A Rice Gene Activation/Knockout Library-

What to expect and surprises

Su-May Yu
余淑美

Institute of Molecular, Academia Sinica

中央研究院 分子生物研究所
 Why choose rice for functional genomics?
1. Rice is a major staple food for ~ 50 % world population.

2. Rice has the smallest genome size among major cereal crops.

Arabidopsis 1.2 x 108 bp 0.3 x

Rice 3.9 x 108 bp 1 x
Corn 2.5 x 109 bp 6 x
Wheat 1.6 x 1010 bp40 x

3.  Rice can be transformed in large scale on a routine basis. 
4. Isolation of genes from rice facilitate isolation of
homologous
genes from other cereal crops.
5. Sequencing of rice genomic DNA is completed.

6. Much molecular and genetic information is available.

 1999
l
Approximately 37,000 non-TE genes in rice
2005
(Institute of Botany)
Institute of Plant and Microbial Biology
 Agrobacterium­mediated 
rice transformation

Chan et al. (2003) Plant Mol Biol 22:491-506.

 Multiple-function T-DNA for
Gene Knockout/Activation Tagging

activation tagging plasmid rescue

selection marker gene
trap
LB RB

35Se*8 hgh’ P35S pBS GUS Do/Ac

Tag 8 (16.00 Kb)

1. Gene knockout
2. Gene activation
3. Gene trap
4. Plasmid rescue
Study of gene function via T-DNA insertional mutagenesis

Gene Knockout (Ko)/Activation (Ac) Tagging

Ko : Loss-of-function Ac : Gain-of-function

Ko Ko Ko

Intergenic Promoter Exon/Intron 3’ un- Intergenic

translated
Ac Ac Ac Ac Ac
Generation and Utility of Rice Gene Knockout/Activation Library

T-DNA
LB T-DNA Tag RB

CaMV35S Hygromycin Promoterless

enhancer resistant gene Gus

Deliver into rice chromosome

via Agrobacterium tumefaciens

T-DNA tag inserted into the rice genome

T-DNA Tag

Rice DNA Rice DNA

T0 Transgenic lines
TAIL-PCR analysis T0 Plants
T-DNA
DNA purified from
Rice DNA Rice DNA transgenic rice leaves

Flanking sequence Collection of T1

tag (FST) transgenic seeds
Identificati
on
FST database of mutantsPropagation of T2
transgenic seeds

Identificatio GUS staining Phenotypic

n screening for screening
of genes gene trapping of mutants

Crop improvement Basic research

through
$ genetic engineering
 Consortium for Generating Rice T-DNA Tagged Mutant Library
Institute of Molecular
Biology
Academia Sinica
T0 transgenic plants
Su-May Yu Flanking sequence analysis
Institute of Plant and
Microbial Biology
Academia Sinica
Yue-Ie
Flanking sequence
Hsing Database/Bioinformatics
TRIM
Research in
Institutes/Universities
Mutant screening &
Characterization
Taiwan Agriculture
Research Institute (TARI)
T0 plants  T1 seeds
Chyr-Guan Chern
Taiwan Agriculture
Research Institute
T1 seeds  T2 seeds
Mutant phenotyping
Sheng-Chung Huang
Ming-Jen Fan
National Plant Genetic
Resource Center
Stock and catalogue of
T1 and T2 seeds
 Academia Sinica

200 Km (125 mi)

TARI
Taichung

Propagation of T1/T2 seeds in TARI

 Storage of TRIM mutant seeds

T1 seeds in aluminum
cans

Mid-term storage (1 °C and 40 % RH)

Long-term storage (-12 °C and 30 % RH)

T2 seeds in metal
TRIM T2 seeds
package

• T2 seeds are shipped to the users upon order.

• Currently limited to domestic users.
• Foreign orders would deliver based on mutual agreements.
 Four largest T-DNA tagged rice mutant libraries worldwide

Flanking
Library Country Rice cultivar Number of lines sequence
tags
Taiwan Rice Insertional Taiwan Tainung 67 10,000 GUS promoter trap 18,000
Mutagenesis (TRIM) Library 45,000 GUS promoter trap + (out of 55,000 lines)
activation tag

POSTECH Rice
Insertion Korea Dong Jin 27,000 GUS promoter trap 33,000
Sequence Database Hwayoug 48,000 GUS promoter trap + (out of 87,000 lines)
(RISD) activation tag
12,000 GUS/GFP promoter trap

Génoplante - Oryza Tag

France Nipponbare 20,000 GUS enhancer trap 27,000
Line (OTL)
3,000 GUS enhancer trap + Ds
(out of 32,000 lines)
9,000 enhancer trap

Rice Mutant Database (RMD) China Zhonghua11 129,000 GUS enhancer trap 15,000
Zhonghua 15 GFP enhancer trap (out of 129,000 lines)

* TRIM library has highest number of T2 seeds for both forward and reverse genetics studies.
 Most transgenic lines contain 1-2 copies of T-DNA

81 %

Facilitates co-segregation analysis of tagged genes with mutant phenotypes

 The TRIM mutant population contains low copy number of
inactive Tos17

)
C
(T
e
nb re
N pon 67

ar
po a
ip g
ip b
N un

NT Transformants

ol
tr
in

on
Lane 1 2 3 4 5 6 7 8 9 10 11 12 3 months 9 months 16 months
Ta

C
Lane 1 2 3

3
copies

Tos17 copy number in T0 Tainung 67 (Oryza sativa cv. Nipponbare)

transgenic rice plants
Hirochika, Plant Mol Biol 35: 231-240 (1997)
No. of Ratio Total copy
Copy no. lines (%) no.
3 91 91 273 Tos17 is activated during tissue cultur
4 6 6 24
5 3 3 15
Total 100 100 312
Average 3.12
* Stable up to 1 year in tissue culture
T-DNA tagging efficiency in the genic region of TRIM library is ~ 80 %

TRIM library

Type of sequences Ratio (%)

Intergenic 20.26
Genic 79.21
Exon 19.08
Intron 29.51
Promoter -(1000 bp) 26.18
3’UTR (+200 bp) 4.44
No Annotation 0.53
Total 100.00
 Enhancers activate genes located nearby T-DNA

Arabidopsis genome 8.2 kb 8.2 kb

E

Effective distance for gene activation

Rice genome 12.5 kb 12.5 kb (17 kb)
E

Gene X 9.9 kb Gene Y 9.9 kb Gene Z

Average distance between genes in the rice genome = 9.9 kb

Correlation:
T-DNA integrations
Gene distribution
Callus EST

T-DNA integrations are less prone to

cold and hot spots than Tos17
TAIL-PCR analysis T0 Plants
T-DNA
DNA purified from
Rice DNA Rice DNA transgenic rice leaves

Flanking sequence Collection of T1

tag (FST) transgenic seeds
Identificati
on
FST database of mutantsPropagation of T2
transgenic seeds

Identificatio GUS staining Phenotypic

n screening for screening
of genes gene trapping of mutants

Crop improvement Basic research

through
$ genetic engineering
 Search by query sequences
keywords
mutant line
numbers
phenotypes

Taiwan Rice Insertional Mutant (TRIM) Database

(http://trim.sinica.edu.tw/)
Pong-Chang Lu/Yue-Ie Hsing
 Search by keyword

Pong-Chang Lu/Yue-Ie Hsing

TAIL-PCR analysis T0 Plants
T-DNA
DNA purified from
Rice DNA Rice DNA transgenic rice leaves

Flanking sequence Collection of T1

tag (FST) transgenic seeds
Identificati
on
FST database of mutantsPropagation of T2
transgenic seeds

Search for GUS staining Phenotypic

tagged screening for screening
genes gene trapping of mutants

Crop improvement Basic research

through
$ genetic engineering
Expression of T-DNA tagged genes in mutants is activated or abolished

RT-PCR Tissue Putative Observed

Line no. Location of T-DNA insertion
analysis assayed function of gene phenotype

(a) ATG
*
W/W
1
T/T
2
M19081 Leaves of Mo25α Semi-dwarf
500 bp
T-DNA
1-month-old Low fertility
(Ac) (3.4 kb upstream of START codon) plants

(b) ATG
*
W/W T/W
1 2
T/T
3
M34845 Leaves of Hypothetical Late flowering
1 kb 3-month-old
T-DNA
(Ac) (1.1 kb downstream of STOP codon) plants

(c) ATG
*
W/W T/W T/T
1 2 3
M47191 Leaves of Gibberellin 2 Dwarf
3-month-old oxidase
500 bp T-DNA
(Ac) (2.1 kb upstream of START codon) plants

W/W T/W
(d) ATG
*
1 2

M48000A Leaves of Cytochrome P450 Semi-dwarf

3-month-old monooxygenase Slightly low
T-DNA 500 bp
(Ac) (2.1 kb upstream of START codon) plants fertility

(e) ATG
W/W T/W T/T
* 1 2 3
M27337 Leaves of Gibberellin 2 Dwarf
500 bp 3-month-old oxidase
(Ac) (23 bp upstream of STOP codon) plants
Expression of T-DNA tagged genes in mutants is activated or abolished

RT-PCR Tissue Putative Observed

Line no. Location of T-DNA insertion
analysis assayed function of gene phenotype

(f) ATG W/W T/W T/T

* 1 2 3
M23454 Leaves of AP2-like Sensitive to
500 bp 3-month-old protein diseases
T-DNA
(Ac) (1.0 kb downstream of STOP codon) plants

(g) ATG
W/W T/W T/W
* Leaves of
M04629 1 2 3 DEAD box Male sterile
2-week-old RNA helicase
(Ko) 500 bp
T-DNA plants
(5th exon)

(h) ATG
* W/W T/W T/T

M04801 1 2 3 Leaves of Tryptophen ?

2-week-old synthase α
500 bp
(Ko) T-DNA plants
(6th exon)

(i) ATG
W/W T/W T/T
* 1 2 3
M00329 Leaves of α-Galactosidase Sensitive
2-week-old to salt
T-DNA 500 bp
(Ko) (1st intron) plants

W/W T/W T/T

1 2 3
(j) ATG
*
M17031 Young Flavanone Semi-dwarf
500 bp panicles 3-β hydroxylase Low fertility
T-DNA
(Ko) (1st intron )
 Shung-Fan Lo( 羅舜芳 )/Liang-Chu Chen( 陳良築

13
A severe dwarf mutant A Day 0 C

12
18 days

11
*
T-DNA

10
(5’ region)

9
500 bp

8
W/WW/WT/W T/T

7
Activation of 1 2 3 4 -GA3 +GA3

6
A GA2ox gene GA2ox M47191

5
Day 4 D

4
18S rRNA

3
2
0
WT M47191

B
-GA3 +GA3
120 days
M47191

Inactive
Day 14 E

Dominant mutation

Active Active +GA3 -GA3 +GA3

WT T/W T/T
Pathway of GA metabolism in plants M47191
M55523 M47191
Figure 8
TAIL-PCR analysis T0 Plants
T-DNA
DNA purified from
Rice DNA Rice DNA transgenic rice leaves

Flanking sequence Collection of T1

tag (FST) transgenic seeds
Identificati
on
FST database of mutantsPropagation of T2
transgenic seeds

Search for GUS staining Phenotypic

tagged genes screening for screening
gene trapping of mutants

Crop improvement Basic research

through
$ genetic engineering
 GUS GUS
Genes active in flowers TATA

Intergenic Promoter Exon/Intron 3’ UTR Intergenic

5’ UTR

Anthers Lower glume and anthers Lodicule

Anther and stigma Anther and style Anther, stigma, Anther and endosperm
style and ovary
 Peng-Kai Sun ( 孫
Genes active in germinating rice embryos 鵬凱 )

GUS GUS
TATA

Intergenic Promoter Exon/Intron 3’ UTR Intergenic

5’ UTR

Shoot apex Root and Embryo Vascular bundle

Scutellum

Embryo Scutellum Scutellum Epithelium

except shoot and root and epithelium
 Peng-Kai Sun ( 孫
Genes active in endosperm of germinating seeds 鵬凱 )

GUS GUS
TATA

Intergenic Promoter Exon/Intron 3’ UTR Intergenic

5’ UTR

Endosperm Endosperm Vascular bundle

and endosperm

Endosperm Vascular bundle Seminal root,

and endosperm embryo and endosperm
Genes active in roots GUS GUS
TATA

Intergenic Promoter Exon/Intron 3’ UTR Intergenic

5’ UTR

Except lateral root tips Lateral roots Lateral roots

Only in lateral root tips Whole roots Vascular bundle

Young root Elongation zone Root hair zone

 Peng-Kai Sun ( 孫
Genes active in various tissues 鵬凱 )

GUS GUS
TATA

Intergenic Promoter Exon/Intron 3’ UTR Intergenic

5’ UTR

M35838 M26527 M03000 M35838

Leaf blade Leaf Calli Leaf sheath

vascular bundle vascular bundle

Root Ro Coleoptile Shoot

sclerenchyma ot
and
Genes responsive to stresses
GUS GUS
TATA

Intergenic Promoter Exon/Intron 3’ un- Intergenic

translated

H2O NaCl (salt) Sorbitol (osmotic)

a b c
Constitutive
M05365 (leaf)

d e f
Salt and osmotic
M05085 stresses repressed
(leaf)

g h i Osmotic
M05159 stress induced
(leaf)
j k l
Salt and osmotic
M05146 stresses induced
(root tip)

Wen-Li Huang ( 黃文理

GUS staining screening identifies stress up- and down-regulated genes

Wen-Li Huang ( 黃文理 )

GUS staining assays identify more leaf-active promoters than root-active promoters

Number in bracket indicates % of total lines.

Increase almost 4X due to enhancers Rachel Ko ( 辜瑞雪 )

TAIL-PCR analysis T0 Plants
T-DNA
DNA purified from
Rice DNA Rice DNA transgenic rice leaves

Flanking sequence Collection of T1

tag (FST) transgenic seeds
Identificati
on
FST database of mutantsPropagation of T2
transgenic seeds

Search for GUS staining Phenotypic

tagged screening for screening
genes gene trapping of mutants

Crop improvement Basic research

through
$ genetic engineering
 A mutant tolerant to osmotic
stress
Wild type Gene knockout mutants

350 mM sorbitol at 28 °C / 24 h Wen-Li Huang

Cultivation of TRIM mutants in TARI
 White stripe leaf Yellow leaf

M23580