Studys On The Expression Conditions,Physiological And Genetic Basis Of Zebra-leaf Phenotype In Rice

B03S,the zebra leaf natural mutant of rice,is obtained from the photo-thermo-sensitive genic male rice lines EFENG1 s,mainly shows horizontal discontinuous green-yellow band zebra stripe on the new leaf and leaf sheath after transplanting.In order to elucidate the genetic basis of this trait,the mutation phenotype of the zebra leaf mutant B03 S with different sowing and growth period was identified.Leaf pigment content,chlorophyll fluorescence and antioxidant enzyme activity were determined and chloroplast ultrastructure was measured during the appearing of the zebra leaf phenotype.This study crossed the mutant with restorer materials to analyze the genetic characteristics.Low temperature treatment and sterility appraisal were used to screen the offspring with marked traits.Meanwhile,transcriptome sequencing,two-dimensional electrophoresis of protein and tandem mass spectrometry(ms)were used to explore some genes and the protein expression differences of the green-yellow band zebra stripe performance period.QPCR and western blot were for the validation of the experiment results of transcriptome sequencing and two-dimensional electrophoresis of protein.The main research results are as follows.1.Zebra leaf mutant is controlled by a pair of recessive gene can inherite steadily.Sterile mutant B03 S was used as the female parent and inbred lines 9311,N22,RM5,TXZ were used as the male parents for hybridization.After transplanting,all single plant of F1 group showed normal green,F2 group fitted with the separation of 3:1.At the same time,there were five consecutive years of low temperature treatment and sterility identifying for the descendants of optimal hybrid combination B03 S x 9311,a fertile material with zebra leaf marker traits was bred and the phenotype of the zebra leaf sterile line was similar with female parent material.2.In recessive homozygous,the trait of zebra leaf need to be stimulated by transplanting process.Mutants in seedling stage showed normal green,but young leaves and leaf sheath showed horizontal discontinuous green-yellow band phenotype approximately one week after transplanting.The mutation phenotype only exhibited on the 2-3 new leaves after transplanting,and the following leaves expressed all normalgreen.All rice leaves was under different sowing and transplanting dates showed consistent zebra trast.3.Zebra leaf belonged to the type of chlorophyll synthesis and chloroplast developmental defect.The leaf pigment content decreased significantly in the process of zebra leaf performance,the chlorophyll a and chlorophyll b and carotenoid content declined 82.8%,81.0% and 67.7% in Z-Y,respectively.The chlorophyll fluorescence value F0,Fm,?PSII and ETR declined more than 22% and 28% in Z-Y,respectively,the decrease of parameters of chlorophyll fluorescence and leaf pigment content were closely related.Transmission electron microscopy(sem)results showed that the chloroplast size of the Z-Y was normal,but the internal structure development was incomplete,and there were a lot of autophagy structure,the lack of thylakoids,the number of granum was decresed significantly and grana lamella were also decreased.Antioxidant enzyme activity test showed that the SOD and POD enzyme activity of the Z-Y were the highest,and the difference in POD enzyme activity among four treatments was significant.4.The transcriptome sequencing of zebra leaf stage obtained 720 DEGs,the expresssion of 303 genes were up-regulated and 417 genes were down-regulated in Z-Y.These genes were mainly rich in the synthesis of secondary metabolites,stress resistance,photosynthesis,ribosome,endoplasmic reticulum protein synthesis process and sugar/starch associated with energy metabolism and amino acids formation,qPCR was used to verify stress resistance and chloroplast formation related genes,the result was consistent with the transcriptome.5.During the process of zebra leaf performance,as it indicated by the two-dimensional electrophoresis of protein,there were 46 different protein spots with expression abundance identified in 2 times or more than 2 times,only 79 and 19 protein expression spots were founded in Z-Y and Z-G,respectively.Combined with previous Rubisco antibody,western blot was used for further results verification of two-dimensional electrophoresis of protein.It was found that Z-G/Z-Y difference ratio at about 1.5 times and these two results were consistent.Tandem mass spectrometry analysis identified 28 differentially expressed proteins,most of the proteins involved in protein,energy metabolism,enzyme activity and photosynthesis related pathways.Meanwhile,at the transcription level,stress resistance,coercion and chloroplast forming related proteins were verified,which showed there were some certain relations between gene transcription level and protein expression level.The experimental results addressed the genetic and physiological mechanisms of zebra leaf,the sterile and fertile zebra leaf morphological marker material.This study alsocontisbuted significantly research on chlorophyll synthesis and chloroplast development.Further study was essential to research the mechanisms to the induction of expression of zebra leaf trait gene and induced expression of its transplanting stimulation.