SPOT1 Gene Is A Key Gene Regulating The Development Of Pollen Lining In Arabidopsis Thaliana

The pollen wall is constituted of the outer layer exine and the inner layer intine.The exine is a layer of special material on the male germ cell surface which not only plays important roles in protecting pollen from various environmental stresses and microorganism attacks,but also in the pollination:recognition,adhesion,hydration and germination.The intine is consist of cellulose,pectin and protein,which is more complex than the normal cell wall.We have the deeper understanding about outer exine genes and their regulation mechanism.However,the mechanism of intine formation remains poorly undestood.In order to study the molecular mechanism of intine development,we have bought 7lines mutants of At5g58100(salk?072486 ? salk?073032 ? salk?041237 ?salk?072490 ?salk?079847? salk?061320c and salk?041228)from the SALK mutant library.We screened T-DNA insertion homozygous mutants from lines of salk?072490?salk?079847 and salk?061320c by SEM(Scanning Electron Microscopy)observation.In these lines,pollen grain is distorted and shrunken.Exine elements appeared to be largely disconnected like spotty.And this phenotype is closely-linked to the T-DNAinsertion.So we called spot1-1(salk?072490),spot1-2(salk?079847),spot1-3(salk?061320c.The main research contents were shown as follows:1.Identification of T-DNA insertion and genetic complementation test:T-DNA insertion site identification showed that the T-DNA was inserted in 3502bp(spot1-1),and 4453bp(spot1-2)and 5657(spot1-3)respectively.The pro SPOT1::SPOT1-GFP/ spot1 transgenic plants can develop pollen grains with normal plllen wall.By quantitative RT-PCR(q RT-PCR)analysis,we found that the expression of SPOT1 gene in spot1 was droped down.While the expression of SPOT1 gene in the pro SPOT1::SPOT1-GFP/ spot1 transgenic plants just as the wild type.This result suggests that the T-DNA insertion is the direct reason to SPOT1 expression and defective pollen wall in spot1 mutants.2.A primary analysis of SPOT1 gene function? The amino acids sequence analysiss: SPOT1 gene contained 2289 bp and2838 bp open reading frame(ORF),encoded 945 am ino acids.SPOT1 was located in cytoplasm by analyzing for subcellular localization.There were only two trans membrane domains.SPOT1 was similarly to Arabidopsis lyrata's and Capsella rubella's homologous protein.And their function was unkown too.? Defective formation of the intine layer in spot1 mutants:Fluorescence microscopy analasys to ultrathin section indicated that there is blue fluorescence of callose of spot1-3 mutant but not yllow-greenish(col-0)at the 6-7 stage of anther development.While the blue fluorescence of cellulose of spot1-3 mutant was out compared to col-0 at 11 stage.Additionally,observed from TEM(Transmission Electron Microscope),the intine of spot1-3 microspores is thiner than col-0 at 11 stage,and the intine structure is changed too.By q RT-PCR analasys,we found that SPOT1 induced CALS5 and CESA1,CESA3,CESA6 expression.Therefor,it could be concluded that SPOT1 has effect on the development of exine and intine through repress CALS5,CESA1,CESA3 and CESA6.