Molecular Mechanism For Anthocyanin Synthesis In Rice Sheath And A New Method For Quickly Identifying Spontaneous Mutant Genes

Part? :anthocyanin is one of the three main pigments in plants attracting insects for pollination,protecting plants from pests and diseases,and preventing the UV damage.The synthesis of anthocyanins is regulated by structural and regulatory genes which share a relatively conservative pathway among different plants.The structural genes encode the catalytic enzymes of the synthesis pathway,while the regulatory genes,mainly including the MYB,bHLH and WD40 transcription factors,regulate the temporal and spatial expression of structural genes.The purple color of rice sheath is due to the synthesis and accumulation of anthocyanins and is the visually identifiable trait controlled by qualitative genes.Here,we have cloned the Rb1 and Rb2 which regulate the anthocyanin synthesis of the rice sheath by forward genetics approach.The main results are as follows:1.Based on the recombinant inbred lines constructed by Zhenshan 97B and Xizang 2,a gene named PSH1?Purple Sheath 1?controlling the sheath purple color on chromosome1 was primary mapped.This gene was then mapped to a 143.9 kb interval by fine mapping.This interval contains 19 annotated genes,4 of which were annotated as regulatory gene for anthocyanin synthesis encoding the Lc protein.2.By comparing the DNA sequence and referring to the annotation information of candidate genes,The Rb2?including the whole sequence between LOC_Os01g39560 and LOC_Os01g39580?was regarded as the candidate gene,which was 26.1 kb in the Nipponbare genome and 27.5 kb in Zhenshan 97B genome.3.Overexpression of the Zhenshan 97B Rb2 allele to the near-isogenic line NIL-XZ2,displayed different degrees of anthocyanin accumulation in the sheath and leaves.The anthocyanin content of the Rb2 knockout lines were also visibly decreased.However,the anthocyanin content of the knockout lines did not reduce to the level of NIL-XZ2,indicating that there might exist other gene?s?regulating the synthesis of sheath anthocyanins in addition to Rb2.4.Using a F₂ population arrived from Rb2 knockout line and the NIL-XZ2,we mapped another gene Rb1,controlling the sheath color near to Rb2.The relative expression analysis showed that the expression of Rb1 was nearly 130 times between near-isogenic lines.However,there was no nucleotide difference in the CDS region of Rb1,indicating that the difference in non-coding region may lead to the difference of gene function.Overexpression of the Zhenshan 97B Rb1 allele to NIL-XZ2 showed almost the same phenotype with the overexpression of Rb2 lines.The sheath color of the Rb1 knocked lines were also weakened.Besides,the sheath color of the rb1rb2 double mutant almost turned to the level of NIL-XZ2,indicating that Rb1 and Rb2 controlling the synthesis of the sheath anthocyanins together.5.Relative expression analysis showed that Rb1 and Rb2 regulated the anthocyanin synthesis through affecting the expression of three genes?F3H,DFR and ANS?of the anthocyanin synthesis pathway.The accumulation of anthocyanins in overexpressed lines was positively correlated with the expression levels of these three genes,while the decrease of anthocyanins in knockout lines was associated with decreased expression of these genes.6.Ectopic expression of the Rb1 and Rb2 could change the pericarp color from colorless to purple,similar to a rice cultivar"purple rice".Although overexpression of these two genes did not affect fertility,it largely affected the rate of seed filling?which dropped to 10.0%?.7.The segregation ratio of purple sheath to green sheath in a F₂ population arrived from Xizang 2 and Minghui 63 coincided with a separation ratio of 9:7,indicated that there were two genes controlling synthesis of sheath anthocyanins in this population.Genetic analysis showed that these two genes were OsC1 and Rb1/Rb2?Rb1 and Rb2 were regarded as one gene since they were tightly linked?.Yeast two-hybridization proved that Rb1,Rb2interacted with OsC1,separately.Part ?:Both somatic mutants induced by tissue culture and natural mutant belong to spontaneous mutants,which are important materials for gene cloning.However,the nucleotides difference between these mutants and the wild-type are too tiny to identify these mutant gene through the isolated populations arrived from the mutants and their corresponding wild-type.Besides,most traits are controlled by quantitative genes,making it difficult to directly identify the mutant genes through crossing with other varieties.We have created a new method for quickly identifying these series of mutant genes.The main results are as follows:1.A mutant from Zhonghua 11 T-DNA mutant database exhibited few tillers and yellowish leaf.While the insertion of T-DNA did not co-segregate with the mutant phenotype.The mutant was regarded as a somatic mutant induced by tissue culture.The Zhonghua 11 treated with EMS but without phenotypic changes?neutral mutant?was set as the male parent and crossed with this somatic mutant.The separation rate of the leaf color in F₂ populations coincided with a ratio of 3:1,indicating that this mutant was caused by single gene mutagenesis.2.30 individuals with mutant phenotype from F₂ population were selected for next-generation DNA sequencing.LOC_Os07g04840 on chromosome 7 was regarded as the mutated gene through biological information analysis.The mutant had 1 bp deletion at the second exon,resulting in a frameshift mutation of PsbP protein.Overexpressed the PsbP gene to the mutant,leading to increase of the tiller number and chlorophyll content,indicating that PsbP was the gene that controlled tiller number and leaf color.3.A natural mutant of semi-rolled leaf was crossed with the 9311 which was mutagenized by EMS but without significant phenotypic changes?neutral mutant?.Followed the same method,we confirmed that LOC_Os07g01240 was the mutant gene.This mutant had 2 bp deletion at the third exon,leading to frameshift mutation.This gene encoded a glycophosphatidylinositol?GPI?-anchored membrane protein,which had been reported controlling semi-rolled leaf.