Genome-wide analysis of laccase genes in moso bamboo ... · Conclusion A total of 23 laccase proteins were identified in moso bamboo. RLM-5′RACE analysis showed that PeLAC10 was

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


Expression of ped-miR397 and PeLAC10 in

different height shoots


Expression of PeLAC10 in leaves under

different treatments


Expression of PeLAC10 in Arabidopsis


Lignin staining in stem of transgenic Arabidopsis

overexpressing PeLAC10

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


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).


Response of transgenic plants overexpressing PeLAC10 to

drought stress.

Col-0

0 d 15 d 20 d

L1

L2


MDA content in transgenic plants

is significantly lower than Col-0.

Response of transgenic plants overexpressing

PeLAC10 to phenolic acid stress.


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!

Documents

Genome-wide analysis of laccase genes in moso bamboo ... · Conclusion A total of 23 laccase proteins were identified in moso bamboo. RLM-5′RACE analysis showed that PeLAC10 was