Yellowing Mutant Bnyl Mapping And Transcriptome Analysis Of Brassica Napus L.

Photosynthesis is the basis for green plants to obtain solar energy to convert them into chemical energy,which provides energy for plant growth and development.Leaf color mutants have reduced photosynthetic efficiency due to the photosynthetic pigment content,which has a severely negative impact on crop growth and economic product yield.The study of leaf color mutants is helpful to clarify the relevant regulatory mechanisms of chloroplast development and photosynthesis.Leaf color mutants are also an ideal material for studying plant photosynthesis and photomorphogenesis.It has importantly theoretical significance for increasing crop yield through genetic engineering.Besides,as a marker of trait,leaf color mutants play an important role in genetic breeding,functional genomics and plant physiology.In this study,yellow leaf mutants of Brassica napus L.obtained by ethyl methyl sulfone（EMS）mutagenesis were used as materials to conduct field phenotypic observation and physiological index determination.At the same time,The BSA method based on the whole genomic re-sequencing technologies was performed to screen the candidate genes,and the regulation mechanism of yellow leaf mutation was analyzed by transcriptome sequencing.The main findings are as follows:1.In this study,phenotypic observations of yellow leaf mutants of B.napus showed that the cotyledons and true leaves of yellow leaf mutants appeared yellow-green after emergence,and were accompanied by reduced height and slower growth,delayed growth and reduced seed yield.2.The spectrophotometer method was used to determine the content of chlorophyll and chlorophyll synthesis precursors on leaves of mutants and ZS 11.The mutants had significantly lower chlorophyll a,chlorophyll b,total chlorophyll,and carotenoid content than the wild type.The content of ALA and PBG in chlorophyll synthesis precursor material was not significantly different from that of wild type,but the content of tetrapyrrole such as Proto IX,Mg-proto IX,and Pchlide was significantly accumulated.ALA is known as the first direct precursor of chlorophyll synthesis,Pchlide is the most direct precursor of chlorophyll synthesis,suggesting that chlorophyll synthesis is hindered at the late stage of synthesis.The reduction of chlorophyll content leads to the yellow leaf phenotype of plant leaves.As a light-sensitive substance,a large amount of free tetrapyrrole is prone to produce singlet oxygen and has a toxic effect on plants.3.Using a fluorescence microscope to observe the cross-sections of mutants and wild-type leaves,it was found that the mutants had sparse chloroplast distribution and weak auto-fluorescence.The transmission electron microscope photographs revealed that the mutant’s chloroplast shape changed from spindle to oval,the number of thylakoid membranes and basal granule stacks was reduced and the shape was extremely irregular,the basal granules were sparse,and the number of euphilia increased significantly,and volume became larger and the starch granules decreased significantly,indicating that the mutant’s chloroplast photosynthetic membrane structure was aging and its function was obviously defective.4.Photosynthetic characteristics of mutant and ZS11 leaves were analyzed.The mutant’s net photosynthetic efficiency,stomatal conductance and transpiration rate were all significantly reduced,but the intercellular CO₂ concentration was similar.It shows that the cause of the decrease in net photosynthetic efficiency is not the stomatal factors such as the decrease in stomatal conductance and insufficient supply of CO₂,but the poor development of chloroplasts inside the mesophyll cells and damage to the photosynthetic membrane organs.Using Flour cam to analyze the fluorescence kinetic parameters of the leaves,it was found that Fo and Fm of the mutant were lower than the wild type,and this difference was more obvious on the flat leaves,which verified that the mutant had a reduced chlorophyll content.The lowerΦPSII and ETR of the mutant indicates that its light energy conversion efficiency is lower than that of the wild type,and the Rfd is slightly lower than that of the wild type indicates that its leaf viability is lower.There is no difference in Fv/Fm between the two,indicating that the plant growth environment is good and not stressed.5.The yellow leaf mutant and WH-60 were crossed to construct a sequencing population,the F₁ generation self-crossed to generate the F₂ generation,plants with yellowing and normal phenotypes in F₂ generation were selected to establish DNA mixed pools for deep resequencing,and sequencing results showed that the candidate gene was located at 2.72Mb between 3.36-6.07Mb of A01 chromosome,a total of 502genes are located in the candidate region,of which 277 genes contain mutated SNP sites that can cause the changes of protein sequence.6.Design new SSR primers in the candidate region obtained by resequencing,and use SDS-polyacrylamide gel electrophoresis to narrow the candidate interval to the vicinity of the marker bna108 through two sets of different hybridization combinations.Mapping the gene Bna A01g07280D which is encoding atypical thioredoxin（ACHT2）,then cloning the Bna A01g07280D gene fragment in the yellowing mutant confirmed that there was a frameshift deletion on the gene fragment,which was consistent with the resequencing results.It is speculated that Bna A01g07280D is a candidate gene for yellowing mutants.7.To analyze changes at the transcriptome level of yellowing mutants,we performed RNA-Seq on leaves of wild-type and yellowing mutants.A total of 1273differentially expressed genes were identified,of which 624 were up-regulated and 649were down-regulated.Through KEGG analysis,it was found that the differential genes were mainly enriched in phenylpropane metabolism and sugar metabolism.Among the differentially expressed genes related to photosynthetic system,the expression level of Bna Anng22920D gene was severely reduced,and the expression levels of Bna C02g42890D and Bna Cnng19490D genes were both reduced to zero.They encode the apoprotein Lhcb that binds to the photosynthetic system II（PSII）,and the important constituent subunits PSAN and PSAF proteins of the photosynthetic system I（PS I）.It is speculated that the mutation of the candidate gene Bna A01g07280D may indirectly affect the expression of chloroplast development-related genes CAB,PSAN and PSAF,resulting in impaired chloroplast photosynthetic system development and reduced chlorophyll synthesis.