| Chloroplast is the main place for photosynthesis in plants,the chlorophyll content change or chloroplast structural defect may directly affect the photosynthetic efficiency and yield potential formation.Therefore,it is of great guiding significance for rice production to study on leaf color mutant with chlorophyll content change or chloroplast structural defect and to explore the molecular mechanism of gene regulating leaf color.In this study,two leaf color mutantses3(t)(early senescence 3)and ahs1(albino in heat stress 1)obtained by EMS(Ethylmethylsulfone)mutagenesis were reported.The molecular mechanism of two leaf color mutation in rice was analyzed with the phenotypic analysis,genetic analysis,chlorophyll content determination,electron microscopic analysis,and identification and cloning of target genes.The main results of the related researches were as follows:1.ES3(t)gene:(1)Phenotype identification: The mutant es3(t)displayed obvious phenotypic characteristics.The mutant es3(t)showed early senescence,shorter plant height,decreased tiller number,shorted panicle length and lower seed setting rate.(2)Physiological and biochemical indexes detection: In the mutant es3(t),the chlorophyll content decreased,the ROS was accumulated excessive in leaves,and the ABA content increased significantly in senescent leaves.(3)Cytological observation: The results of transmission electron microscopy showed that the chloroplast structure was abnormal in the mutant es3(t).(4)Expression analysis: In the mutant es3(t),the expression level of genes related to photosynthesis was down-regulated,and the expression level of transcription factors related to senescence and SAGs was up-regulated.The expression level of genes related to ABA synthesis in the leaf was up-regulated.(5)Gene mapping and prediction: To map the gene,728 individuals with the early senescencephenotype were screened from 5013 F2 segregation population.We narrowed down ES3(t)to the physical distance35 kilobase(kb)regions between markers HK17-23 and HK17-30 in BAC clone AC097624(BAC1).In this region,there were eight predicted open reading frames(ORFs)and all of thoese were not been reported previously,which indicated that ES3(t)is a new gene controlling leaf senescence.2.AHS1gene:(1)Phenotype identification: At 34 and 38℃ ℃,the mutant ahs1 displayed obvious albino.While at 26 and 30℃ ℃,there was no significant difference between the mutant ahs1 and the wild-type NIP.At heat stress,the glumes displayed albino in the mutant ahs1.(2)Physiological and biochemical indexes detection: At 34 and 38℃ ℃,the content of Chl a,Chl b and Car in the leaves of the mutant ahs1 were significantly lower than that of the wildtype NIP.At 26℃,the content of Chl b in the mutant ahs1 leaves was significantly lower than that of the wild-type NIP,while there was no significant difference between the mutant ahs1 and the wild-type in the content of Chl a and Car.At 30℃,the content of Chl a in the mutant ahs1 leaves was significantly lower than that of the wild-type NIP,while there was no significant difference between the mutant ahs1 and the wild-type NIP in the content of Chl b and Car.The ROS was accumulated excessive in leaves of the mutant ahs1.(3)Cytological observation: At 34℃,the results of transmission electron microscopy showed that there was few thylakoid,no unstacked thylakoid structure and the perfectly structured thylakoid membrane and some vesicular structuresin in the white part of leaves in the mutant ahs1.The thylakoid membrane increased in the white and green part compared to the white part in the mutant ahs1.At heat stress,the mutation of AHS1 delayed the development of the chloroplasts.The results of transmission electron microscopy showed that there were only few small chloroplasts in parenchyma cells of spikelets in the mutant ahs1.(4)Gene mapping and complementary test: Using 1778 recessive individuals,AHS1 was finally narrowed down to 22 kb region between markers K5-13 and K5-27.In this region,there are four predicted open reading frames(ORF1-ORF4).The sequencing analysis showed that the mutational gene carries a single nucleotide mutation(G343T)at the ORF2 in mutant ahs1,which result in an amino acid substitution from Gly to Trp.The western blot analysis showed that AHS1 expressed normally in transgenic positive plants.At the same time,the phenotype of the mutant ahs1 rescued completely and the leaf returned to normal green at heat stress in the 8 transgenic lines of pAHS1.(5)Expression pattern: GUS staining analysis showed that strong GUS signals were observed in leaves at the seedling stage and in spikelets,leaves and stems at the heading stage in transgenic positive plants.RT-PCR analysis showed that AHS1 was expressed in all tissues at heading stage,and the expression level was higher in panicles,stems and spikelets,while the expression level was lower in mature leaves and roots,which showed the constitutive expression pattern.(6)Subcellular localization: The results of the confocal laser scanning microscopy showed that p35S: AHS1-GFP fusion protein was co-localized with chloroplast autofluorescence,which indicated that AHS1 is a chloroplast localization protein. |
