Starch Biosynthesis in Rice Grains: Natural Variation and Genetic Improvement
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Transcript of Starch Biosynthesis in Rice Grains: Natural Variation and Genetic Improvement
Starch Biosynthesis in Rice Grains
—— Natural Variation and Genetic Improvement
Qiao-quan Liu(刘巧泉)
College of Agriculture, Yangzhou University, Yangzhou, Jiangsu Province, China
E-mail: [email protected]
Amylose Amylopectin
11th International Gluten Workshop
Outline
1. Allelic diversities in rice starch biosynthesis
and genetic network for rice grain quality
2. Genetic engineering of starch biosynthesis
for high resistant starch (RS) in rice
Determinants of rice grain quality
Milling quality
Appearance quality
Cooking & eating quality
Nutritional quality
Three key physicochemical properties determine
rice cooking and eating quality
Gelatinization
temperature
determines the
time required for
cooking the rice
Gel consistency
measures the
tendency of the
cooked rice to
harden on cooling.
High amylose content grains
cook dry, are less tender, and
become hard upon cooling.
GT GC
AC
Cultivar
type
Maturity
type No.
Amylose content
(%)
Gelatinization
Temperature (ASV)
Gel Consistency
(mm)
Range Mean Range Mean Range Mean
Indica
Early
Medium
Late
8
33
32
23.75-26.60
9.68-30.64
11.64-28.66
25.28
24.16
20.30
3.22-5.22
2.67-6.89
2.00-6.56
4.35
4.89
4.92
30-97
20-120
21-110
53.63
56.21
63.22
Japonica
Early
Medium
Late
13
25
5
10.54-23.09
11.34-18.00
15.64-22.16
15.25
14.77
18.35
5.94-6.91
3.32-7.00
6.00-6.94
6.42
5.98
6.38
51-95
38-108
24-85
71.77
75.64
59.20
Wide diversity of cooking and eating
qualities among rice cultivars
Amylose Amylopectin
SBE
SSS
DBE
Amylopectin Amylose
AGPase
ADPGlc
?
Starch, the major component in rice endosperm
Classification of key enzymes Gene Localization
ADP glucose pyrophosphorylase
(AGPase)
Large subunit 1 AGPL1 Chr 5
Large subunit 2 AGPL2 Chr 6
Small subunit AGPS Chr 9
Granule-bound starch synthase (GBSS)
GBSSI Wx Chr 6
GBSSII GBSSII Chr 7
Soluble starch synthase (SSS)
SSSI SSSI Chr 6
SSSII
SSSII-1 Chr 10
SSSII-2 Chr 2
SSSII-3 Chr 6
SSSIII SSSIII-1 Chr 4
SSSIII-2 Chr 8
SSSIV SSSIV-1 Chr 1
SSSIV-2 Chr 5
Starch branching enzyme (SBE)
SBEI Sbe1 Chr 6
SBEII Sbe3 Chr 2
Sbe4 Chr 4
Starch debranching enzyme (DBE)
Isoamylase (ISA) ISA Chr 8
Pullulanase (PUL) PUL Chr 4
Starch Synthesis Related Genes, SSRGs
1. Natural variation of starch synthesis
To search and identify the allelic
variation of SSRGs among different
rice ecotypes.
To find how these genes controlling
rice cooking and eating qualities.
(Cooperated with Prof. Jiayang Li, IGDB, CAS)
70 varieties with diverse grain qualities
Indica (33)
Japonica (37)
AC GC GT
AC 1.00 -0.91 a *
0.007 b
-0.46
0.779
GC 1.00 0.50
0.326
GT 1.00
a Correlation Coefficients b Pr > F
* The number marked in bold imply the according line and row quality are
correlated with each other
High correlation among AC, GC and GT
Tian et al., PNAS, 2009, 106: 21760-21765
Starch pasting curve of different rice cultivars
-500
500
1500
2500
3500
4500
5500
6500
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0
Time (min)
Vis
cosi
ty (
cP
)
Tm TN1
LTP GCH
9311 WY7
CJ06 NIP
JZXN THN
SYN
Rapid Visco Analyser (RVA)
High AC
Low or intermediate AC
Very low or no AC
16 core varieties selected for sequence analysis of SSRGs
5„UTR
The Wx gDNA alignment among different varieties Varieties
Nipponbare ( japonica )
Chunjiang 06 ( japonica )
Wuyunjing 7 ( japonica )
Jiangzhouxiangnuo ( japonica - glutinous)
Suyunuo ( japonica - glutinous)
Taihunuo ( japonica - glutinous)
9308 ( indica )
9311 ( indica )
Guichao 2( indica )
Longtepu ( indica )
Minghui 63 ( indica )
Taizhongbendi 1( indica )
Zhenshan97B ( indica )
175 298 495 528 771-785 841 926 987 1056 1083 1088
C C A C CT ( 18 ) T G AATT(6) A C A
C C A C CT ( 17 ) T G AATT(6) A C A
C C A C CT ( 17 ) T G AATT(6) A C A
C C A C CT ( 16 ) T G AATT(6) A C A
C C A C CT ( 16 ) T G AATT(6) A C A
C C A C CT ( 16 ) T G AATT(6) A C A
C C A C CT ( 18 ) T G AATT(6) A C G
C C A C CT ( 18 ) T G AATT(6) A C A
C C G T CT ( 11 ) G A AATT(5) G T A
C T G T CT ( 11 ) G A AATT(5) G T A
C C A C CT ( 18 ) T G AATT(6) A C A
C C G T CT ( 11 ) G A AATT(5) G T A
A C G T CT ( 11 ) G A AATT(5) G T A
Exon 2 intron exon intron
Varieties
Nipponbare ( japonica )
Chunjiang 06 ( japonica )
Wuyunjing 7 ( japonica )
Jiangzhouxiangnuo ( japonica - glutinous)
Suyunuo ( japonica - glutinous)
Taihunuo ( japonica - glutinous)
9308 ( indica )
9311 ( indica )
Guichao 2( indica )
Longtepu ( indica )
Minghui 63 ( indica )
Taizhongbendi 1( indica )
Zhenshan97B ( indica )
2111-2112 3019 3097 3804 4078 4211 4235 4244-4246 4282 4285
------------------------ C C T C G G ATA A G
------------------------ C C T C G G ATA A G
------------------------ C C T C G G ATA A G
ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G
ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G
ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G
------------------------ C C T C G G ATA A G
------------------------ C C T C G G ATA A G
------------------------ - T C T A A --- G A
------------------------ - T C T A A --- G A
------------------------ C C T C G G ATA A G
------------------------ - T C T A A --- G A
------------------------ - T C T A A --- G A
Wx gene alignment
Varieties 111-112 172 1086 1243
Nipponbare ( japonica ) ---------------------------------------- T C
Chunjiang 06 ( japonica ) ---------------------------------------- T C
Wuyunjing 7 ( japonica ) ---------------------------------------- T C
Jiangzhouxiangnuo ( japonica - glutinous) ACGGGTTCCAGGGCCTCAAGCCC TGA
Suyunuo ( japonica - glutinous) ACGGGTTCCAGGGCCTCAAGCCC TGA
Taihunuo ( japonica - glutinous) ACGGGTTCCAGGGCCTCAAGCCC TGA
9308 ( indica ) ---------------------------------------- T C
9311 ( indica ) ---------------------------------------- T C
Guichao2( indica ) ---------------------------------------- C T
Longtepu ( indica ) ---------------------------------------- C T
Minghui 63 ( indica ) ---------------------------------------- T C
Taizhongbendi 1( indica ) ---------------------------------------- C T
Zhenshan97B ( indica ) ---------------------------------------- C T
Wx gene alignment The cDNA Alignment Among Different Varieties
Stop
codon
• The diversities of the coding sequences were much
lower than those of whole genes in all SSRGs.
• The diversities of the nonsynonymous substitution
were lower than the synonymous.
• This result suggested that these SSRGs had likely
undergone artificial selection during domestication
Tian et al., PNAS, 2009, 106: 21760-21765
Association analysis
? How many major and minor genes control grain
cooking and eating quality
? Are AC, GC, and/or GT controlled by one or
multiple genes
? What is the relationship among these genes
? …
— e.g. Who control AC? Association analysis
2111-2112 3019 3097 3804 4078 4211 4235 4244-4246 4282 4285
------------------------ C C T C G G ATA A G
------------------------ C C T C G G ATA A G
------------------------ C C T C G G ATA A G
ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G
ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G
ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G
------------------------ C C T C G G ATA A G
------------------------ C C T C G G ATA A G
------------------------ - T C T A A --- G A
------------------------ - T C T A A --- G A
------------------------ C C T C G G ATA A G
------------------------ - T C T A A --- G A
------------------------ - T C T A A --- G A
Wx I
Wx II
Wx III
Wx I
Wx III Wx II Wx II Wx III
0.00
5.00
10.00
15.00
20.00
25.00
30.00
Wx I Wx II Wx III
Am
ylo
se c
on
ten
t (%
)
A
— e.g. Who control AC? Association analysis
Major
0.00
5.00
10.00
15.00
20.00
25.00
30.00
Wx I Wx II Wx III SBE3 I SBE3 II
Am
ylo
se c
on
ten
t (%
)
A B
SSII-3 I SSII-3 II
C
SSIII-2 I SSIII-2 II
D
SSIV-2 I SSIV-2 II
E
Am
ylo
se c
on
ten
t (%
)
Wx I
SBE3 I SBE3 II
Wx II
SBE3 I SBE3 II
Wx III
SBE3 I SBE3 II
F
0.00
5.00
10.00
15.00
20.00
25.00
30.00
A
— e.g. Who control AC? Association analysis
Major Minor Minor Minor
Minor Interaction
Tian et al., PNAS, 2009, 106: 21760-21765
SSRGs form a network controlling rice cooking and eating quality
Tian et al., PNAS, 2009, 106: 21760-21765
Wx and SSII-3 are central in determining
grain quality by affecting all three properties
Ttwo genes affect two properties
simultaneously, both ISA and SBE3 affect
GC and GT.
Several minor genes are specific for single
properties, SSIII-2, AGPlar, PUL, and SSI
for AC, AGPiso for GC, and SSIV-2 for GT.
The correlations among AC, GC, and GT
were caused by the joint action of these
associated genes and unequal haplotype
combination.
Fig. Summary of genes
controlling rice grain quality
Verification of SSRGs
Donor (s) ╳
F1 Receptor ╳
BCnF1
MAS
Receptor
BCnF2(3)
Transgenic tests Near-isogenic lines
Down-regulation Over-expression
Verification of the major gene for AC, Wx
(Transgenic)
Verification of the minor gene for AC, SBE3
(Transgenic)
Breeding of NILs
BC6F3
LTF × 9311
F1 × LTF
BC1F1
BC6F1
SSSI i SSSI j
0
500
1000
1500
2000
2500
3000
3500
0 200 400 600 800
Time(Sec)
Vis
cosi
ty (
cP
)
NILs
( SSSI j )
LTF
( SSSI i )
RVA profiles of NILs
LTF-NIL-SSSI j
Verification of the minor gene, SSSI
(Near-isogenic lines)
0
500
1000
1500
2000
2500
3000
3500
4000
0 200 400 600 800
Time(sec)
Vis
cosi
ty(c
P)
LTF (SSSI i)
RNAi
0
500
1000
1500
2000
2500
3000
3500
4000
0 200 400 600 800
Time(sec)
Vis
co
sity
(cP
)
Nipponbare (SSSI j)
RNAi
WT RNAi lines
LTF (SSSI i)
WT RNAi lines
Nipponbare (SSSI j)
The starch quality of RNAi transgenic lines
containing different SSSI allele
0
2
4
6
8
10
12
WXJ9 GLXN ZS97 LTP
Ex
press
ion
lev
el
rela
tiv
e t
o A
cti
n
SSSI j SSSI i
SSSI
13193 bp
TGASSS IATG
GUS
SSSI
13193 bp
TGASSS IATG
GUS
SSSI j-GUS
SSSI i-GUS 0
1
0 100 200 300 400 500
GUS activityGUS activity in developing seeds of transgenic rice
Q-RT-PCR analysis in developing rice seeds
The transcriptional level of
SSSI j allele is much lower
than that of SSSI i allele in
rice endosperm
Liu et al., unpublished
GOI Ter Promoter
Transgenic regulation
BC6F3
Receptor × Donor
F1 × Receptor
BC1F1
BC6F1
Allele i Allele j
Marker-assisted selection (MAS)
MAS
Molecular improvement of rice grain/starch quality
Functional SSRGs‟ markers for MAS
M Nip LTF 9311 9308 SYN
MAS
Tian et al., Chinese Sci Bull., 2010. 55: 3768-3777
Improvement of cooking and eating quality
of the female line Longtefu by MAS
Line Wx allele AC
(%)
GC
(cm)
GT
(ASV)
LTF Wxa Wxa 27.81 6.00 7.00
LTF-TT-1 Wxb Wxb 15.30 11.75 2.50
LTF-TT-3 Wxb Wxb 17.91 11.05 3.00
LTF-TT-5 Wxb Wxb 15.56 10.35 5.00
MAS
Liu et al., Crop Science, 2006; Yu et al., J Cereal Sci, 2009
Wxb J1 J3 J4 J5
Wxa I1 I5 I6 wx
Down of AC by transformation of antisense Wx gene
Northern blot
0
5
10
15
20
25
30
Am
ylo
se c
on
ten
t (%
)
WY7 WY8 WX LTF QLZ TQ
Japonica Indica
Wild type
Transgenic
Liu et al., Mol Breed, 2005; Yu et al., J Cereal Sci, 2009
Summary
Rice grain cooking and eating qualities are
regulated by starch synthesis related genes
(SSRGs) in a network.
Transgenic and near-isogenic studies with
selected major and/or minor SSRGs have
verified the above results, and which shown that
genetic modification with SSRGs will improve
rice grain qualities as desired.
Outline
1. Allelic diversities in rice starch biosynthesis
and genetic network for rice grain quality
2. Genetic engineering of starch biosynthesis
for high resistant starch (RS) in rice
Resistant Starch (RS)
Butyrate production
Prebiotic-stimulate growth
Inhibit cancer
Boost immune system
Reduce glycemic response
(slower insulin release)
Low calorie intake
Starch that escapes degradation in the small intestine,
and, therefore, is available for bacterial fermentation in the
large intestine.
Christer Jansson, Bioproducts, Nov. 2008
Potato
Oat
Corn
Wheat
Pea
Taro
Millet
Buck wheat
Rice
Bean
Sweet potato
Resistant starch
Source Resistant Non-Resistant
starch starch
Content of resistant starch in different starch sources
High amylose content is a source of
resistant starch (RS) R
es
ista
nt
sta
rch
(%
)
Zhu et al., Carbohydrate Polymers, 2011, 86: 1751-1759
Effects of regulation of different SSRGs on high-amylose production
Zhu et al., Plant Biotech J, 2012, 10: 353-362
Very-high-amylose rice grain with a high
level of RS and total dietary fiber
Zhu et al., Plant Biotech J, 2012, 10: 353-362
(Wild type: Indica, high AC)
J Agri & Food Chem, 2010, 58: 1224; 2010, 58:11946
WT
WT
RS
RS
Starch granule morphology of RS-rich rice
Polygonal granules with sharp
angles and edges
Irregularly large voluminous starch granules and
sausage-like elongated small starch granules
Fine structure of starches from RS-rich rice
Zhu et al., Plant Biotech J, 2012, 10: 353
(Increase of B-chains)
WT
RS
(High-amylose)
RS-WT
High-resistant starch rice Regular rice
RS-rich rice highly resistant to alkali
digestion and gelatinization
Wei et al., J Agri Food Chem, 2010, 2011
(Intact milled rice soaked in 5% KOH solution for 16 hours)
50 oC
70 oC
75 oC
80 oC
90 oC
RS WT
Wei et al., Food Chemistry,
2011, 128: 645-652
Resistant to
gelatinization
during heating
in water
200
240
280
320
360
1 3 5 7 9 11 13 15 17 19 21 23
Feeding time (d)
Bo
dy
wei
gh
t (g
)
Regular rice group
RS rice group
Zhu et al., Plant Biotech J, 2012, 10: 353-362
Improvement in indices of animal health
in rats by RS-rich rice meal
0
50
100
150
200
250
乙酸 丙酸 丁酸 短链脂肪酸
Con
ten
t (u
mole
/g)
WT RS
Acetic Propionic Butyric Total
acid acid acid SCFA
The rats consuming the RS-rich rice excreted more total short
chain fatty acids (SCFAs) than those fed the regular rice
Improvement in indices of animal health
in rats by RS-rich rice meal
Zhu et al., Plant Biotech J, 2012, 10: 353-362
Reduce of blood glucose response in diabetic
Zucker fatty rats fed the RS-rich rice starch
4.0
6.0
8.0
10.0
12.0
14.0
16.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
Time (h)
Glu
cose
lev
el
WT
RS
Acute oral rice tolerance test (ORTT) in type II diabetic rats
Zhu et al., Plant Biotech J, 2012, 10: 353-362
Summary
A high-amylose (64.8%) rice enriched with resistant
starch (14.6%) was developed by transgenic regulation
of starch biosynthesis.
RS-rich rice starches highly resistant to digestion and
gelatinization
Consumption of the RS-rich rice had improved in
indices of animal health in both normal and diabetic rats.
Acknowledgements
Collaborators:
Prof. Jiayang Li (Inst. Genet. Develop. Biol., CAS)
Prof. Mengming Hong (Shanghai Inst. Plant Physiol. Eco., CAS)
Prof. Qian Qian (Chinese Rice Research Institute)
Prof. Yongcheng Shi (Kansas State University, USA)
……
Supported by: National Natural Science Foundation of China (NSFC)
National Key Basic Research Projects (“973” project)
National Major Projects for Transgenic Research
Thank you !