Map-based Cloning And Gene Functional Analysis Of A Dwarf And High-tillering Mutant Mz3 And A Leaf Rolling Mutant Crl3 In Rice

This article contains two research projects,namely map-based cloning and genetic functional analysis of dwarf and high-tilling mutant and leaf rolling mutant crl3 in rice.In this article,We found a dwarf and high-tilling mutant and a leaf rolling mutant crl3 which were respectively obtained from Zhonghuall and Nanjing5055 induced by Ethyl methane sulfonate(EMS).The analysis and summarize of the breeders for many years show that the plant height and moderate leaf rolling are important constituent factors of the ideal plant type of super high-yielding rice.A "green revolution" of rice in the 1950s,through dwarf breeding,has led to a significant increase in rice production.The moderate leaf rolling can improve the ventilation and light transmission of the population,increase the photosynthetic efficiency of the population,and improve the biological yield.Compared with the wild-type,in the whole growth period,the more significant phenotype of the dwarf and high-tilling mutant mz3,was the reduce plant height and increased tiller numbers,and the agronomic traits were very significant differences such as grain length,width,thickness,1000-grain weight,grain numbers per panide and etc.Genetic analysis showed that the mutant was controlled by a pair of recessive genes.The F2 population was developed by crossing incida rice variety Nanjing 11 with mz3,for mapping of the mutant gene,which was ultimately restricted within between RM19353 and RM510 SSR markers on rice chromosome 6,with a physical distance of about 747 kb.Gene prediction showed that D3,a gene conferring rice plant height and tiller number located in this positioned interval.Comprehensive phenotypic analysis suggested that the mutant gene and the D3 gene may be a pair of alleles.Sequencing results identified that the D3 gene in mz3 had a single G to A nucleic acid substitution at the position 636,after the start codon and the codon TGG was mutated the termination codon TGA,leading to early termination of translation.Subcelluar location showed that mutant D3 protein was located in the nuclei as the wild type D3 protein.BiFC analysis indicated that the mutant D3 protein could not interact with D14 protein for lacking of essential amino acids that interacts with D14,hindering transduction of SLs signaling.So we speculated that the mutant phenotype of mz3 was probably caused by D3 gene mutation.This article described a rice leaf rolling mutant crl3,which had incurved leaves and displayed narrow leaves and reduced plant height.The flag leaves showed curling along the axial direction,and obvious irregular,different degrees of wrinkles.Statistical analysis indicated that,compared with wild-type leaves,the leaf rolling index(LRI)of the flag leaves of the crl3 mutant was about 5-fold of the wild-type at the mature period.Genetic analysis showed that the mutant was controlled by a pair of recessive genes.An F2 population was developed by crossing indica rice cultivar N22 with crl3 for mapping of the mutant gene,which was ultimately located between SSR marker RL3 and InDell on rice chromosome 3,and the physical distance was about 146.5kb.The ORF prediction analysis of the 146.5 kb locus showed that 29 open reading frames were included in this positional interval,and all the gene information and its protein function were used to predict the gene and the primers were designed.The sequencing results of crl3 and Nanjing 5055 showed that the first bases of the fourth intron of the gene encoding ATP-dependent Clp protease proteolytic subunit(Os03g0308200)was mutated from G to A,which was located in the mRNA precursor intron cleavage site.Sequencing of 10 randomly selected recessive plants from crl3/N22 mapping population indicated the same mutation site as crl3 carried by the 10 individuals.It proved again that the single base mutation of the first base of the fourth intron of the gene(Os03g0308200)affected the normal cleavage of the gene in the mutant,resulting in a transcript that differred from the wild type,which in turn affected the protein function,as well as the appeared mutant phenotype.It was found that crl3 and the SRL2 gene of the reported semi-rolled leaf 2(srl2)were a pair of alleles.