Gene Mapping And Cloning Of Leaf Color Mutant Yl1 Of Guizhou Landrace Cultivar Lailong In Rice (Oryza Sativa L.)

Leaf color mutants have important value in the research of chlorophyll synthesis and metabolism,photosynthesis process and so on,it also be used as screening markers in heterosis.In this study,yellow leaf mutant?yl1?was obtained from the EMS mutant library in LaiLong.The chlorophyll,chlorophyll synthesis precursors,chlorophyll anabolism-related gene expression and chloroplast development-related gene expression in the three growth stages of mutants were analyzed to explore the phenotypic characteristics of yl1 mutants.And gene mapping by MutMap technology.The results obtained as follows:1.Analysis of the formation mechanism of yl1 mutantThe chlorophyll content of yellow leaf mutant yl1 and wild type seedling,tillering and maturity were determined.It was found that yl1 chlorophyll a and chlorophyll b were significantly lower than wild type in three growth stages.However,the chlorophyll a/b ratio of yl1 in the seedling stage was higher than the wild type,and the tillering period was not much different,and the maturity stage was lower than the wild type.Precursor materials for chlorophyll synthesis in mutant and wild type leaves were assayed.It was found that the precursor substance content of the yl1mutant in the three chlorophyll synthesis stages from porphobilinogen?PBG?to protoporphyrin IX?Proto IX?was higher than that of the wild type,synthesized from protoporphyrin IX?Proto IX?and thereafter.The precursor content of all stages was significantly lower than that of the wild type.Moreover,the expression of CHLD gene in the yl1 mutant encoding Pro-IX synthetic Mg-ProxoIX key enzyme magnesium hydrazone D subunit was significantly decreased,and the expression of the synthetase-encoding gene in the pathway after Mg-ProxoIX synthesis was decreased.It is speculated that the yellow leaf trait of yl1 is due to the decrease of the expression of CHLD encoding the D subunit of magnesium hydrazine synthase,which leads to the inhibition of Proto IX synthesis of Mg-ProxoIX,which reduces the chlorophyll synthesis of yl1.Ultrastructural observation of yl1 mutants and wild-type leaves revealed that the chloroplast boundaries were not obvious in the mutants,and the matrix sheets showed varying degrees of looseness.At the same time,the expression level of the key gene Ftsz in the first stage of chloroplast development was also significantly lower than that in the wild type,but there was no significant change in the expression of key enzyme genes in the latter two developmental stages.It is speculated that the down-regulation of the first stage of chloroplast development in yl1 mutants leads to the influence of chloroplast development.2.Fine mapping of yl1 and screening of candidate genesUsing yl1 and wild-type backcross F₂ as the target population,MutMap technique was used to study the gene mapping of yl1.Re-sequencing and SNP locus analysis were performed on yellow leaves and normal leaf color progeny in the F₂population.Two candidate genes located on chromosome 3 were obtained,the one is LOC_Os03g31210 which encoding 6 glucose dehydrogenase and the other one is LOC_Os03g36760 which is unknown function.The base 1432 of LOC_Os03g31210?located on the second exon?was mutated from C to T,resulting in the mutation of amino acid 478 from valine to leucine.On the second exon of LOC_Os03g36760,the2717th base was mutated from A to G,resulting in the amino acid at position 906being changed from lysine to glutamic acid.3.Cloning and functional verification of yl1 candidate geneThe plant overexpression vector pCam-35S-YL1-1 and pCam-35S-YL1-2 were constructed by pCambia1301,the 35S promoter and the 10kDa prolamin termination sequence.Two gene overexpression vectors were genetically transformed into yl1mutants using Agrobacterium-mediated rice stem tips.The results showed that the yl1transfected with LOC_Os03g31210 gene did not turn green,but the yl1 transformed with LOC_Os03g36760 gene returned to the wild type phenotype.Therefore,we believe that:LOC_Os03g36760 may be the key gene leading to yellowing of yl1yellow leaf mutant leaves.