Studies On The Screening Of An Al-Sensitive Rice Mutant, Identifying The Gene And The Mechanism Of Antioxidant Responses To Aluminum Stress

Aluminum(M) toxicity is considered as one of the primary causes of low rice productivity on acid upland and lowland sulfate soils.In this study,we chose 1635 isolated rice(Oryza Sativa L.Subsp,Japonica,CV.Nipponbare) mutants as material from the T-DNA inserted the mutant system derived from gene transformation mediated via Agrobacterium tumefacieas.We isolated an aluminum-sensitive mutant by T-DNA insertion in rice through screening in water cultivation.We primarily analyzed this mutant and explored the sensitive mechanism in rice.The results we obtained should be useful for cloning the target Gene and knowing the sensitive mechanism.The main conclusions are as follows:(1)Isolation and primary characterization of a T-DNA insertion rice mutant sensitive to AlBy screening 1635 of enhancer trap lines in rice,we isolated a T-DNA insertion mutant-T9610 which was sensitive to A1 toxicity.At 50μM Al³⁺(pH 4.5),root elongation of Nipponbare was inhibited by 10%for 24h,while that of the aluminum sensitive mutant was inhibited by 52%.Genetic analysis of the mutant showed that the phenotype of relative root elongation in the segregating populations derived from T-DNA heterozygotes fit the ratio of 3:1.Test for hygromycin resistance and using PCR method,the aluminum sensitivity plants were resistant and the ratio of resistant and susceptible plants was nearly 3:1,which indicated that the aluminum sensitive:mutant was co-segregated with hygromicin resistant.By southern blots,T-DNA was single-copy inserted in this mutant.With PCR-walking,we could rescue its flanking rice sequences,and the results indicated that the flanking sequence showed 100%identity at the nucleotide level to the sequence of clone OSJNBa0003G23 of rice chromosome 3.Form it,we could know that it encoded a putative stress-induction protein which was TPR protein families.The analysis of nucleotide acid, By the means of biotechnology,T-DNA was inserted in the fourth exons in this gene.Owing to T-DNA insertion,the gene had no ability to perform it,and the phenotype of mutant was more sensitive to A1 toxicity than WT's.(2)The different antioxidant to the mechanism of antioxidant responses to A1 stress of the sensitive-mutantWe analyzed the different antioxidant responses to aluminum stress in Al-sensitive mutant rice seedlings.Our results indicated that ROS can produce and accumulate in the leaves of the mutant and wild type after A1 stress.The content of MDA of mutant and wild all increased when A1 concentration increasing;the degree of membrane lipid peroxidation of the Al-sensitive mutant rice was the highest after 3 days of 500μM A1 treatment. Furthermore,we observed a rapid Al-induced H₂O₂ increase in the mutant leaves,which induced abnormally activity in antioxidant enzymes such as superoxide dismutase(SOD). However,we found lower catalase(CAT) activities in the mutant leaves.By contrast, peroxidase(POD) activities were higher in the mutant than in the wild type when it had high A1 concentration.Together with the A1 toxicity-induced decline of early responsive enzymatic activities in vivo,especially CAT,the inability of mutants to scavenge accumulated H₂O₂ led to higher lipid peroxide levels.H₂O₂ might strengthen the expression of POD as a signaling molecule.Thus,based on our research presented here,we conclude that the decline of antioxidant enzymes activities is a physiological explanation for A1 sensitivity in the mutant and POD activity increased.