The Map-based Cloning And Functional Verification Of A Rice Mesocotyl Elongation Gene OsEIN2L

Rice mesocotyl indicates the part between the coleoptile and the basal part of the root of seed.The progress of mesocotyl elongation provides the initial energe for rice seedling breaking through soil.Only a few genes are reported to control mesocotyl elongation.The molecular mechanisms regulating the mesocotyl elongation are still unclear.To figure out the genetic and molecular mechanisms of rice mesocotyl elongation,a population consisting of 146 Nip/ZS chromosomal segment substitution lines（CSSL）and a rice core-collection of 252 rice varieties were used for the analysis of mesocotyl quantitative trait loci（QTL）.Here,we reported the main results of the map-based cloning and functional study of Os EIN2L as follows:1.There are 24 QTLs detected for rice mesocotyl elongation through genome-wide association study in the 252 rice varieties.And 18 QTLs controlling rice mesocotyl elongation are detected in the Nip/ZS CSSLs.Of them,a QTL located in 23 Mb on chromosome 2 and q Mel3 located nearby 28 Mb on chromosome 3 are repeatedly detected in both two populations.2.q Mel3 were narrowed into 38.3 kb between MP0322 and XF9 by using CSSL derived F2 population and progeny test of recombinants.There are 5 candidate genes in this region.Expression and bioinformation analysis suggested LOC＿Os0349400 was the most likely candidate gene for q Mel3.3.Transgenic complemental test and Crispr analysis revealed that LOC＿Os0349400 was q Mel3,and the allele from Nip could suppress mesocotyl elongation.Considering LOC＿Os03g49400 encoding an ethylene insensitive protein,we designated it Os EIN2L.4.The positive RNAi transgenic lines showed a longer mesocotyl phenotype than wild type.A T-DNA insertion in Os EIN2L promoter led to an increas of the Os EIN2L expression,and a shorten mesocotyl.These results indicate that Os EIN2L negatively controls rice mesocotyl elongation.5.Expressing profile analysis showed that Os EIN2L was constitutively expressed,and Os EIN2L^ZS showed much higher expression level than Os EIN2L^Nip.Subcellular localization revealed that the Os EIN2L^Nip-GFP fusion protein is predominantly localized to the nuclei,while the Os EIN2L^ZS-GFP fusion protein is predominantly localized to the ER membrane.Plant hormone treatment experiment indicated that Os EIN2L^Nip was much more sensitive to ACC（Aminocyclopropane carboxylic acid）than Os EIN2L^ZS.Expression analysis of several genes in the ethylene signaling pathway indicated that Os EIN2L is involved in the ethylene signalling.6.Comparison of the 10 kb sequence of Os EIN2L^Nip and Os EIN2L^ZS,showed a large number of SNPs in the promoter and coding regions.Compared with Os EIN2L^Nip,there were 62 bp and 17 bp deletion in the 1374～-1310 and-1094～-1077 upstream of the ATG of Os EIN2L^ZS.In the coding region,there were 14 SNPs variation,and 7 of them could lead to the predicted amino acid change.The 252 rice varieties were classed into 5 major haplotypes by the 7 non-synonymous SNPs.H1 and H2 were mainly detected in indica,H4 and H5 were mainly detected in japonica.Only C to T variation at SNP2208 was found between H1 and H2,and only A to C varaition at SNP122 detected between H4and H5.ANOVA analysis of the mesocotyl length among the major haplotypes indicated that the A/C variation in SNP122 could significantly regulate rice mesocotyl elongation.Transgenic test of the SNP122 mutation revealed that SNP122^Areduced the rice mesocotyl elongation.7.Expression analysis of Os EIN2L at the seedling stage in 45 varieties with SNP122^Crevealed that two In Del in the promoter suppressed Os EIN2L expression,however,the Os EIN2L expression level was not significantly associated with the variation of mesocotyl length.Bioinformation study indicated that a FIP/Harbinger MITE existed in this promotor region,and the 62 bp deletion in Os EIN2L^ZS promoter led to the MITE deletion.Luciferase activity analysis suggested that the MITE region may suppress Os EIN2L expression.There were no significant difference in methylation between Os EIN2L^Nip and Os EIN2L^ZS,indicating that the difference is not due to the DNA methylation nearby the MITE.8.The germination test with NILs,transgenic complementary and Crispr lines in the sowing depth of 3 cm and 6 cm,showed that Os EIN2L has no effect on growth vigor and seedling establishment under the 3 cm sowing depth,however Os EIN2L^ZS could promote seedling break through the soil in the 6 cm sowing depth.In addition,Os EIN2L^Nip could delay heading date and increase panicle spikelet number compared with Os EIN2L^ZS.