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Research On The Function Of Arabidopsis SKS Family Genes In Pollen Development

Posted on:2022-06-21Degree:MasterType:Thesis
Country:ChinaCandidate:Y W WuFull Text:PDF
GTID:2480306347992169Subject:Genetics
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Pollen development is a complex and delicate process,which is regulated by many genes.The SKS(SKU5-similar)family is a class of proteins that are similar in sequence to the Multicopper Oxidase(MCO)protein,but they lack the histidine residues necessary to bind copper ions,so they do not use the enzyme features.At present,Nineteen members of the SKS family have been identified in Arabidopsis thaliana,including SKU5 and SKS1-SKS18.Among them,AtSKS11-AtSKS14 is the flower-specific expression SKS gene.In this paper,semi-quantitative and quantitative RT-PCR,subcellular localization analysis,T-DNA insertion mutant phenotype analysis,etc.are used for their functions in pollen development and polar growth of pollen tubes.Preliminary studies have been carried out on the molecular mechanism and the main research results are as follows:(1)AtSKS11-AtSKS14 gene is vegetative cell specific geneWe verified that AtSKS12/13/14 was specifically expressed in floral organs by RT-PCR.Using bioinformatics analysis,it was found that its promoter region contained the cis-regulatory element of the late pollen gene "pollen Lat 52",which should function in the late stage of pollen development.We constructed a fusion expression vector of AtSKS12 promoter and H2B-GFP gene,transformed Arabidopsis thaliana by Agrobacterium-mediated flower soaking method,and obtained homozygous transgenic plants through resistance screening and PCR verification.GFP fluorescence observation results showed that,driven by the AtSKS12 promoter,H2B-GFP was specifically expressed in vegetative cells from the twin stage,and its expression continued until the pollen matured and the pollen tube grew after pollination.(2)AtSKS12?AtSKS13 are involved in pollen maturation,pollen germination and pollen tube growth.In order to explore the function of AtSKS12-AtSKS14,we used sks12,sks13 mutant as materials to observe the phenotype caused by the deletion of the SKS gene.The results showed that there was no obvious phenotype for the single mutations of these two genes,indicating that these genes may have functional redundancy.Therefore,we crossed these two single mutants and tested the segregation ratio of the F2 generation.The results showed that the sks12 sks13 double mutant genes transmitted through the male gametophyte were defective,which meant that AtSKS12 and AtSKS13 played an important role in male reproductive development.After a lot of selfing screening on the F3 generation,we finally got the homozygous double mutant of sks12 sks13.Observing the phenotype of the sks12 sks13 double mutants,it was found that the vegetative growth of the mutant plants was normal,but the number of seeds in the silique was reduced,and there were a certain degree of underfilled seeds,indicating that the double mutants had a certain degree of reduced fertility.In order to further explore the reasons for the decreased fertility,we observed the pollen morphology and mitotic development process of the sks12 sks13 double mutants.The results showed that there was no obvious abnormality in pollen morphology and cell division,and normal triplet pollen could be formed.The pollen was stained with FDA/PI to investigate its pollen activity.The results showed that the pollen of sks12 sks13 double mutants had strong activity.Observing the pollen tube orientation results also showed that the pollen of these mutants could enter the ovule correctly.But in vitro pollen germination and growth experiments showed that the pollen germination rate of sks12 sks13 double mutants decreased,the growth of pollen became slower,and the arrangement of callose plugs was irregular.Further in vivo pollen tube growth survey results also showed that the growth rate of sks12 sks13 double mutants pollen in the stigma was much lower than that of the wild type.These results all indicate that AtSKS12?AtSKS13 play an important role in pollen maturation,pollen germination and pollen tube growth.(3)AtSKS12 can interact with the proteins encoded by pectin esterase and late pollen gene Lat52In previous studies in the laboratory,it was discovered through Co-IP MS experiments that cotton SKS protein GhPSP231 can form protein complexes with many proteins.Therefore,we conducted homology comparisons through bioinformatics,isolated the genes encoding these proteins in Arabidopsis thaliana,and used the BiFC experiment for protein interaction analysis.The results showed that AtSKS12 can interact with the proteins encoded by pectin esterase and late pollen gene Lat52.The interaction of AtSKS 11?AtSKS 13?AtSKS 14 with these two proteins is still under verification.(4)Creation and detection of gene edited mutantsJudging from the results of our functional analysis experiments and the expression patterns of the four SKS genes AtSKS11?AtSKS12?AtSKS13?AtSKS14,they have functional redundancy.However,SKS11 and SKS13 are closely linked on the same chromosome,and SKS1 2 and SKS 14 are closely linked on the same chromosome.Multiple mutants cannot be obtained by hybridization.Therefore,in order to obtain sks11 sks12 sks13,sks12 sks13 sks14 three mutants and sks11 sks12 sks13 sks14 four mutations We use the CRISPR Cas9 gene editing system to mutate AtSKS11 and AtSKS14 in the context of sks11 sks13 double mutants.At present,we have obtained related heterozygous triple mutants,and homozygous strains are being separated and identified.
Keywords/Search Tags:Arabidopsis thaliana, SKS gene, pollen development, pollen tube growth
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