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

Posted on:2024-01-10Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y XinFull Text:PDF
GTID:1523307187965719Subject:Vegetable science
Abstract/Summary:PDF Full Text Request
Chinese cabbage(Brassica rapa L.ssp.pekinensis)is a vegetable crop commonly cultivated in East Asian countries and regions.The leaf is photosynthetic organ and main product organ of Chinese cabbage.Leaf width contributes to the formation and development of the leafy head,and leaf crinkling affects photosynthesis and light energy and premature leaf senescence reduces the quality and yield of leafy head.Therefore,the study of leaf morphology and development has been the focus.Our team used 0.8%EMS aqueous solution to mutagenize the germinated seeds of Chinese cabbage double haploid line(DH line)‘FT’,and created a mutant library containing 718 stable genetic mutation materials.In this study,three kinds of leaf development mutants including narrow leaf mutant lhd,crinkled leaf mutant lcm and leaf premature senescence mutant pls were selected.Based on the analysis of the genetic characteristics of mutant traits,the mutant genes were identified and cloned using Mut Map/BSA-seq combined with KASP genotyping.The function of the candidate gene was verified by allelic variation analysis/Arabidopsis transformation.The main findings in this paper are as follows:1.Chinese cabbage Br AN encoding ANGUSTIFOLIA was cloned,and its function in regulating leafy head formation was verified by allelic mutantsTwo narrow leaf mutants lhd1 and lhd2 were screened from the EMS mutant library of heading Chinese cabbage.They were crossed with the wild type respectively to obtain lhd1 F1and lhd2 F1,then lhd1 F1was crossed with lhd2 F1.The results suggested that the mutant traits of both were controlled by a single recessive nuclear gene.Using Mut Map combined with KASP genotyping,Bra A10g000480.3C was predicted to be the candidate gene.This gene encodes a C-terminal-binding protein ANGUSTIFOLIA,which is involved in cell expansion in the leaf-width direction,and it was named Br AN.The cloning and sequencing results of the candidate gene showed that a base substitution(G→A)was occurred in the second intron of Br AN,resulting in a 98 bp second intron sequence retained in the CDS sequence,and the retained sequence contained a TGA stop codon,causing early termination of amino acid coding in lhd1.In lhd2,a base substitution(G→A)occurred at base 1855 of the fourth exon of Br AN,resulting in a stop codon(TAG)and premature termination of amino acid coding.The results of the two alleles mutants confirm that the narrow-leaf and non-heading phenotypes were caused by Br AN mutations.2.The results of Arabidopsis transformation experiments demonstrated that Br AN influences cell expansion direction and regulates leaf width by regulating microtubule arrangementThe promoter of Br AN,Br AN in‘FT’and Bran in lhd1 were used to construct recombinant vectors and transform to Arabidopsis mutant an-t1.The results proved that the wild-type gene Br AN could restore the narrow leaf phenotype of an-t1,while the mutant gene Bran could not.Further observation of cell morphology and microtubule arrangement using laser confocal microscopy revealed that pavement cells were jigsaw-shaped with randomly arranged intracellular microtubules in Col-0,while shape of cells was more regular,and the microtubules were arranged parallel to leaf width direction in an-t1.Br AN could restore adnormal cell morphology and microtubule arrangement of an-t1,but Bran could not.These results indicate that Br AN regulates leaf width by regulating the arrangement of microtubules and affecting the expansion of cells in the leaf width direction.3.The candidate gene for leaf crinkle mutant lcm was identified as Br SCRM by BSA-Seq combined with KASP genotypingThe crinkled leaf mutant lcm was dwarfed and had shrunken leaves.Genetic analysis proved that the crinkled leaf trait was controlled by a pair of incompletely dominant gene.Combining BSA-Seq with KASP genotyping,Bra A06g038100.3C was predicted as a candidate gene,which encodes the transcription factor SCRM and regulates stomatal development,and it was named Br SCRM.The results of cloning and sequencing showed that a G→A substitution occurred in the first exon of Br SCRM,resulting in the conversion of arginine to histidine.Br SCRM was expressed in all organs in‘FT’.The results of subcellular localization showed that Br SCRM localized in the nucleus.4.The results of Arabidopsis transformation experiments proved that Br SCRM mutation caused a surge in stomatal number and affect leaf expansionCytological observations showed that compared with the wild type,the stomatal density on the leaves of the mutant lcm was significantly increased.Stomatal density on the leaves of the F1 generation plants was intermediate between the two parents,verifying that the mutant trait was controlled by a pair of incompletely dominant gene.When the mutant gene Brscrm was transferred into Arabidopsis wild type Col-0,the stomatal density on the leaves of the transgenic plants increased significantly,and the leaves were shrunken and curled.These results further proved that the overdevelopment of stomata affected leaf expansion and resulted in shrunken leaves of the whole plant.5.Chinese cabbage premature leaf senescence gene Br SRFR1 was cloned,and its function was verified by sequence variation analysis of allelic mutantsAt the late heading stage,the outer leaves of Chinese cabbage gradually senescent and yellowed,while the leaves of the premature senescence mutants pls1 and pls2 showed senescence and yellowing phenotype at the early heading stage.Genetic analysis showed that the mutation trait was controlled by a single recessive nuclear gene,and the mutant trait was controlled by the same gene in two mutants.Using the Mut Map and KASP genotyping verification,Bra A01g001400.3C was predicted to be a candidate gene for pls1,which encodes the protein SRFR1 containing the tetratricopeptide repeat domain and involves in the regulation of immune response.It was named Br SRFR1.Cloning and sequencing results showed that a C→T base substitution occurred in exon 7 of Br SRFR1,resulting in a stop codon TGA and early termination of amino acid coding in pls1.A base substitution G→A occurred in exon 16of Br SRFR1,leading to a stop codon TGA and premature termination of amino acid coding in pls2.6.Immune regulator gene Br SRFR1 was found to negatively regulate leaf premature senescence in Chinese cabbage for the first timeAt SRFR1 was an immune adapter protein that plays a negative role in immune regulation.In the Arabidopsis accession RLD,mutation in At SRFR1 enhanced plant resistance to Spodoptera exigua(S.exigua).The results of insect resistance identification in our study showed that pls mutants had significantly higher resistance to S.exigua than the wild type,verifying its role in immune regulation.pls mutants leaves showed earlier senescence than the wild type,chloroplast disintegration and a significant decrease in chlorophyll content.Expression levels of Br SRFR1 increased gradually with leaf development and was highest at the late heading stage.RNA-Seq analysis showed that multiple senescence-related genes were up-regulated in the mutant,a large number of photosynthesis-related genes were down-regulated,and multiple key genes in jasmonic acid(JA)and salicylic acid(SA)signaling pathways were differentially expressed.The content of JA and SA in the mutant leaves was significantly higher than that in the wild type.These results suggested that the premature-senescence phenotype of pls mutants might be related to the synthesis of JA and SA signal transduction pathways.
Keywords/Search Tags:Chinese cabbage, leaves, leafy head, stomata, premature senescence
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