Genome-wide analysis of laccase genes in moso bamboo ... Conclusion A total of 23 laccase proteins were

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  • Genome-wide analysis of laccase genes in moso

    bamboo highlights PeLAC10 involved in lignin

    biosynthesis and in response to abiotic stresses

    Dr. Zhimin Gao

    International Center for Bamboo and Rattan

    State Forestry and Grassland Administration of China

    2019.10.02. Sao Paulo, Brazil

  • Content  Introduction

     Materials and methods

     Result and analysis

     Conclusion

     Acknowledgements

  • Introduction

  •  Woody bamboo is a wonderful gift of nature

    widely used for many things, which is

    considered as a good alternative to wood.

    Introduction

  •  Distribution of all woody bamboo

     The woody bamboos are much more widely distributed all

    over the world, there are 1521 woody bamboo species.

    Introduction

  •  China has more than 30 genera and more than 500 bamboo

    species, with is 6.01 million hectares bamboo forest area.

     Bamboo wood substitutes ~20% timber.

    Introduction

  • Woody bamboo has fine features

    Introduction

  • Excellent materiality

    Introduction

  •  The graded structure: vascular bundle and

    parenchyma

     The components: lignin, cellulose and hemicellulose

    Introduction

  •  The draft genome of moso bamboo

    Introduction

  • http://gigadb.org/dataset/100498

    Contigs N50=53.293 Kb Scaffolds N50=79.90 Mb

    51,074 Genes

    Introduction

    GigaScience, 2018, 7(10): giy115.

     Resequencing of moso bamboo

  • Lignin synthesis pathway

    Introduction

  • Materials and methods Plant materials  Phyllostachys edulis

  • Materials and methods Plant materials

     Arabidopsis thaliana (Col-0)

  • Materials and methods  Methods

     Bioinformatics

     Molecular biology

  • The family number in lignin biosynthesis pathway

     A total of 12 families in lignin biosynthesis pathway were identified using four

    genomes of A. thaliana, B. distachyon, O. sativa and P. edulis.

    Result and analysis

    Family A. thaliana B. distachyon O. sativa P. edulis 4CL: 4-coumarate CoA ligase 12 13 12 15 C3H: Coumarate 3-hdroxylase 3 1 1 3 C4H: Cinnamate 4-hydroxylase 1 2 3 6

    CAD: Cinnamyl alcohol dehydrogenase 9 7 10 14

    CCoAOMT: Caffeoyl-CoA 3-O-methyltransferase 4 7 6 9

    CCR: Cinnamoyl-CoA reductase 3 9 12 17

    COMT: Caffeic acid 3-O-methyltransferase 11 4 6 4

    F5H: Ferulate 5-hydroxylase 1 4 5 16 HCT:Hydroxycinnamoyl-CoA 3 12 6 16

    LAC: Laccase 16 22 20 41 PAL: Phenylalanine ammonia lyase 4 9 8 12

    CHS: Chalcone synthase 4 7 17 12

    POD: Peroxidase 45 44 37 77

    Total 116 141 143 242

  • Result and analysis Phylogenetic analysis of LACs

    P e L A

    C 3

    96

    AtLAC7

     A total of 23 laccase

    proteins were identified

    in moso bamboo and

    clustered into five large

    groups according to the

    phylogenetic tree

    constructed on the basis

    of LAC sequences of

    Arabidopsis, rice,

    Brachypodium and

    moso bamboo.

  • Result and analysis Gene structure analysis of PeLACs

     23 PeLACs have diverse gene structures

  • Result and analysis Expression profiles of PeLACs

    S1: Leaves; S2: The panicles at the early stage; S3: The panicles at the flowering

    stage; S4: Roots; S5: Rhizomes; S6: 20 cm shoots; S7: 50 cm shoots.

     23 PeLACs expressed in the tissues with diverse profiles

  • PeLAC10 was isolated by RT-PCR

     PeLAC10 was 1692 bp, encoding a putative peptide of

    487 amino acids with molecular weight of 52.75 kDa

    and theoretic isoelectric point of 6.859 .

    Semi-quantitative PCR of PeLAC10

    1 2 3 4

    PeLAC10

    PeActin

    1: Roots;

    2: Stems;

    3: Leaf blades;

    4: Leaf sheaths.

    Result and analysis Functional analysis of PeLAC10

  • ped-miR397-mediated cleavage site identification

     The result of sequencing showed that there were nine

    clones with cleavage sites between the 10th-11th bases,

    and only one sample with a cleavage site between the 12th

    -13th bases in the ten sequenced clones.

    RNA ligase-mediated 5 ′RACE

    9/10

    A M 1 B

    250 bp

    100 bp

    U C A U U G A G U G C A G C G U U G A U G

    A G C A A C U C A C G U C G C A A C U A G - 5'3'-

    - 3'5'-ped-miR397

    PeLAC10

    Result and analysis Functional analysis of PeLAC10

  • Expression of ped-miR397 and PeLAC10 in

    different height shoots

    Result and analysis Functional analysis of PeLAC10

  • Expression of PeLAC10 in leaves under

    different treatments

    Result and analysis Functional analysis of PeLAC10

  • Expression of PeLAC10 in Arabidopsis

    Result and analysis Functional analysis of PeLAC10

  • Lignin staining in stem of transgenic Arabidopsis

    overexpressing PeLAC10

    co, cortex; if, interfascicular fiber; ph, phloem; xy, xylem. Bar = 50 μm.

    Result and analysis Functional analysis of PeLAC10

  • Lignin content in stem in stem of transgenic

    Arabidopsis overexpressing PeLAC10

    Asterisks indicate a

    significant difference

    between transgenic

    plants and Col-0 (*p

    < 0.05; **p < 0.01).

    Result and analysis Functional analysis of PeLAC10

  •  Response of transgenic plants overexpressing PeLAC10 to

    drought stress.

    Col-0

    0 d 15 d 20 d

    L1

    L2

    Result and analysis Functional analysis of PeLAC10

    MDA content in transgenic plants

    is significantly lower than Col-0.

  •  Response of transgenic plants overexpressing

    PeLAC10 to phenolic acid stress.

    Result and analysis Functional analysis of PeLAC10

  • Conclusion

     A total of 23 laccase proteins were identified in moso bamboo.

     RLM-5′ RACE analysis showed that PeLAC10 was regulated

    by ped-miR397 after transcription.

     PeLAC10 was up-regulated obviously under the treatments of

    ABA (100 μmol·L–1) and NaCl (400 mmol·L–1), and it was

    down-regulated under the treatment of GA3 (100 μmol·L –1).

     Overexpression of PeLAC10 was helpful to increase the

    content of lignin in transgenic Arabidopsis plants and improve

    their adaptability to drought and phenolic acid stresses.

  • Acknowledgements

     The work was supported by Special Funds for

    Fundamental Scientific Research on Professional Work

    Supported by the International Center for Bamboo and

    Rattan (No. 1632019008) and the Special Fund for

    Forest Scientific Research in the Public Welfare from

    State Forestry Administration of China (No.

    201504106).

  • Thank you for

    your attention!