on the concentration of sucrose,boric acid and calcium chloride.The influence of bentazon treatment on the pollen viability of Mc526,which included bsl gene, was researched by using pollen bourgeoned and I₂-KI pigmentation's measurement.The results shown that in early period(1～4 days) after bentazon treatment Mc526's pollen viability was close to comparison and in later period(5～8 days) Mc526's pollen viability was a bit lower than comparison,but the difference was not salience.The setting rate results showed that bentazon treatment had no salient influence on the setting rate of sterile line.This indicates that bentazon does not effect on bsl rice's pollen viability immediately, which provides the physiological basis for the feasibility of applying the method of mixed-planting seeds production in hybridize rice.In addition,this study investigated the differences of bentazon injure at different light intensities and temperatures in order to provide theoretical basis for properly regulating bentazon dosage in hybridize rice seeds production.'Mc526' was employed in this study. The research of the influence of temperature on bentazon injure indicated that there was significant positive correlation between temperature and bentazon injure degree in the range of 21℃～36℃.As for 1℃increase of temperature,the lethal concentration of bentazon declined 74.09 mg·L^-1,namely bentazon injure degree increased 0.7%.Related physiological research showed that the increases of water stress and Oxygen free radical stress were the main cause for the increase of bentazon injures in high temperature.The same results were obtained from the field experiments.The research of the influence of light intensity on bentazon injure indicated that bentazon injures in strong light(100μmol·m^-2·s^-1) or weak light(25μmol·m^-2·s^-1) were significantly increased compared with bentazon injure in medium light(50μmol·m^-2·s^-1).Related physiological research showed that the increases of Oxygen free radical stress was the main cause for the increasing bentazon injure degree in strong light.Finally,a reversed phase HPLC method was determined for analyzing bentazon and its metabolites—6-hydroxy-bentazon and 8-hydroxy-bentazon in this study.We used the HPLC method to detect the bentazon residue and degradeation behaveior in the field of hybrid rice maxed-planting seed production,in order to scientifically evaluate the possibility of environmental threat induced by the application of hybrid rice maxed-planting seed production.The results showed that the herbicide residues in the grain and plant of sterile line were lower than the minimum detectable concentrateion. However,there were more residues in the grain and plant of restorer line.The analysis of bentazon degradeation behaveior indicated that bentazon in soil could be rapidly degraded.