Map-based Cloning And Functional Analysis Of CPP1 Gene In Rice (Oryza Sativa L. Ssp. Indica)

Rice（Oryza sativa L.）is one of the most important food crops in the world,and it is also a model plant to study the functional genes of monocotyledons.Carbohydrates synthesized by photosynthesis in rice leaves are transported to non-photosynthetic organs through the phloem afterwards,and forming sucrose to maintain normal growth and development or storage in the form of starch.Once there is an obstacle to the distribution of carbohydrates,it will have an important impact on plant growth,development and yield.Therefore,elucidating the molecular mechanism of carbohydrate partitioning in plants,especially crops,is not only helpful to understanding plant growth and development,but also providing some evidences for improving crop yield through molecular breeding design.In this paper,we treated indica rice maintainer Xinong 1B with chemical mutagen ethyl methanesulfonate（EMS）,and screened a carbohydrate partitioning protein 1（cpp1）mutant from the library.The phenotypic observation and agronomic character analysis of the cpp1 mutant were carried out,and the map-based cloning,complementary verification,expression pattern analysis and functional analysis of CPP1 gene were carried out.The main results are as follows:1.Carbohydrate partitioning defects in cpp1 mutantcpp1 mutant showed yellowing leaf accompanied by premature senescence from the seedling stage and showed late heading.The main agronomic characters（including plant height,tillering number,effective panicle number,filled grain number of per panicle,seed setting rate,1000-grain weight,etc.）were significantly lower than those of the wild type.The photosynthetic pigment content and net photosynthetic rate of cpp1 mutant were significantly lower than those of wild type.The expression of chlorophyll degradation genes and senescence associated genes in the cpp1 mutant were significantly higher than those in wild type.Through iodine-potassium iodide（I₂/KI）staining,it was found that a large amount of starch was accumulated in the mutant leaves not only at the end of the day but also at the end of the night.Further transmission electron microscopy（TEM）observed more starch granules were accumulated in the mesophyll cells of the cpp1 mutant than in the wild type,which destroyed the chloroplast structure.Finally,the contents of starch,sucrose and glucose in source leaves and sink leaves were determined,respectively.The results showed that the contents of starch,sucrose and glucose in the source leaves of the cpp1 mutant were significantly higher than those of the wild type,while the contents of sucrose and glucose in the sink leaves of the cpp1 mutant were significantly lower than those of the wild type.These results showed that the accumulation of carbohydrates led to leaf yellowing and premature senescence,and had an effect on the main agronomic characters and heading date.2.Genetic analysis and molecular mapping of CPP1 geneGenetic analysis populations were derived from the cross between the cpp1 mutant and 56S.The F₁ plants displayed the characteristics of premature senescence,which indicates that the cpp1 phenotype was controlled by a dominant gene.This was confirmed in the F₂population,in which the segregating ratio of the mutant phenotype（2135 individuals）to the wild type phenotype（693 individuals）was 3:1 in a total of2828 individuals(χ²=1.56<χ_0.05²=3.84).This further suggests that the cpp1 trait was completely controlled by a single dominant nuclear gene.The recessive single plant of F₂ generation obtained from the cross between 56S and cpp1 mutants was used as the mapping population,and the CPP1 gene was located to a 71kb region between makers S3 and S6 on the short arm of chromosome 6.3.Cloning and functional complementary verification of CPP1 geneThe full-length sequencing of the genomic sequences of 10 candidate genes in the location region showed that in the cpp1 mutant,only Loc＿Os06g03380 carried a single-nucleotide substitution（A to G,at the position 365 bp from the ATG start codon）,which resulted in an amino acid change from glutamic acid（Glu）to glycine（Gly）in cpp1 plants compared with the wild type plants.Therefore,this gene is predetermined as the candidate gene of CPP1 gene.To confirm that it was indeed the CPP1 gene,a5923-bp mutant genomic fragment of Loc＿Os06g03380,including a 3050-bp sequence upstream of the start codon and a 503-bp sequence downstream of the terminal codon,was introduced in WT background.The transgenic plants exhibited phenotype similar to the cpp1 mutant.Besides,Iodine-potassium iodide（I₂/KI）staining also showed that the transgenic plant leaves were more deeply stained than the WT leaves,like the cpp1leaves.These results indicate that Loc＿Os06g03380 is the CPP1 gene.4.Analysis of expression pattern of CPP1 geneThe expression pattern of CPP1 gene was analyzed by quantitative reverse transcription-PCR（qRT-PCR）.The expression of CPP1 in the root,stem,young leaf,fully spreading leaf and panicle were analyzed by qRT-PCR with the expression of rice Ubiquitin as a housekeeping gene.It was found that CPP1 gene was mainly expressed in young leaves.5.Protein analysis encoded by CPP1 geneThe CPP1 genome contains no intron and consists of only an exon,which encodes a protein with 789 amino acids residues.Using the CPP1 protein sequence to blast on the NCBI protein database,CPP1 was found to be unrelated to any known proteins,and to contain two conserved domains of unknown function,DUF4220 and DUF594.Through homologous protein search and phylogenetic analysis,the results showed that no homologous sequence of CPP1 was found in lower plants such as moss,peat moss,green algae,dunaliella salina,microcytic algae and so on.The phylogenetic analysis showed that CPP1 in rice may be descended from CPP1 of sorghum.The fusion protein of CPP1-GFP was constructed and transiently co-expressed with PDLP1-m Cherry fusion in N.benthamiana leaves by Agrobacterium tumefaciens.The results showed that the green fluorescence signal was co-located with the RFP signal,indicating that CPP1 protein was located in the plasmodesmata（PD）.Yeast two-hybrid（Y2H）and bimolecular fluorescence complementary（Bi FC）experiments showed that CPP1 interacted with expansin EXPB4.6.CPP1 gene affects the expression of genes in carbohydrate metabolism pathwayThe expression level of genes related to starch metabolism and sugar metabolism pathway were detected by quantitative reverse transcription-PCR（qRT-PCR）in cpp1mutant and wild type.The results showed that the expression of sucrose biosynthesis related genes cFBP1,c FBP2,SPS1,SPS2 and sucrose transport related genes SUT1and SUT2 were all downregulated in cpp1 mutant compared to the wild type,while the transcript leavels of starch synthesis pathway related genes AGPL1 and AGPS1 in cpp1mutant were higher than that in wild type,the expression of starch degradation pathway gene MEX1 was decreased in cpp1 mutant.However,the expression levels of sucrose transport related genes SWEET11 and SWEET14 were significantly increased in cpp1 sink leaves.At the same time,the expression level of PD negative regulator GSD1 in sink leaves of the cpp1 was increased compared to the WT plant,which indicates that the permeability of the PD might be lower in the cpp1 mutant compared to the WT plant.Therefore,the apoplastic pathway was induced and the rate of sucrose transport increased in sink leaves.Sucrose synthesis and export were decreased,the rate of starch synthesis increased,while the rate of starch degradation decreased,in cpp1 source leaves compared to WT source leaves.7.CPP1 gene affects the PD permeabilityIn view of the fact that a large amount of carbohydrates were accumulated in the source leaves of the mutant,while the content in the sink leaves decreased,and the expression level of PD negative regulatory factor in the cpp1 sink leaves was significantly higher than that in the wild type,which indicates that the permeability of PD in the sink leaves may be affected.Therefore,the permeability of N.benthamiana leaves with transient expression of CPP1^E122G was analyzed by Drop-ANd-See（DANS）method.The results showed that the diffusion area of fluorescence signal on the abaxial surface of the leaf was significantly lower than that of the control,and the relative diffusion area was only 49%of the control.It is suggested that CPP1^E122Gaffects the PD permeability.