Map-based Cloning And Functional Analysis Of A Rolling And Erect Leaf Gene,REL2

Rice is one of the most important corps in the world.How to improve the yield of rice has become a research focus.A key point of increasing rice yield is how to construct an ideal plant structure.Rice leaves are crucial organ of energy sources.An optimal leaf structure is beneficial for rice leaf blades to capture light and exchange gas,thus improving the yield of rice.Some studies indicated that moderate leaf rolling and relatively erect plant architecture could contribute to the high yield of rice.But the mechanism of leaf rolling still remains unclear.In this study,we identified and analyzed a rolled and erect leaf blade mutant with the genetic background of Nipponbare.This mutant was obtained by EMS treatment and named it as rel2(rolling and erect leaf 2).Histological analysis showed that the bulliform cell is increased whereas the size of bulliform cells is decreased in rel2 mutant,indicating that the rolling leaves should be caused by the abnormal development of bulliform cells.We firstly mapped REL2 to a 35-kb physical region on rice chromosome 10 by using SSR,In Del and SNP molecular markers.In this region,there are six candidate genes.Gene sequencing results indicated that there are only a 4-bp deletion in 5’-UTR and two nucleotide substitutions in the first exon of Os10g0562700 locus.Therefore,Os10g0562700 should be the target gene.Sequence analysis gave the information that REL2 encodes a DUF630 and DUF632 domains contained protein.Further analysis illustrated that there is no transcription expression level differences of REL2 between WT and rel2,implying that the 4-bp deletion at 5’-UTR does not cause the expression changes,which indicated that the two nucleotide substitutions in conservative DUF630 domain should lead to the loss-function of REL2.The function of REL2 could be confirmed by complementary expression of REL2 in rel2 mutant.Subcellular localization showed that REL2 protein mainly distributes on plasma membrane.However,the peptides containing DUF630 domain and DUF632 domain alone exhibits no special localization.Combing with the results of multiple sequences alignment and evolution analysis,DUF630 domain and DUF632 domain may always bind together to implement their functions.Tissue expression analysis indicated that REL2 has a higher expression in relatively young leaves in rice.The results from quantitative RT-PCR analysis indicated that REL2 functioning in the leaf shape formation might be coordinated with many genes associated with the bulliform cells development,auxin synthesis and transport,etc.In addition,REL2 is the DUF domains contained protein which involves in the control of leaf rolling.Moreover,this study displayed a new leaf rolling control gene in rice and this gene contains DUF630 andDUF632 domains.Our study could provide a primary reference to the function of DUF domains due to relatively less researches on DUF domains’ function.