| Chinese cabbage is one of the most important vegetables in China.In the growth process of Chinese cabbage,the leaves of seedling stage and rosette stage spread out and grow,while the leaves of heading stage grow upright and embrace inward to form the head leaf In recent years,with the development of molecular biology technology,the leaf growth mechanism of Chinese cabbage has been understood,but the regulation mechanism of leaf growth and development is still unclear.The leaf curl growth of del1(downward-curling leaf 1)mutant of Chinese cabbage grew slowly during the seedling stage.The correlation between leaf curling and smalland photosynthetic rate and hormone content in Chinese cabbage was revealed by phenotype identification and physiological index determination.Genetic analysis of roll leaf and small traits was carried out by F2 isolation population,candidate genes were located by MutMap and KASP genotyping techniques,and transcriptome sequencing techniques were used to analyze the expression differences of different genes in different developmental stages at the transcription level.The main results are as follow:1.At the seedling stage,the leaves of the mutant del1 were curled outward and downward.The earlier the leaves developed,the higher the curling index was.The leaf length and leaf width of del1 were significantly lower than wild-type WT,and the plant height,plant spread,head height and head weight were also significantly lower than wild-type WT.In terms of physiological characteristics,the total contents of chlorophyll a,chlorophyll b and chlorophyll were not significantly different from that of the wild type.The net photosynthetic rate,transpiration rate,transpiration and stomatal conductance were all lower than those of the wild type,but there was no difference in intercellular CO2 concentration.The contents of abscisic acid,auxin and salicylic acid in the curly leaves of seedling stage were significantly lower than that of wild type.2.According to F1 traits,the curling leaf and small plant of the mutant are semi-dominant heredity.The curling leaf traits about F2 separation population of the mutant was investigated,which conforms to the separation ratio of 3:1 and the BC1 conforms to the separation ratio of 1:1.MutMap technology was used to sequence the individual hybrid pool of the F2 isolated population with extreme traits,and the candidate gene range was defined as 292291-391666 bases on A06,and the candidate gene was determined as Bra06g003880.3C by KASP typing.3.F2 separation population was created by using the inbred line parents 85-1 and dcl1 commonly used in breeding,and gene location of small traits was conducted together with the previously constructed dcl1 and wild-type F2 population to obtain the same candidate gene range as the leaf curl.By KASP detection,the traits of Bra06g003880.3c in small F2 population were consistent with the genotype.It was speculated that the genes of small and roll leaf in dcl1 were the same gene.4.The DNA of Bra06g003880.3c gene is 1226 in length,and the 442nd base changes from A to G.This mutation occurs at the selective splicing site of the gene,resulting in an error in gene splicing,the retention of introns containing terminators,and early termination of gene translation.5.Transcriptome sampling was carried out according to different periods of leaf growth.At the early stage of leaf development,GO enrichment resulted in significant differences in photosynthesis-related classifications such as thylakoid membrane and thylakoid.KEGG enrichment results showed that PsbQ and other genes were down-regulated in the photosynthetic pathway of dcl1.The transcriptional levels of the enzymes involved in the photosynthetic carbon sequestration pathway were down-regulated.This may result in a decrease in the amount of dry matter accumulated in the leaves.At the late stage of leaf growth,the transcriptional level of material transporter-related genes changed significantly.The damage of photosynthetic performance may be the key to the significant reduction of dcl1 plants.6.The second period of transcriptome sampling is the roll generation period,during which auxin and abscisic acid synthesis and response are active,especially the up-regulation of auxin synthesis YUCCA,which can lead to auxin accumulation.In the auxin response pathway,21 genes were enriched and 20 transcription levels were significantly up-regulated.After the injection of exogenous auxin,the leaves of Chinese cabbage were curled downward.After the injection of auxin transport inhibitors,the leaves changed from upright to flat.Therefore,auxin accumulation and transport may be the key to leaf curl. |
