| Leaf color mutants are ideal materials for studying photosynthesis and photosynthesis in chloroplast development.Mutations in chloroplast development-related genes cause defects in chloroplast development and lead to decreased leaf color mutation and photosynthesis,while chloroplast development also has great influences in fruit quality.In the early study,our reasearch team has did fine mapping of yellow-green leaf trait in melon.Based on this study,we successfully screened and cloned melon green leaves gene CmGLK by biosequence analysis,and constructed an overexpression vector to transform Arabidopsis glklglk2 mutant in order to achieve functional complementation verification.These results of this study not only can enrich and improve the molecular mechanism of GLK transcription factors in regulating chloroplast formation,but also can provide new ideas and directions for melon fruit quality improvement.The main findings as follow:(1)We resequenced the melon parents DHL92 and M68 and found that there were six exons in the candidate segment.Due to the deletion of one base C in the third exon,the gene has a frameshift mutation.As a result,a yellow-green trait appeared,and the candidate gene was successfully located in the candidate segment.Further using the dCAPS marker developed by the mutation site,genotypic analysis of F2 population and natural population was performed,and the result showed that dCAPS and leaf color phenotype showed co-segregation,indicating that the gene is the candidate gene of controlling melon leaf color yellow-green traits CmGLK(Gene ID:XP 008455954).The ORF of CmGLK gene is 1128bp,which encodes 450 amino acids.(2)By constructing overexpression vectors of CmGLK,cmglk and AtGLKl and transforming them into Arabidopsis glklglk2 mutants respectively,we found that CmGLK and AtGLKl genes can restore the phenotype of yellow leaves in Arabidopsis glklglk2 mutant..The yellow leaf turned green in 35S:CmGLK::glklglk2 and 35S:AtGLK::glklglk2 transgenic plants,while overexpress cmglk can not restore the phenotype of the Arabidopsis glklglk2 mutant.Besides,by constructing GFP fusion expression vector of CmGLK,cmglk and AtGLK1 and transiently transforming Arabidopsis protoplasts,it was confirmed thatK,cmglk and AtGLK1 were located in the nucleus.(3)By measuring the photosynthetic rate of pure strains of 35S:CmGLK,we found that the photosynthetic rate of plants was increased in 35S:CmGLK::glklglk2 plants.Besides,by measuring the chlorophyll fluorescence dynamic parameters of 35S:CmGLK::glklglk2,35S:AtGLK1::glklglk2 plants,we found that F0,Fm,qN-lss,qP-lss were improved to different extents in 35S:CmGLK::glk1glk2,35S:AtGLK1::glklglk2 plants.Compared with glklglk2 double mutant,the ability of Arabidopsis to consume excess light energy and the open ratio photoreaction center II(PSII)were significantly improved.(4)Statistical analysis of the seed germination rate of Arabidopsis wild type Col-0,glk1glk2 mutant and different transgenic plants under simulated drought and salt stress was performed,and we found that the germination rate of the mutant was lower than that of the wild type.The germination rate of overexpressing lines was higher than mutants.It indicated that GLK gene plays a positive regulatory role in the process of Arabidopsis seed germination.(5)Through cluster analysis of GLK homologous genes in various plants,it was found that GLK genes were distributed in lower to higher plants:GLK gene first appeared in green algae,It was distributed in bryophyte,stone pine gate,monocotyledon and dicot plants;this gene is present in pairs in most plants,but it was present in a single copy in diploid melon crops. |
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