| Isoetes,the only existing genus of Isoetaceae in Lycopodiophyta,is one of the earliest representative groups of vascular plants and occupies an important position in the whole phylogenetic history of plants.Compared with Lycopodium and Selaginella,previous studies have shown that Isoetaceae have experienced a completely different evolutionary history,from the huge scale Protolepidodendrales woody plants in Carboniferous,via Pleuomeia or Annalepis plant in Triassic,by extremely shortened globular of the stem,vegetative leaves disappear and a series of degradation type evolution,now turned into herbs.Morphological characteristics showed that the adaptive evolution of Isoetes was realized by degenerative evolution of plant size.However,what the corresponding molecular evolutionary events in the process of the stem of Isoetes degenerating from the tall and tough woody stem to the short and woody herbal stem are,whether these genes related to stem growth and development adapted to the environment through changes in the expression regulation pattern or through genetic variation pattern during the evolution of Isoetaceae,related scientific problems need further study.This research studied in three different developmental stages in the stem of Isoetes yunguiensis: seedling stage,growth stage and maturity stage.By using transcriptome method,we discussed the gene expression patterns of stem in different periods,cloned and identified the key functional genes,sequenced and analyzed the structures of related genes,so as to provide a basis for further research.1.Studies on gene expression patterns of stem tissues of Isoetes yunguiensis in different periods(1)15711,14809,16881 Unigene were obtained at seedling stage,growth stage and maturity stage,respectively.There were 4,554 Unigene with period specificity and 13,870 Unigene with constitutive expression.702,632,2131 were expressed only in seedling stage,growth stage and maturity stage,respectively.881 Unigene genes were turned on and 1,333 Unigene genes were turned off during the transition from seedling stage to growth stage,and 2,762 Unigene genes were turned on and 1,140 Unigene genes were turned off during the transition from growth stage to maturity stage.(2)The plant stem tissue present strong synergy in seedling stage,the growth period and mature period showed many advantages function way,the advantage of growth phase function way were translation,the ribosome structure and biological form,post-translational modifications and protein folding,partner proteins,carbohydrates,in addition to the six advantage function way in growth phase,the advantage of mature phase function way was energy metabolism and transformation of energy.In the process of plant from seedling stage to mature stage,molecular level involved in DNA replication,recombination and repair,and translation,posttranslational modifications,protein folding,the ribosome structure and biologynesis,cellular level involved in intracellular transport secretion,transport and metabolism of smaller molecules,the biosynthesis of secondary metabolites,transportation and catabolism,biological process involved in plant response to stimulation,reproduction,growth,energy production,which the number of gene present an increasing trend;The number of gene participate in RNA processing and modification,cell cycle regulation,cell division,chromosomes,defense mechanism and signal transduction mechanism,biological process of cell structure,were a decrease in the growth period and in turn increased after entering mature period,the above differences to a certain extent reflected the plant stem tissue in seedling period and mature period has a strong ability of cell proliferation and differentiation of strong,intercellular contact,intracellular signal transduction,regulation and plant active defence external stimulate biological pathways,This might be related to the more sophisticated morphological regulation of stem tissue in the seedling stage and the more complex reproductive regulation in the mature stage.(3)According to the prediction analysis results of transcription factors,a total of 1534 transcription factors were predicted,mainly clustered into 11 families,most of which,such as AP2/ ERF-ERF,MYB,BHLH,SET and BZIP,were related to vascular tissue development.This indicates that the development of vascular tissue plays an important role in the development of plant stem tissue.2.Analysis and identification of key functional genes for vascular development in stem tissue of Isoetes yunguiensis(1)In this experiment,27 homologous genes related to the development of stem vascular structure were annotated from the stem tissue transcriptome data of Isoetes yunguiensis at different periods,including 7 GRAS family homologous genes: SCARECROW-LIKE 30 ? SCARECROW-LIKE 28 ? SCARECROW-LIKE 18 ?SCARECROW-LIKE 11?SCARECROW-LIKE 7 and SCARECROW;NAC family noted to 5 : NAC105?NAC083?NAC072?NAC071 and NAC002;MYB family notes to 6: MYB3R4,MYB3R-1,MYB30,MYB3,APL2,APL-LIKE;There are three noted in the ARF family: AGD3,GNOM,ARF2A;HD-ZIP III TF family noted to 3: ATHB8,ATHB3,ATHB2;PIN family noted to 2: PIN4,PIN3;RECEPTOR PROTEIN family annotated to 1: WOL/CRE1/AHK4;The LRR-RECEPTOR KINASE family had two annotations: BRI1 and BRI1-LIKE.(2)TA cloning experiment was conducted to obtain the full length of the NAC002 and SCARECROW homologous genes in the stem of Isoetes yunguiensis.Named as Iy NAC002 and Iy SCARECROW respectively.Sequence analysis showed that Iy NAC002 homologous gene of NAC family was composed of 873 bp nucleotide and encoded 291 amino acids.Iy NAC002 homologous genes had the highest homology with Cynara cardunculus and Helianthus annuus in the level of nucleotide sequence and amino acid sequence.In addition,phylogenetic analysis showed that Iy NAC002 homologous genes also clustered together with Cynara cardunculus and Helianthus annuus at the level of nucleotide and amino acid sequence.Whether this indicates that Iy NAC002 homologous genes in isoetes are related to their underdeveloped secondary growth and repeated adaptation process on land and water remains to be further proved.The Iy SCARECROW homologous gene was composed of 1839 bp nucleotide and encodes 613 amino acids.Iy SCARECROW homologous genes had the highest homology with highly developed stem gymnosperms Pinus radiata and Pinus sylvestris,but its coding sequence of amino acids had the highest homology with Selaginella moellendorffii,phylogenetic analysis revealed that Iy SCARECROW homologous gene,at the nucleotide level,and gymnosperms,gathered into a large branch,in the amino acid level,still more near to proximal species Selaginella moellendorffii.Through comparison with Arabidopsis thaliana,nearly 40 amino acid sequence lengths were found to be missing at the 5 'end of the gene,which may be related to the nonsense mutation of the 5' end codon of the gene and the formation of the UAA termination codon,Does this imply that the Iy SCARECROW homologous gene in Isoetes once had similar functions to the gymnosperms with relatively developed vascular tissues,but its gene structure changed during the adaptive evolution process,leading to great changes in the biological functions of proteins,thus leading to the degenerative evolution of stem? Relevant research needs to be further proved.3.This experiment had cloned CCoAOMT from genomic DNA and c DNA in stem tissue of Isoetes yunguiensis,structure analysis showed that: the genome DNA of CCoAOMT contains four introns,five exons,from the initiation codon ATG to termination codon TAG,consistence of 774 nucleotides,encoding 257 amino acids,the molecular weight is 29020.25,theory of protein isoelectric point IP value is 5.20,the three dimensional structure contains two SAM domain and two calcium binding sites.This structure is highly conserved in different plants,and phylogenetic relationship shows that the amino acid sequence of this gene is most similar to that of the closest taxa,Selaginella.CCoAOMT homologous gene plays an important role in lignin synthesis,and its protein structure is highly conserved.In the regressive evolution process of Isoetes plants,the lignin content of the Isoetes was significantly reduced from the trees to the herbs.However,how the Iy CCoAOMT homologous genes regulated the expression in Isoetes still needs to be further proved by relevant studies. |
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