Caught red-handed Rc encodes a basic helix-loop-helix protei(7)

3-Mb region on rice chromosome 7,surrounding Rc .The intron–exon structures of In1and Rc are similar,but not identical.Rc has seven exons,whereas In1has nine,but in both genes the bHLH domain spans an intron–exon boundary.Although the phylogeny of Rc and In1and the positions of the 14homologous genes support orthology,In1and Rc have different functions.In1is a

negative regulator of anthocyanin synthesis,which gives an intense purple color when mutated,whereas Rc is a positive regulator of proanthocyanidin synthesis,which has no color when the function is lost (Burr et al.,1996).All of the other genes on the tree whose functions have been established are positive regula-tors,suggesting that the ancestral function is positive regulation (Goff et al.,1992;Nesi et al.,2000;Spelt et al.,2000;Sakamoto et al.,2001;Bernhardt et al.,2003;Elomaa et al.,2003).

It is possible that two orthologous genes,Rc and In1,have evolved separate functions since the divergence of the common ancestor of rice and maize.To further test the hypothesis of orthology,we compared the nucleotide divergence (Ks)values between these genes and two other pairs of genes from bin 7.02and the region around Rc (see Supplemental Figure 2online).The Ks values for the Rc –In1comparison are consistent with the expected values of orthologous gene pairs from these taxa.The surrounding genes had Ks values above and below those from the Rc –In1comparison,showing that Rc and In1were not changing faster or slower than other genes in the homologous region.Given the homologous positions of these loci and Ks values that are in agreement with expected values for two of the three genes analyzed,there is no evidence that contradicts the orthologous relationship between Rc and In1.

Thus,it appears that the rice and maize bHLH genes associ-ated with pigment production in plants have evolved separate functions over time.The rice genes found in different clades have specialized,becoming part of either the proanthocyanidin or anthocyanin pathway,whereas in maize,the duplicated genes have become positive and negative regulators of the anthocya-nin pathway.

DISCUSSION

QTL analysis was used to identify the location of rg7.1,a locus for red grain,near the centromere on rice chromosome 7.A com-bination of ?ne-mapping,mutant analysis,and sequence com-parisons demonstrated that a bHLH protein corresponding to the gene LOC_Os07g11020.1was responsible for rg7.1as well as for the classically de?ned mutant alleles Rc and Rc-s .

Allelic Variation at Rc

The functional nucleotide polymorphism,a 14-bp deletion that knocked out gene function,was suf?cient to explain the change in seed color between O.ru?pogon and cv Jefferson.It remains to be seen whether independent mutations in this gene or other genes also give rise to the white phenotype in other lineages.We are currently undertaking an association study to determine the predictive power of these mutations for pericarp color in rice.When comparing the rc and Rc-s alleles,it is not immediately apparent why the 14-bp deletion that frame shifts the bHLH domain should result in no pigment production and why a premature stop codon before the bHLH domain would give an allele conditioning light red color.In petunia and maize,an1and b1mutants lacking the bHLH domain are able to promote anthocyanin synthesis,much like the Rc-s allele (Liu et al.,1998;Spelt et al.,2002).Insertions that cause a frame shift

within

Figure 5.Phylogenetic Analysis of Rc and Other bHLH Proteins.Topology derived from Bayesian analysis using the GTR þG model after 5million generations (saving 5001trees),discarding the ?rst 100trees as burnin.It was compared with a single most parsimonious tree of 3629steps found after a heuristic search using 100random addition sequence replicates holding a maximum of 100,000trees (maximum not reached).The parsimony tree differed from the Bayesian analysis.The parsimony-preferred positions of taxa are shown by arrows where they connect to other branches;for example,in the parsimony tree,JAF13and Delila are grouped together to the exclusion of MYC1.Parsimony bootstrap per-centages (from 1000pseudoreplicates)are shown above the branches,whereas posterior probabilities from the HKY model and the GTR þG model are shown,left and right,below the branches (after 100burnin trees of 5001trees were discarded).Thicker branches have >80%bootstrap and >0.95posterior probability in each analysis.The topology should be considered unrooted.Genes that have been shown to function as regulators of the anthocyanin or proanthocyanidin pathways are named;additional sequences were retrieved from BLAST searches using Rc as the query and align across more than just the bHLH domain of ;60amino acids.The pound sign indicates a known negative regulator.

Rc Encodes bHLH Protein Red Rice 289

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