Map-Based Cloning And Functional Analysis Of The Chlorophyll-Deficient Gene BnaA02.YTG1 In Brassica Napus

Chloroplast is the place for photosynthesis,its biological process is very complex and its regulatory mechanism is still elusive up to now.It is very critical to the growth and development of plants whether the chloroplast function is normal or not.The yellow-leaf mutants have important basic research values,they are ideal materials for studying chlorophyll metabolism,chloroplast structure and functions,which helps people to better explore and use the mechanism of photosynthesis.It is the most common type of leaf-color mutants.In this study,we obtained a chlorophyll-deficient mutant of Brassica napus,named ytg.Subsequently,the gene,named BnaA02.YTG1,was successfully cloned by phenotypic analysis,genetic analysis,and gene mapping.Then functional analysis of it was performed.The main findings were as follows:1.Phenotype and agronomic traits characterization of ytg mutantytg,a chlorophyll-deficient mutant isolated in the progenies of the restorer recurrent selection population.Compared with 4608 with normal leaf color,the cotyledons and newly emerged true leaves of the mutant were yellowish and became greening with development.The mature leaves could recover the green color to a certain extent just like4608.Compared with 4608 leaves at the same stage,the chlorophyll(Chla and Chlb)and carotenoid content of the newly emerged true leaves in ytg mutant were significantly reduced and then gradually increased with the growth and development of plants,and finally similar to that of mature leaves(top fourth and fifth leaves).In 4608,the chloroplasts showed a fusiform shape and had well-structured thylakoid membranes in both the top first and fourth leaves.However,in the mutant,the thylakoid membranes in the chloroplasts of the top first leaves were much less abundant and chloroplast development was strunked.In contrast,the thylakoid membrane organization and abundance in the chloroplasts of the top fourth leaf of the mutant were comparable to those in 4608.Similarly,the buds and siliques exhibited a delayed greening phenotype.Plants with yellow leaves(NIL-ytg)and normal leaves(NIL-4608)in the BC₄F₁population were investigated for agronomic traits.The results showed that the TSW,PH,LMI,SN,BN,oil content of NIL-ytg were significantly lower than those of NIL-4608 and LA content was the opposite.2.The mapping and knockout of BnaA02.YTG1Genetic analysis revealed that yellowish-leaf phenotype of the ytg mutant conferred by a single gene mutation coding by nuclear.The segregation populations were developed by crossing ytg mutant and 4608,and the gene was further located within a physical region of 9.9 kb,which included three putative genes.The nearest markers were 02-20 and 02-21.Three complementary vectors were constructed,respectively.Genetic transformation experiments proved that BnaA02g10480D,the ortholog of at WTG1(AT5G53080),corresponds to BnaA02.YTG1 which controls the leaf color phenotype and was named according to the phenotype of the ytg mutant.According to the ZS11 reference genome information,BnaA02.YTG1 and its homologous copy,BnaC02.YTG1,were 89.6%and 90.4%identical at the nucleotide and protein levels,respectively.Two single guide RNAs(sg RNAs)that target the coding regions of the BnaA02.YTG1 and BnaC02.YTG1 were designed.The results showed that transgenic lines,only edited in BnaA02.YTG1 but not BnaC02.YTG1,showed phenotypes similar to the ytg mutant.These results demonstrated that BnaA02.YTG1 was responsible for the leaf phenotype in ytg mutant.3.The expression pattern of BnaA02.YTG1According to the results of qPCR and GUS staining,BnaA02.YTG1 was mainly expressed in young tissues and lower expressed in relatively mature tissues.Corresponding to leaf color phenotypes,BnaA02.YTG1 was not expressed in the mature siliques and seeds.The above results indicate that BnaA02.YTG1 mainly played a role in the young tissues.RNA-seq was performed on the first(HB1)and fourth leaves(HB4)of the transgenic-positive plants,the first(hb1)and fourth leaves(hb4)of the transgenic-negative plants in the transgenic complementary PT1 line.Analysis of differentially expressed genes(DEGs)and found that DEGs in the top first leaves were mainly mapped to terms related to chloroplast membrane systems.qPCR confirmed the reliability of the transcriptome results.In addition,the subcellular results indicated that BnaA02.YTG1 was located in the chloroplast.4.BnaA02.YTG1 might be involved in chloroplast RNA editingBnaA02.YTG1 encodes a tetratricopeptide repeat(TPR)protein.The yeast two-hybrid technology was performed and found that BnaA02.YTG1 could interact with RNA editing factors MORF2,MORF8,OZ1,and chloroplast membrane transporters Toc33,Toc34,and Tic22-III.These results were also verified by SLC experiments.To predicte the RNA editing sites in rapeseed chloroplast genome,homologous alignment method was selected and 29 sites were found.We detected RNA editing level of HB1 and hb1 and found that the editing level of ndh D-2,ndh F-290,pet L-5,and ndh G-50 in hb1 was significantly lower than that of HB1,while the editing level of hb4was increased,which was similar to that of HB1.This indicated that RNA editing was affected in ytg mutant and BnaA02.YTG1 may affect RNA editing.5.Function study of BnaC02.YTG1The homologous copy of BnaA02.YTG1 in Brassica napus is BnaC02.YTG1.We comparatively sequenced BnaC02.YTG1 in 4608 and ytg mutant.A 698-bp h AT transposon insertion was found in the third exon.The transcription of BnaC02.YTG1 was started from the position after the insertion of the h AT transposon,indicating that the transcription of BnaC02.YTG1 was abnormal in parents.Overexpression of BnaC02.YTG1 in ytg mutant rescued the leaf color phenotype.The subcellular results indicated that BnaC02.YTG1 was located in the chloroplast.These results suggested that BnaC02.YTG1 was also required for early chloroplast growth and development and probably showed functional redundancy with BnaA02.YTG1.