Cloning And Functional Analysis Of NOG1 Controlling Grain Yield In Rice

Rice yield is a complex agronomic trait which is determined by three main components:panicles per plant,grain number per panicle and grain weight.Grain yield has always been the most important target during rice domestication and improvement,and it is one of the most important indicators to measure the quality of rice varieties.In previous studies,we constructed one set of introgression lines(ILs)using Dongxiang common wild rice as a donor,and an elite rice cultivar Guichao2 as a recipient.In this study,we selected one introgression line SIL176 with low grain yield,to identify the QTL(NOG1)that controls grain yield in SIL 176 by map-based cloning.The main results were described as follows:1.Compared with Guichao 2,the grain number per main panicle and grain yield per plant of SIL176 decreased by 39.6%and 44.3%,respectively.A genetic linkage analysis of F2 individuals derived from the cross between SIL176 and Guichao 2 showed that there is a major QTL,NOG1(NUMBER OF GRAINS 1),which locates on chromosome 1.Using a larger segregation population,NOG1 was delimited to a 4.3-kb region which harbors the promoter(cis-regulatory region)and the first three exons of LOC_Os01g54860.Transformation test demonstrated that the grain number per main panicle and grain yield per plant of the complemented transgenic plants using the construct containing the entire gene region and 2504-bp promoter region of LOC_Os01g54860 from Guichao 2,increased by-50%compared with the control plants(SIL 176),while grain number per main panicle and grain yield per plant decreased in the RNAi transgenic plants compared with control plants(Guichao 2),indicating that LOC_Os01g54860 is the NOG1 gene which controls the grain number per panicle and grain yield per plant in rice.2.NOG1 expression were detected in various rice organs,including root,tiller base,culm,eustipes,pulvinus,leaf,and the young panicles,and NOG1 relative expression levels in the leaf and young panicles of Guichao 2 were significantly higher than that of SIL 176.RNA in situ hybridization signals of NOG1 in young panicles indicated that NOG1 is expressed in primordia of branches.3.NOG1 encodes enoyl-CoA hydratase/isomerase(ECH),a key enzyme in fatty acid ?-oxidation pathway.The detection of protein eukaryotic expression product confirmed that the NOG1 protein has ECH enzyme activity.Sequence analysis showed that nogl cDNA of SIL176 has a 3-bp insertion in the exon compared with NOG1 cDNA of Guichao2,and results in one amino acid insertion after translation.However,the insertion-deletion(In-Del)of one amino acid between Guichao2 and SIL 176 did not change the protein function of NOG1.Sequence analysis of the promoter region showed that there was one 12-bp InDel and 15 SNPs between Guichao 2 and SIL 176 in the promoter region of NOGl.Two closely connected copies of the 12-bp in Guichao 2 but only one copy of the 12-bp in SIL 176.4.An association test of 158 cultivars showed that the strongest signal was present at the 12-bp In-Del between grain number per main panicle and sequence variations.Further examination showed that the insertion of the 12-bp in the NOG1 promoter region up-regulated the NOGI gene expression levels,increased the ECH levels,reduced the content of total fatty acids and linolenic acid,decreased the levels of endogenous JA,and enhances the grain number and grain yield in rice.5.We sequenced NOG1 promoter in 158 varieties of cultivated rice and 75 accessions of wild rice.The results showed that 75 accessions of wild rice all harbor one copy of the 12-bp functional site.However,among 158 varieties of cultivated rice,61 varieties(48 indica and 13 japonica varieties)harbor two copies of the 12-bp functional sites,and 97 varieties(36 indica and 61 japonica varieties)harbor one copy of 12-bp,indicating that the mutation of the 12-bp insertion in NOG1 of cultivated rice might occur after the transition from wild rice to cultivated rice,and the 12-bp variation in NOG1 promoter region might have originated in the indica cultivars and was later introduced into japonica cultivars.6.To explore the application potential of NOG1 in rice breeding,we introduced the complementary construction which contained the entire gene region of NOG1 from Guichao 2 into japonica cultivar Zhonghual7,which contains one copy of 12-bp.The NOG1 complemented transgenic plants exhibited an obvious increase in grain number per panicle and grain yield per plant than control.Field estimation of grain yield showed that the NOG1 complemented transgenic plants increased grain yield by 25.8%compared with the control.We further introduced an overexpression construct into a high yielding indica culticar Teqing which harbors two copies of the 12-bp.Compared with Teqing as control,the NOG1 overexpression transgenic plants increased grain yield by 19.5%.Compared with the control,there were no significant differences in yield-related traits such as panicle numberper plant,grain weight,seed setting rate,and heading date.The cloning of NOG1 not only provides an important new gene for the cultivation of high-yield rice varieties,but also provides new clues for revealing the molecular mechanism of rice yield traits.