Cloning And Functional Analysis Of The Mutant Gene In Pale-green Leaf Wheat Chli

Common wheat（Triticum aestivum L.）is one of the most stable crops on which human beings depend for survival and also the third largest food crop after maize and rice.Wheat provides food,about 20 percent of calories,for about 40 percent of the world’s population.In the past half century,photosynthetic efficiency improvement in crop has played only a small role in the increase of grain yield,although the yield of grain has been greatly improved.However,the further increase of crop yield in the future will be largely dependent on the improvement of photosynthesis.Chloroplasts in leaves are the main organ for photosynthesis,which is closely affected by chlorophyll content.Wheat leaf color mutants are easy to observe,and also excellent materials for the plant chlorophyll biosynthesis and metabolism study,which have great potential theoretical for improving wheat yield.chli was a stable chlorophyll deficient mutant at seedling stage which was identified from variety shaannong 33.In this study,the chlorophyll content in chli and shaannong 33at different stages were measured,as well as the main agronomic traits at mature period.The leaf and chloroplast structures were analyzed by paraffin section and TEM.The genetic model of chlorophyll deficiency and the mutant gene were revealed by F₂population of cross chli×ZM 895 and ZM 895×chli.Furthermore,the mutant genes were located and cloned by using wheat 66ok SNP chip.On the basis of cloned mutant gene,we firstly analyze the size of mutated protein,as well as the protein distribution location in the cell by using prokaryotic expression and subcellular localization techniques.Based on the yeast two-hybridization,arabidopsis mutants mediated gene function complementation test,BiFC and FERT.we preliminarily revealed the cause of chlorophyll deficiency in chli.The main results of this paper are as follows:1.Phenotype identification.The mutant chli showed an obvious chlorophyll deficiency at the seedling stage,but the phenotype differences between chli and shaannong33 could not be distinguished at the heading stage.Especially at the one-leaf stage,the contents of chlorophyll a,chlorophyll b and carotenoids in chli were significantly lower than those in control shaannong 33.There were only 68.2%,43.2%and 62.5%of shaannong33,respectively,and the proportion increased to 84.6%,76.6%and 80.4%of shaannong 33 at three-leaf stage.At the time of ripening and harvesting,plant height,panicle length,seed numbers per panicle and seed setting rate was not significant difference between chli and shaannong 33,excepting panicle numbers per plant which was only 5 in average.Either at one-leaf stage or three-leaf stage,the leaf structure between chi and shaannong 33 did not show difference.But the chloroplast structure is stunted significantly in chli at one-leaf stage.Meanwhile,the number of hylakoids in chloroplast stroma is significantly less than that in shaannong 33.When they grow to the trifoliate stage,the number and the structure of hylakoids was same between them.These results suggested that mutant gene impaired the hylakoids development in chli.2.Genetic analysis.In the F₂population of the cross and back cross between chli and zhongmai 895 indicated that the ratio of pale green plants to green plants is fit to 1:3.According to the phenotypic separation of F_2:3lines,the corresponding F₂genotype was inferred,and the results showed that pale-green leaf homozygote plants:green-leaf heterozygote plants:green-leaf homozygote plants are 1:2:1.These result demonstrated that the pale green phenotype is control by a pair of recessive gene.3.Cloning and functional analysisof mutant proten Tachli-7A.The mutant gene was further located within a 670-680 Mb interval at wheat chromosome 7A by wheat 660k SNP chip.After the comparison and analysis of the predicted gene in this region with the genes related to the chlorophyll synthesis pathway of Arabidopsis thaliana and rice,the transcriptional Traes CS7A01G480700 was predicted as the candidated gene which was named Tachli-7A.The full length CDS of TaCHLI-7A in shannong 33 and chli was 1266 bp.The sequence result revealed a SNP between chli and shaannong 33.The gene TaCHLI-7A of shaannong 33 have a nucleotide G at position 664,but mutant gene Tachli-7A of chli have a A at same position.This single nucleotide mutation resulted in an amino acid substitution（D221N）which was in the highly conserved domain of TaCHLI-7A protein.4.The characteristics of mutant protein Tachli-7A.Through prokaryotic expression and SDS-PAGE electrophoresis,we found that the size of TaCHLI-7A protein was about 40k Da.The TaCHLI-7A was found to be located on the chloroplast by subcellular localization.The yeast two-hybrid test showed that the mutant protein Tachli-7A could not interact with the normal TaCHLI-7A due to the change of amino acid residues at position 221.The FERT test showed that protein Tachli-7A could interact with the normal TaCHLI-7A,but the interaction between Tachli-7A and TaCHLI-7A was significantly weaker than that between TaCHLI-7A and TaCHLI-7A,which indicated that the amino acid mutation reduced the activity of the protein Tachli-7A.In addition,TaCHLI-7A could restore the chlorophyll efficiency phenotype of Arabidopsis thaliana mutant SALK＿050029,while the mutant gene Tachli-7A could not.These results indicated that the mutation of gene Tachli-7A was the main cause of chlorophyll deficiency in chli at seedling stage.5.Expression pattern analysis of gene TaCHLI.The q RT-PCR results in different tissues of shaannong 33 showed that the expression pattern of TaCHLI showed obvious tissue specificity.The expression level of TaCHLI in green tissue was obviously higher than that in non-green tissue.The expression pattern between chli and shaannong 33analyses showed that there was no significant difference in the expression of TaCHLI at the one-leaf stage.However,although the expression level of TaCHLI was decreased at third-leaf stage in both materials,the expression of TaCHLI in chli was higher than that in shaannong 33.The high expression in chli at three-leaf stage may be one of the main reasons for leaf restore of chli at the later growth stage.6.Transcriptome analysis of mutant chli.Transcriptome analysis was performed on the first leaf of the mutant chli and the control shaannong 33 at one-leaf stage and third-leaf stages.The results showed that 486 differentially expressed genes were identified at one-leaf stage.Compared with chli,there were 295 genes up-regulated and 191 genes down-regulated in shannong 33.At the three-leaf stage,a total of 735 differentially expressed genes were identified.Compared with chli,there were 413 genes up-regulated and 322 genes down-regulated.Functional annotation and analysis of these differentially expressed genes revealed that these differentially expressed genes were mainly involved in the lipid metabolism of cell membrane at the one-leaf stage.In the mutant chli,the differences in these pathways may be related to the formation of pale-green leaf phenotypes in chli.