Cloning And Identification Of Leaf Wrinkled Genes In Chinese Cabbage(Brassica Rapa L.ssp.pekinensis)

Leaf is the most important photosynthetic organ in plants.Its shape and function directly determine crop productivity by affecting plant architecture,respiration,transpiration and nutrient absorption.The leaves of Chinese cabbage（Brassica rapa L.ssp.pekinensis）are not only photosynthetic organs but also the main product organs,and properly wrinkled leaves can improve the yield and quality of the plant by improving its light energy utilization efficiency.Given that mutants are important materials for exploring gene functions,this study used EMS to induce the DH line’FT’of Chinese cabbage and obtained three leaf wrinkled mutants,cwm,lwm,and bwm.The mutant genes were mapped based on BSR-Seq or Mut Map methods,and the candidate genes were predicted based on gene function annotation information.The functions of candidate genes were validated using Arabidopsis heterologous transformation or parallel variation analysis.q RT-PCR was used to analyze the expression patterns of mutant or candidate genes.1.The mutant gene Brcwm of wrinkled leaves,which encodes the protein TON1related to cortical microtubule tissue,was cloned from Chinese cabbage.The mutant cwm of Chinese cabbage obtained by EMS mutagenesis showed wrinkled leaves,compact architecture and dwarf phenotype.Genetic analysis manifested that the mutant trait was controlled by a single recessive nuclear gene.Through BSR-seq analysis,the mutation gene Brcwm was initially located within 3 intervals on A07 chromosome and 1interval on A05 chromosome.Further,SSR and Indel markers were used to anchor mutation genes into the 205.66kb interval between Indel 12 and Indel 21 on chromosome A07,which contains 39 genes.Considering that there were no available molecular markers in the candidate interval,the whole-genome resequencing was used to explore SNP variations.The results showed that only the fourth exon of Bra A07g021970.3C in the target interval had a non-synonymous base substitution from C to T,resulting in the replacement of proline（Pro）with serine（Ser）.The results of co-segregation validation indicated that the mutant trait was co-segregated with this SNP,thus predicting Bra A07g021970.3C as the candidate gene.Bra A07g021970.3C is a homologous gene with AT3G55000,which encodes the cortical microtubule tissue related protein TON1 in Arabidopsis.Dwarfing and wrinkled leaves phenotypes similar to cwm were observed in the recessive homozygous Arabidopsis mutant cwm-f1 with TON1 deletion.Overexpression vector 35S:Br CWM:GFP was constructed and heterologous transformation was performed in Arabidopsis.Using the c DNA of T3generation transgenic plants as a template and using c DNA clone primers of Bra A07g021970.3C,PCR amplification was performed to identify T3 generation transgenic plants that stably express Bra A07g021970.3C.Ectopic overexpression detection of the Arabidopsis mutant cwm-f1 revealed that the transgenic line Br CWM OX/cwm-f1 reverted to the wild-type Arabidopsis phenotype,indicating that Bra A07g021970.3C was the target gene for the trait of wrinkled leaves in the cwm.The results of q RT-PCR showed that the expression of Bra A07g021970.3C in cwm leaves was significantly lower than that in‘FT’leaves.Paraffin section observation revealed that compared to the wild-type’FT’,the mutant cwm had more vascular bundles in the mesophyll cells,thicker leaves,and larger gaps between sponge cells and palisade cells.At present,there is only research on cortical microtubule related protein in the roots,and the lack of function of this protein can cause short and small plant roots.This study provides the first evidence that the functional loss of cortical microtubule related proteins can bring about wrinkled leaves.2.The mutant gene Brlwm of wrinkled leaves,which encodes H⁺-ATPase2,was cloned from Chinese cabbage.The mutant cwm of Chinese cabbage obtained by EMS mutagenesis showed wrinkled leaves,compact architecture and dwarf phenotype.Genetic analysis manifested that the mutant trait was controlled by a single recessive nuclear gene.By Mutmap technology,the candidate regions related to mutation traits were reduced to 3.41 Mb on A01 chromosome,with 4 non synonymous SNPs distributed across 4 genes.The KASP validation results indicated that the mutant trait was co-segregated with Bra A01g007510.3C,and it was predicted to be the candidate gene for the mutant trait.Sequence analysis showed that the Bra A01g007510.3C of mutant had a non-synonymous SNP mutation from G to A in exon 4,resulting in a substitution of threonine（Thr）to isoleucine（Ile）.The Bra A01g007510.3C homologous gene in Arabidopsis is AHA2,encoding H⁺-ATPase2,which is closely related to plant growth and development.In the early stage,our team used another mutant lcm similar to lwm with wrinkled leaves in Chinese cabbage for gene mapping,and also predicted that Bra A01g00751.03C was the candidate gene for wrinkled leaves.In this study,hybridization between lcm and lwm found that the F₁ generation still exhibited wrinkled leaves,indicating that the mutant genes of lcm and lwm were allelic,and Bra A01g00751.03C was responsible for the mutant phenotype.The results of q RT-PCR analysis revealed that the expression of Bra A01g007510.3C in lwm leaves was significantly higher than that in wild-type’FT’leaves.3.The mutant gene Brbwm of wrinkled leaves,which encodes cytokinin oxidase/dehydrogenase CKX,was finely mapped in Chinese cabbage.The mutant bwm of Chinese cabbage obtained by EMS mutagenesis showed wrinkled leaves,compact architecture and dwarf phenotype.Genetic analysis manifested that the mutant trait was controlled by a single recessive nuclear gene.The results of Mutmap gene mapping indicated that the mutant gene was located in a 2.11 Mb interval on chromosome A04,with only one non-synonymous SNP in this region.The KASP validation results indicated that the mutant trait was co-segregated with Bra A04g029320.3C,and it was predicted that Bra A04g029320.3C was the candidate gene.Bra A04g029320.3C encodes cytokinin oxidase/dehydrogenase（CKX）,which is a key enzyme regulating plant growth and development.There was a C-T mutation in the first exon of Bra A04g029320.3C,resulting in the replacement of proline（Pro）with serine（Ser）.The results of q RT-PCR revealed that the expression of Bra A04g029320.3C in bwm leaves was significantly lower than that in‘FT’leaves.