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 methodsPlant materials Phyllostachys edulis
Materials and methodsPlant 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 15C3H: Coumarate 3-hdroxylase 3 1 1 3C4H: 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 16HCT:Hydroxycinnamoyl-CoA 3 12 6 16
LAC: Laccase 16 22 20 41PAL: 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 analysisPhylogenetic analysis of LACs
PeLA
C3
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 analysisGene structure analysis of PeLACs
23 PeLACs have diverse gene structures
Result and analysisExpression 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 analysisFunctional 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 analysisFunctional analysis of PeLAC10
Expression of ped-miR397 and PeLAC10 in
different height shoots
Result and analysisFunctional analysis of PeLAC10
Expression of PeLAC10 in leaves under
different treatments
Result and analysisFunctional analysis of PeLAC10
Expression of PeLAC10 in Arabidopsis
Result and analysisFunctional 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 analysisFunctional 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 analysisFunctional analysis of PeLAC10
Response of transgenic plants overexpressing PeLAC10 to
drought stress.
Col-0
0 d 15 d 20 d
L1
L2
Result and analysisFunctional 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 analysisFunctional 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!