Opaque7 Encodes an Acyl-Activating Enzyme-Like Protein That Affects Storage Protein Synthesis in Maize Endosperm - Genetics
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INVESTIGATION
Opaque7 Encodes an Acyl-Activating Enzyme-Like
Protein That Affects Storage Protein
Synthesis in Maize Endosperm
Gang Wang,*,1 Xiaoliang Sun,*,1 Guifeng Wang,* Fei Wang,* Qiang Gao,* Xin Sun,* Yuanping Tang,*
Chong Chang,* Jinsheng Lai,† Lihuang Zhu,‡ Zhengkai Xu,* and Rentao Song*,2
*Shanghai Key Laboratory of Bioenergy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, China,
†State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100101, China, and
‡The State Key Laboratory of Plant Genomics and National Center for Plant Gene Research,
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,
Beijing 100101, China
ABSTRACT In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins,
the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins
accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation
and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional
complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation
of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7
mutant was produced by the reduction of protein body size and number caused by a decrease in the a-zeins concentrations. Analysis
of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting a-ketoglutaric acid and
oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower
amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal
crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for
the genetic manipulation of storage protein contents in cereal seeds.
a number of opaque or floury endosperm mutants that affect
T HE texture and protein quality of maize (Zea mays L.)
endosperm are important factors affecting grain ship-
ping, insect and fungal pathogen resistance, and nutritional
the texture and protein quality of endosperm by altering
zeins accumulation. Our understanding of the underlying
quality. Much evidence indicates that the reduction in the mechanisms determining zeins accumulation comes from
amount of zeins in the endosperm leads to a decrease in the the study of seed mutants.
endosperm hardness and an increase in lysine content There are .18 mutants that can exhibit an opaque or
(Mertz et al. 1964; Misra et al. 1972; Schmidt et al. 1987; floury endosperm (Thompson and Larkins 1994; Hunter
Dombrink-Kurtzman and Bietz 1993; Holding and Larkins et al. 2002). Among them are the recessive opaque mutants
2006; Wu and Messing 2010; Wu et al. 2010). Maize have (o1, o2, o5, o7, o9-o11, and o13-o17), the semidominant
floury mutants (fl1, fl2, and fl3), and the dominant mutants
Copyright © 2011 by the Genetics Society of America Mucronate (Mc) and Defective endosperm B30 (De-B30) (Motto
doi: 10.1534/genetics.111.133967
Manuscript received August 18, 2011; accepted for publication September 21, 2011
et al. 1996; Gibbon and Larkins 2005). The cloning and char-
Supporting information is available online at http://www.genetics.org/content/ acterization of some of the opaque mutants has revealed im-
suppl/2011/09/27/genetics.111.133967.DC1. portant regulatory mechanisms for zeins accumulation in
Sequence data have been deposited to EMBL/GenBank under accession nos.
JN578265, JN578266, and HQ234502. maize endosperm. The O2 gene, which encodes a defective
1
2
These authors contributed equally to this study. basic-domain-leucine-zipper transcription factor, regulates
Corresponding author: Shanghai Key Laboratory of Bioenergy Crops, School of Life
Sciences, Shanghai University, 333 Nanchen Rd., Shanghai 200444, China.
several endosperm-expressed genes, in particular the 22-kDa
E-mail: rentaosong@staff.shu.edu.cn a-zeins (Schmidt et al. 1987, 1990; Damerval and De Vienne
Genetics, Vol. 189, 1281–1295 December 2011 1281
1993; Habben et al. 1993). The o2 mutant reduces the pro- ing F2 seeds were derived from a cross between o7/o7 in the
duction of zeins by repressing the expression of 22-kDa a-zein W22 background and an unknown flint line. Selfed ears of
genes (Schmidt et al. 1987, 1990). Other mutants, such as fl2, plants from these F2 seeds were selected for solid opaque
De-B30, and Mc, have structural defects in the zeins them- phenotype segregation and verification of the expected seg-
selves, which results in improper zeins deposition, protein body regation ratio. At least two more rounds of selection were
deformation, and the “unfolded protein response” (UPR) carried out to obtain suitable parental lines to construct pop-
(Lending and Larkins 1992; Coleman et al. 1995; Gillikin ulations for genetic mapping. The backcross (BC) population
et al. 1997; Hunter et al. 2002; Kim et al. 2004, 2006), caus- was prepared from a cross between o7/o7 and o7/+ plants.
ing a general translational repression of zeins. However, Genomic DNA samples from o7/o7, o7/+ and the 13,933
mechanisms other than reduced zeins do exist. For example, descendants of the BC population were extracted from leaf
the fl1 mutation does not affect the amount and composition tissue by the CTAB method (Murray and Thompson 1980) at
of zeins; it only changes the location of 22-kDa a-zeins within the seedling stage and used for marker analysis.
the protein body (Holding et al. 2010). The o5 mutant phe- Seeds of the Hi-II parent A (pA) and Hi-II parent B (pB)
notype is caused by a reduction in the galactolipid content of lines for maize genetic transformation were initially
the maize endosperm, with no change in zeins (Myers et al. obtained from the Maize Genetic Cooperation Stock Center
2011). Cloning and characterization of additional mutants and amplified on the campus of Shanghai University. F2
could reveal novel regulatory mechanisms for opacity forma- immature zygotic embryos (1.5–2.0 mm) of the maize Hi-
tion, zeins synthesis, and accumulation in maize endosperm. II hybrid genotype (Armstrong et al. 1991) were aseptically
The opaque7 (o7) is an important opaque mutant with dissected from ears harvested 10–12 days after pollination
large effects on zeins accumulation (Burr and Burr 1982). It (DAP) and were used for genetic transformation.
is considered one of the three most valuable high-lysine
opaque mutants, along with the aforementioned o2 and Scanning electron microscopy and transmission electron
fl2. The o7 mutant arose from a spontaneous mutation in microscopy analysis
W22 (Tsai and Dalby 1974) and has a starchy endosperm at For scanning electron microscopy (SEM) analysis, wt and o7
maturity. The o7 mutant induces a reduction in endosperm mutant kernels were prepared as described previously (Lending
weight and total protein content (Di Fonzo et al. 1979) and and Larkins 1992), with some modifications. Briefly, maize
exhibits a general inhibition of the synthesis of all zein clas- kernels were excised with a razor and immediately placed in
ses (Misra et al. 1972; Lee et al. 1976; Burr and Burr, 1982; 2.5% glutaraldehyde. Mature samples were critical-point dried,
Hartings et al. 2011). Previous studies suggested that o7 and 21-DAP samples were cryofractured, sputter coated with
might have a very different mechanism for zeins regulation gold in an E-100 ion sputter, and observed with a scanning
compared to that of o2 and fl2. Studying this mechanism electron microscope (S3400N; Hitachi).
could help us to understand the regulatory mechanisms of For transmission electron microscopy (TEM) analysis,
zeins accumulation and endosperm opacity. However, the o7 previously published methods were used on immature
mutant is more difficult to study because its mutant pheno- kernels from wt and o7 mutant with some modifications
type is only stable in a very narrow genetic background (Lending and Larkins 1992). Briefly, 21-DAP kernels of wt
(McWhirter and Brink 1978). and mutant were fixed with paraformaldehyde and post-
In this study, we cloned the O7 gene by map-based clon- fixed in osmium tetraoxide. Fixed samples were dehydrated
ing and confirmed its identity by transgenic functional com- in an ethanol gradient up to 100% and then transferred to
plementation. The O7 gene encodes an acyl-activating a propylene oxide solution and slowly embedded in acrylic
enzyme like (AAE3-like) protein, and the o7 mutant allele resin (London Resin Company), where they were allowed to
differs from the wild-type (wt) allele by a 12-bp in-frame polymerize for at least 48 hr. Thin sections (70 nm) were
deletion. The deletion does not affect o7 transcript levels, taken using a diamond knife microtome (Reichert Ultracut
but it greatly reduces the amount of o7 protein in the endo- E). Sections were placed on 100-mesh copper grids and
sperm. Interestingly, transgenic RNAi targeting the O7 gene stained for 30 min with uranyl acetate and for 15 min with
was effective in downregulating seed protein content and lead citrate. Sections were visualized using a Hitachi H7600
producing opaque endosperm both in maize and rice, sug- transmission electron microscope.
gesting that the function of O7 is conserved among different
cereal crops. The O7 gene could serve as a target for the Measurement of starch, lipid, and protein contents
genetic manipulation of seed proteins in cereal crops. Starch analysis: Thirty-gram mature kernels from wt and
mutant were collected from well-filled, mature ears. The
samples were analyzed by the Cereal Quality Supervision
Materials and Methods and Testing Center, Ministry of Agriculture, People’s Repub-
lic of China. The analysis method was based on the GB7684-
Plant materials
87 and GB5006-85 protocols developed by the Ministry of
The o7 mutant stock was kindly provided by Frances Burr of Agriculture, People’s Republic of China. The starch analysis
Brookhaven National Laboratory, Upton, NY. These segregat- was replicated three times for the wt and opaque kernels.
1282 G. Wang et al.
Lipid analysis: Five mature kernels of the wt or mutant were sense PCR fragment, rice intron, and antisense PCR fragment
collected from well-filled, mature ears. One hundred milli- were cloned into the SacI site of the pHB vector (Mao et al.
grams of dried endosperm flour were used for lipid extraction. 2005). The O7 gene promoter was then amplified by the
The extraction procedure followed a standard protocol for lipid primer pair O7 promoter F/promoter R and used to replace
extraction from Arabidopsis seeds (http://www.k-state.edu/ the 2·35S promoter of pHB by cloning into the EcoRI and
lipid/lipidomics/AT-seed-extraction.html). The extracted sam- BamHI sites. The resulting pHB–O7RNAi construct was intro-
ples were dried completely, stored in vials filled with nitrogen duced into the Agrobacterium strain LBA4404. Agrobacterium-
gas, and shipped to the Kansas Lipidomics Research Center for mediated maize transformation using F2 immature zygotic
analysis. The lipids analysis was done according to lipid pro- embryos of the Hi-II hybrid (pApB) was carried out accord-
filing protocols established by the facility (http://www.k-state. ing to Frame et al. (2002). For the molecular identification
edu/lipid/lipidomics/profiling.htm). The lipid analysis was of T0 transgenic plants, a marker was designed to identify
replicated three times for the wt and mutant kernels. the O7 gene with the primer pair rice intron F and maize
P3 R (which bind the rice intron and O7 gene, respectively).
Protein analysis: Twenty mature kernels of the wt or mutant
were collected from well-filled, mature ears. Total protein, RT–PCR and real-time quantitative PCR analysis
zein, and nonzein proteins were extracted from 40 mg of Root, stem, leaf, husk, silk, tassel, ear, and kernels (3 DAP–
dried endosperm flour, according to the method of Wallace 36 DAP) from adult W22 plants were collected and flash
et al. (1990). Protein quantification analyses of the total ex- frozen in liquid nitrogen. Kernels (12 DAP, 18 DAP, and
tract, zeins, and nonzein fractions were performed as de- 24 DAP) from adult wt and o7 mutant plants were also
scribed by Smith et al. (1985) using a BCA standard kit collected and flash frozen in liquid nitrogen. RNA extraction,
(Pierce). SDS–PAGE was performed in 12% polyacrylamide purification, and quantification were performed according
gels, and the gels were stained with Coomassie brilliant blue to Feng et al. (2009) and Wang et al. (2010, 2011). For
R250 (Bradford 1976). RT–PCR and real-time PCR, 2 mg of total RNA was reverse
transcribed to cDNA with oligo d(T)18 primer using Rever-
Complementation test Tra Ace reverse transcriptase (Toyobo). The expression pat-
For functional complementation tests, a 4066-bp genomic tern was analyzed using real-time quantitative RT–PCR with
DNA fragment containing the entire coding region, an 1856-bp SYBR Green Realtime PCR Master Mix (Toyobo). The reac-
upstream sequence, and a 507-bp downstream sequence tion was performed on a Mastercycler ep realplex 2 (Eppen-
were isolated from BAC clone ZMMBBb0342E21 (GenBank dorf) with the primer 6837Q, and Ubiquitin (GenBank
accession no. HQ234502) by digesting with BamHI and accession no. BT018032) was used as a reference gene (Ta-
KpnI. The genomic DNA was cloned into pTF102 (Frame ble S1). All of the real-time RT–PCR experiments were per-
et al. 2002), which carries a bar resistance marker and formed with two independent sets of RNA samples.
a CaMV 35S promoter. The resulting pTF102-O7 gene con- Quantification of the relative changes in gene expression
struct was introduced into the Agrobacterium tumefaciens was performed using the 2-DDCt method as described pre-
strain LBA4404. Agrobacterium-mediated maize transforma- viously (Livak and Schmittgen 2001). The specificity of the
tion using F2 immature zygotic embryos of the Hi-II hybrid PCR amplification procedures was verified after PCR by us-
(pApB) was carried out according to Frame et al. (2002). For ing a heat dissociation protocol (from 65 to 95) to ensure
the molecular identification of transgenic plants, a marker that each amplicon was a single product.
was designed to identify the O7 gene with the primer pair
Sequence homology analysis
Trans6837 F3 and Trans6837 R3, which bind the 35S pro-
moter and O7 gene, respectively (see Supporting Informa- Related sequences were identified in the NCBI nonredun-
tion, Table S1 for primer sequences). The marker Indel6819, dant (nr) protein sequences database by performing
linked tightly with the O7 locus, was designed to identify the a BLASTp search (Camacho et al. 2009) with O7 protein
genotype of the O7 gene on chromosome 10. sequences. Selected sequences were aligned using ClustalW
in the MEGA5 package (Kumar et al. 2008). Conserved res-
Maize RNAi transformation idues in the alignment were shaded using the SMS2
To generate the RNAi construct, a PCR fragment containing program (http://www.bioinformatics.org/sms2/color_align_
303 bp from the cDNA of the O7 gene (nucleotides 1277– cons.html) (Kumar et al. 2008). The phylogenetic tree was
1579) was amplified using the first-strand cDNA derived produced with MEGA5 using the bootstrap analysis (1000
from W22 mixed endosperm (12 DAP, 15 DAP, and 18 replicates), although neighbor joining gave an identical to-
DAP) as the template and the following pairs of primers pology (Tamura et al. 2007).
maize P3 F and maize P3 R. The PCR fragment was sequen-
O7 antibody preparation
tially cloned into the SpeI/KpnI and BamHI/SacI sites of the
pTCK303 (Wang et al. 2004) vector in both the sense and The full-length O7 ORF was amplified from cDNA derived
antisense orientations. Then the fragment containing the from W22 mixed endosperm (12 DAP, 15 DAP, and 18 DAP)
The Cloning and Analysis of Maize Opaque7 1283
Figure 1 Phenotype of the BC ear, o7, and wt kernels.
using primers 6837FLcDNA F and 6837FLcDNA R. The se- modification. Briefly, dried extracts were dissolved in mixed
quence correctness of the fragment was confirmed and was solution of water/isopropanol/methanol (1:3:6, v/v). The
digested with BamHI and EcoRI and ligated into pGEX-4T-1 analysis of extracts by mass spectrometry was carried out
(Amersham Biosciences) to create an in-frame fusion with according to Broeckling et al. (2005). The a-ketoglutaric
GST. The selected clones were sequenced to exclude the acid (a-KG) and oxaloacetate (OAA) analyses were each
possibility of PCR-induced errors, and a single clone was replicated three times for the wt and opaque kernels.
subsequently used for large-scale induction of the fusion pro-
tein. The fusion protein was purified using a standard tech- Rice RNAi analysis
nique (Amersham Biosciences). Antibodies were produced in To generate the RNAi construct, a PCR fragment contain-
white rabbits at Shanghai ImmunoGen Biological Technol- ing 258 bp (nucleotides 967–1224) of the cDNA of
ogy. The polyclonal antibodies were affinity purified using Os04g0683700 (OsAAE3) was amplified from the first-
their respective bacterially expressed GST fusion proteins strand cDNA derived from Nipponbare young panicle by
according to standard procedures (Harlow and Lane 1988). using the following pair of primers: RiceP3 F and RiceP3
R. The PCR fragment was sequentially cloned into the
Total endosperm protein extraction and SpeI/KpnI and BamHI/SacI sites of the pTCK303 vector
immunoblot analysis
(Wang et al. 2004) in both the sense and antisense orienta-
Developing endosperms (12 DAP, 15 DAP, 18 DAP, and 21 tions, yielding the pTCK303–OsAAE3 RNAi vector. The
DAP) were dissected as described above. Total protein from pTCK303–OsAAE3 RNAi construct was introduced into the
wt and mutant endosperms was extracted according to the Agrobacterium strain LBA4404. Agrobacterium-mediated rice
method of Bernard et al. (1994). Protein samples were transformation using the calli of Kitaake (a japonica rice
loaded onto SDS gels (10% acrylamide) and run using the variety) was carried out according to Wang et al. (2004).
Mini-protean system (Bio-Rad). Proteins were then blotted The regenerated plants were further confirmed by PCR us-
onto nitrocellulose using the Mini-transblot system (Bio-Rad) ing rice intron F and rice P3 R.
according to standard protocols. The immunoblot analyses
with the O7 antibody, a-tubulin antibody (Sigma-Aldrich), Results
and BiP antibody (Santa Cruz Biotechnology) were per-
Characterization of the o7 mutant endosperm
formed according to Holding et al. (2007).
BC ears with progenies exhibiting 1:1 segregation of opaque
Soluble amino acids analysis (o7/o7) and wt (o7/+) (vitreous) kernel phenotype were
used for cytological analysis (Figure 1). SEM analysis
The soluble amino acids (SAAs) were analyzed according to
the method of Holding et al. (2010) with some modification.
Briefly, 3-mg aliquots of each sample were refluxed for 24 hr
in 6N HCl. Samples are typically hydrolyzed at 110 for
24 hr in a vacuum or inert atmosphere to prevent oxidation.
Hydrolysates were evaporated to dryness. The residue was
dissolved in 10 ml of pH 2.2 citrate buffer and the amino
acids were profiled on a Hitachi-L8900 amino acid analyzer
at the Instrumental Analysis Center of Shanghai Jiaotong
University. Amino acid analyses were replicated three times
on the wt and opaque kernels. The SAAs of 21-DAP endo-
sperm samples were analyzed by the same methods.
a-Ketoglutaric acid and oxaloacetate analysis
Three wt or mutant kernels (21 DAP and 24 DAP) were
collected from each of three wt or mutant ears. One hundred Figure 2 Scanning electron microscopy of wt and o7 endosperm. (A) o7
milligrams of dried endosperm flour were used for metab- kernel at 21 DAP (·5000). (B) Wt kernel at 21 DAP (·5000). (C) Mature o7
olite extraction according to Chen et al. (2011) with some kernel (·3000). (D) Mature wt kernel (·3000). st, starch; pb, protein body.
1284 G. Wang et al.
Figure 3 Transmission electron microscopy of wt and o7
endosperm. (A) o7 kernel at 21 DAP (·2500). (B) Wt kernel
at 21 DAP (·2500). (C) o7 kernel at 21 DAP (·6000). (D)
Wt kernels at 21 DAP (·6000). cw, cell wall; pb, protein
body; st, starch; rER, rough endoplasmic reticulum; m,
mitochondrion. (E) Comparison of protein body numbers
per 100 mm2 in o7 and wt 21-DAP endosperms (o7,
protein body counts ¼ 36.87 6 6.30; wt, protein body
count ¼ 26.20 6 4.21. *P , 0.05, Student’s t-test). (F)
Comparison of protein body area in o7 and wt 21-DAP
endosperms (o7, protein body area ¼ 0.547 6 0.345; n ¼
203. wt, protein body area ¼ 0.624 6 0.246; n ¼ 270.
**P , 0.01, Student’s t-test).
revealed that the protein bodies (PBs) and proteinaceous significant decrease, P , 0.05, Student’s t-test). We then
cytoskeletal matrix of o7 immature endosperm (21 DAP) analyzed starch (amylose, amylopectin, and total starch)
were much smaller than those of the wt (Figure 2, A and (Figure S1) and lipid content (Figure S2) per dry weight
B), resulting in loosely packed starch granules. SEM exam- of opaque and wt kernels, and we found no obvious differ-
ination of mature kernels showed that the mutant kernel ences between both opaque and wt kernels. However, the
had smooth, spherical starch grains with very little matrix protein content was found to be dramatically lower in opa-
surrounding them, whereas the wt kernel contained polyg- que kernels (Figure 4, A and B). Interestingly, there is a gen-
onal starch grains with a very dense proteinaceous matrix eral reduction in zeins, nonzeins, and total proteins in opaque
surrounding them (Figure 2, C and D). TEM analysis re- kernels (Figure 4, A and B). Quantitative analysis showed that
vealed that in the mutant endosperm, there is a significant in the mutant kernels, zeins had the most severe decrease
reduction in PB numbers (reduced 29%) and PB size (re- (33.4%), greater than that of nonzeins (15.1%) or total pro-
duced 13%) compared to the wt endosperm (Figure 3, A–F). teins (20.7%). This result is consistent with the cytological
To determine the underlying biochemical reasons for the analysis, which showed that the PBs numbers and size of o7
opaque phenotype of the mutant, we examined the major were reduced about one-third. From the SDS–PAGE, we
components in the endosperms of the mutant and wt found that there is a dramatic decrease of both 19-kDa and
kernels. First, we found that the 100-seed weight of opaque 22-kDa a-zeins in o7 kernels (Figure 4A). The result showed
kernels was just 93% of the weight of wt kernels (obviously that o7 does not downregulate the 19-kDa a-zein specifically.
The Cloning and Analysis of Maize Opaque7 1285Figure 4 Protein analysis of wt and o7 endosperm. (A) SDS–PAGE analysis of total proteins from wt and o7 endosperm. (B) Comparison of zein,
nonzein, and total proteins from wt and o7 endosperm. Values are the mean values with standard errors (*P , 0.05; **P , 0.01, Student’s t-test).
Map-based cloning of the Opaque7 gene Functionally complement the o7 mutant by transgenic
candidate gene
The O7 gene was mapped between the markers umc2021
and umc1038 on the long arm of chromosome 10 by an We used a functional complementation test involving genetic
analysis of 400 BC individuals (Figure 5A). Additional mo- transformation to test the cloned candidate gene. In total, 13
lecular markers were then developed by comparing genomic independent transgenic lines were obtained. These transgenic
sequence fragments near the O7 locus in both parental lines lines were then backcrossed to o7/o7 mutant plants for at
(Table S1). After characterizing a BC population with a total least two more generations to obtain individuals homozygous
of 13,933 individuals, the O7 gene was eventually mapped at the o7 locus. We characterized four independent o7/o7
between the marker LM4 and the marker RM1, a region transgenic lines. In Figure 6, we present the analysis of one
encompassing a physical distance of 23 kb (Figure 5A). of the transgenic lines. A randomly selected set of 20 vitreous
Sequence analysis of this interval identified two predicted and 10 opaque kernels (Figure 6A) from one transgenic line
genes (gene1 and gene2) (Figure 5A), but only gene2 had were genotyped using molecular marker Indel6819 to confirm
support from expressed sequence tags (ESTs). We se- that the kernels were homozygous at the o7 locus (Figure 6B).
quenced the 23-kb regions from the mutant and wt haplo- The primer set Trans6837 F3/Trans6837 R3 was used to de-
types, and we found no sequence polymorphism within tect kernels containing the O7 transgene. Without exception,
gene1. However, there was a 12-nucleotide in-frame dele- all 20 vitreous kernels were found to be transgenic, whereas
tion in the predicted second exon (codons 350–353) (Figure none of the 10 opaque kernels were transgenic (Figure 6C).
5B) of the o7 allele of gene2. Thus, gene2 appeared to be Biochemical analysis of kernel proteins from transgenic
the strongest candidate for the O7 gene. functional complementation ears by SDS–PAGE showed that
Figure 5 Positional cloning of the O7 gene. (A) Fine map-
ping of the O7 gene on chromosome 10. Names of the
molecular markers, contig, BAC, and the recombinants
are indicated (n ¼ 13,933). ZMMBBb0166J07 and
ZMMBBb0342E21 (abbreviated as b0166J07 and b0342E21)
are the names of BAC clones covering this locus. The O7
locus was mapped to a 23-kb region containing gene1
and gene2 between the molecular markers LM4 and RM1.
(B) Schematic representation of the gene structure of O7.
The mutant sequence has a 12-nucleotide in-frame dele-
tion in the second exon. ATG and TGA represent the start
and stop codons, respectively.
1286 G. Wang et al.Figure 6 Phenotypic and molecular characterization of transgenic kernels from functional complementation experiment. (A) The phenotype of 20
vitreous kernels and 10 opaque kernels, randomly selected from a transgenic line with homozygous o7 locus on chromosome 10. (B) Genotyping the 30
kernels displayed in A for o7 locus on chromosome 10 using the molecular marker Indel6819. All 30 kernels are homozygous at the o7 locus on
chromosome 10. (C) Characterization of transgenic status of the 30 kernels displayed in A using the molecular marker Trans6837F3R3. The vitreous
kernels (lanes 1–20) are transgenic, and the opaque kernels (lanes 21–30) are nontransformed. 2, H2O control; 1, pTF102–O7Gene vector. (D) SDS–PAGE
analysis of total proteins from different kernels. T1, transgenic and o7/o7 homozygous kernel; T2, nontransformed and o7/o7 homozygous kernel. (E)
Comparison of zein protein content in wt, o7, pApB, transgenic homozygote o7/o7 (T1), and nontransformed homozygote o7/o7 kernels (T2).
the total proteins and zeins levels in functionally comple- strated that the candidate gene2, which was identified by
mented transgenic kernels were recovered to the protein map-based cloning, was in fact the O7 gene.
levels of wt kernels and that the levels recorded in nontrans-
RNAi against O7 reproduces the o7 phenotype
genic kernels were similar to those of the mutant (Figure
6, D and E). This result indicated that the o7 mutant phe- To further verify the function of the cloned O7 gene, we
notype was fully rescued by transgenic candidate gene. The constructed an RNAi transgenic vector to target the O7 gene
analysis of another three independent transgenic lines gave sequence, and we transformed the construct into maize.
the same results (data not shown). Therefore, the o7 mutant Seven independent transgenic events were obtained from
allele was functionally complemented by the wt O7 allele self-pollination. We characterized the seeds from two inde-
through stable genetic transformation. This result demon- pendent RNAi transgenic lines with opaque and vitreous
The Cloning and Analysis of Maize Opaque7 1287Figure 7 Phenotypic and molecular characterization of transgenic kernels from maize RNAi transgenic experiment. (A) The vitreous and opaque
phenotype of the 20 randomly selected kernels from an RNAi transgenic line. (B) Characterization of the transgenic status of the 20 kernels displayed
in A using the molecular marker defined by the primer pair rice intron F/maize P3 R. The opaque kernels (lanes 1–10) are transgenic, and the vitreous
kernels (lanes 11–20) are nontransformed. 2, H2O control; 1, pHB–O7RNAi vector. (C) SDS–PAGE analysis of total proteins from different kernels.
R12CK and R32CK, nontransformed vitreous kernels from transgenic lines R1 and R3; R11 and R31, transgenic opaque kernels from transgenic lines
R1 and R3. (D) Comparison of zein protein content in pApB, R12CK, R32CK, R11, and R31 kernels.
kernel segregation (Figure 7A). In one of the transgenic lines, 7, A–D). The results confirmed that the opaque phenotype
10 vitreous kernels and 10 opaque kernels selected randomly was indeed caused by the suppression of O7 gene function.
were analyzed with the primer set rice intron F/maize P3 R to
O7 encodes an AAE3-like protein
determine whether the kernels contained the transgenic RNAi
construct. The result indicated that all 10 opaque kernels We obtained the full-length cDNAs of the O7 and o7 genes of
were RNAi transgenic, whereas none of the 10 vitreous ker- W22 by rapid amplification of cDNA ends (RACE) RT–PCR
nels were RNAi transgenic (Figure 7B). (GenBank accession nos. JN578265 and JN578266). We found
Analysis of the kernel proteins from RNAi transgenic that the W22 O7 cDNA is 1884 bp long, containing an open
events indicated a decreased level of zeins compared to pApB reading frame of 1584 bp, encoding a polypeptide of 527
control seeds (R1 decreased 14.6%, R3 decreased 16.9%; amino acids. Sequence comparison between the genomic
Figure 7, C and D). These results showed that the o7 pheno- DNA and cDNA indicated that the O7 gene contains two exons.
type could be caused by the quantitative reduction in zeins. BLAST searches using the O7 protein sequence indicated
Therefore, in both RNAi transgenic events, the seeds bearing that O7 belonged to the acyl-CoA synthetase (ACS) super-
the RNAi construct produced the opaque phenotype (Figure family and contained the AMP-binding and ACS conserved
1288 G. Wang et al.expression levels of O7 and o7 at 12 DAP, 18 DAP, and 24
DAP and found no significant difference in transcript levels
between wt and mutant alleles (Figure 10C).
Significant reduction of o7 protein in the
mutant endosperm
We further analyzed the O7 gene product of immature ker-
nels (12 DAP, 15 DAP, 18 DAP, and 21 DAP) from the wt and
mutant at the protein level. Immunoblot analysis employing
antibody against a-tubulin was used to ensure equal loading
of total proteins for each sample. Western blot analysis
employing O7 protein antibody showed that the quantity of
o7 protein in the mutant kernel was significantly lower than
the O7 protein in the wt kernel (Figure 11, A and B). There-
Figure 8 Phylogenetic relationships of O7 (ZmAAE3) and its homologs.
The O7 gene product (shown as Zm AAE3) was aligned with AAE3 fore, the 12-bp deletion in the o7 mutant allele caused sig-
proteins from Capsicum annuum (AF354454.1), Vitis vinifera (XP nificant reduction of o7 protein level in developing kernels.
002267459), Populus trichocarpa (XP 002322473), Ricinus communis At the same time, we analyzed the O7 protein from
(XP 002509782), Arabidopsis thaliana (NP 190468), A. lyrata subsp. lyrata the 18-DAP kernels of transgenic homozygous o7/o7 plants
(XP 002877640), Oryza sativa Japonica (NP 001054304), Hordeum vul-
and nontransformed homozygous o7/o7 kernels from the
gare subsp. vulgare (BAK00674), Zea mays (NP 001152269.1), Sorghum
bicolor (XP 002448800), A. thaliana AAE13 (At AAE13, NM 112487), and functional complementation tests, and using the O7 anti-
A. thaliana AAE14 (At AAE14, NM 102789). body, we found that the quantity of O7 protein in the trans-
genic homozygous o7/o7 kernels (vitreous phenotype) was
domains (Black et al. 1992, 1997; Weimar et al. 2002; recovered but not in nontransformed homozygous o7/o7
Shockey et al. 2003; Black and Dirusson 2006). We con- kernels (opaque phenotype; Figure 11, C and D). We also
structed an ACS superfamily phylogenetic tree on the basis analyzed the O7 protein in developing kernels (18 DAP)
of the O7 protein sequence and the ACS proteins of Arabi- from the transgenic RNAi experiment. Indeed, the O7 pro-
dopsis thaliana. The O7 protein was most similar to AAE3 in tein was significantly reduced in kernels bearing the trans-
this phylogenetic tree (Figure S3A). We then constructed an genic RNAi construct (Figure 11, E and F).
AAE3 family phylogenetic tree on the basis of homologs
from fungi, bacteria, and plants (Figure S3B). In this phylo- Normal UPR in the o7 endosperm
genetic tree, we found that the AAE3 proteins of plants were Because the UPR can cause a general translational repression
monophyletic. In this tree, there were AAE3 homologs from of zeins, we investigated whether zein reduction in the o7
other grass species, such as Sorghum bicolor (XP 002448800), endosperm was caused by UPR. We used immunoblot analy-
Oryza sativa (NP 001054304 and Os04g0683700), and Hor- sis to compare the level of the ER lumen binding protein (BiP)
deum vulgare (BAK00674) (Figure 8). A detailed sequence (Kim et al. 2004) in 12-DAP, 15-DAP, and 18-DAP kernels
alignment of these AAE3 homologs showed a highly con- from wt and o7 plants by BiP antibody. The immunoblot
served AMP-binding domain and ACS domains (Figure 9). showed that BiP level was not obviously different between
The four-amino-acid deletion of o7 was also found to lie in wt and o7 (Figure 12, A and B). The result indicated that
a conserved sequence region (Figure 9). The deleted Val and zeins reduction and phenotype of o7 was not caused by UPR.
Gly residues in the o7 protein were highly conserved among
the analyzed AAE3 homologs in plants (Figure 9). Soluble amino acids and key metabolites analysis of the
o7 endosperm
Expression analysis of the O7 gene
Amino acid supply was proposed as a possible cause for
Real-time quantitative RT–PCR (real-time qRT–PCR) analy- downregulation of zeins (Motto et al. 1996). To observe the
sis revealed that O7 is expressed in a broad range of tissues, changes in amino acid composition in o7 endosperm, we
including root, stem, leaf, husk, silk, tassel, ear, and kernel analyzed the SAAs from wt and o7 mature seeds. SAAs anal-
(Figure 10A). The strongest expression was detected in im- ysis showed that the mutant seeds have a higher content of
mature seeds. However, root and tassel also had relatively Asx (Asp + Asn) (+21.9%) and Lys (+38.2%), but a lower
high expression levels (Figure 10A). Because the o7 mutant content of Glx (Glu + Gln) (214.5%) compared to wt seeds
phenotype is related to maize endosperm, we further exam- (Figure 13A). To understand the changes that contribute to
ined the expression profile of O7 during seed development. the final concentrations of SAAs in mature endosperm, we
The O7 transcript was first detectable at 3 DAP and sharply characterized the SAA levels and composition of Asx (Asp +
increased in abundance, peaking at 7 DAP (Figure 10B). A Asn), Lys and Glx (Glu + Gln) of 21-DAP kernels. The results
relatively small but still significant amount of O7 expression showed that mutants have a higher content of Asx (+10.6%)
was detected from 18 DAP to 36 DAP. We also compared the and Lys (+12.8%) and have a lower content of Glx (210.2%)
The Cloning and Analysis of Maize Opaque7 1289Figure 9 Protein sequence alignment of plant
AAE3 proteins. ClustalW amino acid alignment
of Zm AAE3 protein and its homologs. The
black shading with white lettering indicates res-
idues conserved in all 10 members, whereas
gray shading indicates conservation between
two or more of the family members. The 10
sequences listed are from Zea mays (NP
001152269.1), Oryza sativa (NP 001054304),
Arabidopsis thaliana (NP 190468), Sorghum bi-
color (XP 002448800), Hordeum vulgare subsp.
vulgare (BAK00674), Ricinus communis (XP
002509782), Capsicum annuum (AF354454.1),
Populus trichocarpa (XP 002322473), Vitis vinif-
era (XP 002267459) and A. lyrata subsp. lyrata
(XP 002877640).
compared to wt (Figure 13B). The data were highly consistent targeting OsAAE3 in the japonica rice variety Kitaake and
between developing and mature endosperm. obtained six independent transgenic lines. These transgenic
Because amino acid biosynthesis is tightly linked to the plants showed segregation of the opaque and vitreous seed
concentrations of certain metabolites, we analyzed some key phenotypes (Figure 14, A and B). We randomly selected 10
metabolites of the TCA cycle at 21 DAP and 24 DAP. The vitreous kernels and 10 opaque kernels from an RNAi trans-
analysis showed that mutant kernels have a higher content genic line and examined the transgenic RNAi construct by
of OAA, the precursor substrate for Asx and Lys biosynthesis, the primer pair rice intron F/rice P3 R. On the basis of this
but they have a lower content of a-KG acid, the precursor analysis, all 10 opaque kernels were RNAi transgenic and all
substrate for Glx biosynthesis (Figure 13, C and D). 10 vitreous kernels were not RNAi transgenic (Figure 14C).
Total seed proteins were analyzed by SDS–PAGE, and there
O7 gene function is conserved between maize and rice
was an apparent decrease of total seed proteins in trans-
The synteny and phylogenetic tree analysis showed that the genic kernels compared to nontransgenic kernels (Figure
OsAAE3 (Os04g0683700) of rice is the orthologous gene of 14D). SEM examination revealed that the opaque pheno-
maize O7 (Figure 8). We generated RNAi transgenic plants type kernel had loosely packed starch granules (Figure 14,
1290 G. Wang et al.Figure 10 Expression analysis of the O7 gene. (A) Real-time qRT–PCR analysis of O7 gene expression in root, stem, leaf, silk, tassel, ear, and kernel (7
DAP). (B) Real-time qRT–PCR analysis of O7 gene expression from 3-DAP to 36-DAP kernels in the W22 background. (C) Real-time qRT–PCR analysis of
O7 gene expression in 12-DAP, 18-DAP, and 24-DAP samples from wt and o7 mutant kernels endosperm.
E and F). These results showed that RNAi-based repression shift mutation or premature termination, but it removes four
of OsAAE3 resulted in storage protein reduction and an opa- amino acids from the O7 protein. Due to the unknown func-
que endosperm phenotype in rice, as was observed in the tion of the O7 protein, we were unable to determine the
maize o7 mutant and O7 RNAi transgenic endosperm. These effect of the deletion on its AAE activity. Transcript quanti-
results suggested that the gene function is conserved be- fication by real-time qRT–PCR analysis indicated that the
tween maize and rice. expression of O7 was not different from o7 at the transcript
level. However, at the protein level, we found a dramatic
decrease of o7 protein in the mutant endosperm. This in-
Discussion
dicated that the four-amino-acid deletion caused low accu-
In this study, we cloned the O7 gene by map-based cloning mulation of o7 protein in vivo. Thus, the molecular basis of
and confirmed it by functional complementation test. The o7 the o7 phenotype is a dramatic decrease in o7 protein that
allele is a recessive mutant, suggesting a loss-of-function leads ultimately to a loss of O7 function.
mutation. This is further supported by the result from the The O7 gene was annotated as the AAE gene. In Arabi-
RNAi experiment with knocked-down O7, which reproduced dopsis, there are 44 genes belonging to the family, which can
the o7 mutant phenotype. Sequence analysis indicated that be divided into seven groups on the basis of phylogenetic
the o7 mutant has a 12-nucleotide in-frame deletion in the analysis (Shockey and Browse 2011; Shockey et al. 2003).
second exon. The 12-bp deletion does not create a frame- However, only half of them have been assigned definitive
Figure 11 Immunoblot analysis of O7 pro-
tein from endosperms of wt, o7, functional
complementation transgenic line, and RNAi
transgenic line. (A) Immunoblot comparing
the accumulation of O7 protein in 12-DAP,
15-DAP, 18-DAP, and 21-DAP kernels from
wt and o7 plants with an antibody against
O7 protein. (B) Immunoblot analysis using
an a-tubulin antibody. (C) Immunoblot
comparing the accumulation of O7 protein
in 18-DAP kernels with an antibody against
O7 protein. T1, transgenic homozygote o7/
o7; T2, nontransformed homozygote o7/
o7. (D) Immunoblot analysis using an a-tu-
bulin antibody. T1, transgenic homozygote
o7/o7; T2, nontransformed homozygote
o7/o7. (E) Immunoblot comparing the accumulation of O7 protein in the 18-DAP kernels with an antibody against O7 protein. R12CK and R32CK,
nontransformed kernels from transgenic lines R1 and R3; R1+ and R3+, transgenic kernels from transgenic lines R1 and R3. (F) Immunoblot analysis
using an a-tubulin antibody. R12CK and R32CK:, nontransformed kernels from transgenic lines R1 and R3; R1+ and R3+, transgenic kernels from
transgenic lines R1 and R3.
The Cloning and Analysis of Maize Opaque7 1291Figure 12 Immunoblot analysis of BiP from wt and
o7 endosperms. (A) Immunoblot comparing the
accumulation of BiP protein in 12-DAP, 15-DAP,
and 18-DAP wt and o7/o7 kernels by using BiP
antibody. (B) Immunoblot analysis using a-tubulin
antibody.
biochemical functions. On the basis of the phylogenetic anal- not determine the exact biochemical function of maize AAE3
ysis, O7 is a maize ortholog of AAE3 and belongs in clade VII (O7), the characterization of the o7 mutant phenotype
(Shockey et al. 2003). In Arabidopsis, clade VII contains clearly indicated that the AAE3 gene function is closely re-
three genes, AAE3, AAE13, and AAE14 (Shockey et al. lated to storage protein synthesis in the endosperm tissue in
2003). The AAE14 and AAE13 genes encode the o-succinyl maize.
benzoyl-CoA ligase and malonyl-CoA synthetase respectively During endosperm development, starch granules and
(Kim et al. 2008; Chen et al. 2011). Although AAE3 was protein bodies are embedded in a proteinaceous cytoskeletal
annotated as an ACS, its exact enzymatic activity is uncer- matrix (Clore et al. 1996; Gibbon et al. 2003). The protein-
tain (Shockey et al. 2003). In Saccharomyces cerevisiae, aceous matrix is made up of protein bodies and clear, viscous
Pcs60/Fat2, the ortholog of AAE3, showed no ACS activity cytoplasm. Therefore, any change in protein body size,
on any of the saturated and unsaturated fatty acids tested quantity, or shape or the composition of the cytoplasm could
(Blobel and Erdmann 1996). In Capsicum annuum, the AAE3 produce loosely packed starch granules and an opaque phe-
homolog was found to be rapidly upregulated by treatment notype. Several maize mutants, such as o2, fl2, De-B30, and
with salicylic acid or the pathogenic bacterium Xanthomonas Mc (Schmidt et al. 1987; Gillikin et al. 1997; Kim et al. 2004,
campestris (Lee et al. 2001). In this study, although we could 2006), which have opaque kernels due to zeins reduction
Figure 13 The analysis of soluble Asx, Lys, Glx, a-ketoglutaric acid, and oxaloacetate from wt and o7 endosperms. Values are the means and standard
errors (n = 3; *P , 0.05; **P , 0.01, Student’s t-test). (A and B) Soluble Asp 1 Asn, Lys and Glu 1 Gln in mature and 21-DAP kernels of wt and o7.
(C and D) a-Ketoglutaric acid and oxaloacetate analysis of 21-DAP and 24-DAP kernels of wt and o7.
1292 G. Wang et al.Figure 14 Phenotypic and molecular characterization of transgenic kernels from rice RNAi transgenic experiment. (A) The vitreous and opaque
phenotype of the 20 randomly selected kernels from the RNAi transgenics. (B) Transverse sections of vitreous (top) and opaque (bottom) rice kernels.
(C) Characterization of the transgenic status of the 20 kernels displayed in A using the molecular marker defined by the primer pair rice intron F/rice P3
R. The opaque kernels (lanes 1–10) are transgenic, and the vitreous kernels (lanes 11–20) are nontransformed. 2, H2O control; 1, pTCK303–OsAAE3
RNAi vector. (D) SDS–PAGE analysis of total proteins from different kernels. Lane 1, Kitaake kernel; lanes 2–4, nontransformed kernels; lanes 5–7,
transgenic kernels. (E and F) SEM (·350) of a nontransformed vitreous rice kernel (E) and a transgenic opaque rice kernel (F).
and small, misshapen protein bodies, support the relation- that a reduction in zein protein synthesis could be caused
ship between vitreous endosperm and zeins content. In o7 by limiting the supply of tyrosine (Holding et al. 2010).
mutant endosperm, there is a significant reduction of zein The o7 mutant is different from the o2, fl2, De-B30, and
proteins. In tandem with the reduction in zeins, the mutant Mc mutants. The O7 gene encodes neither a regulatory gene
endosperm also shows significantly reduced PB size and nor a structural gene for zeins and its endosperm has a nor-
number. The cytological and biochemical analysis indicated mal UPR. However, we did observe changes in amino acid
that the thinner proteinaceous cytoskeletal matrix in o7 mu- biosynthesis in the mutant endosperm, particularly for Glx,
tant endosperm caused by PB size and number reduction Asx, and Lys. The cue for such a change could lie upstream
was the direct reason of the opaque phenotype. of amino acid biosynthesis because the corresponding met-
Previous studies have suggested that most of the opaque abolic precursors for these amino acids, such as a-KG and
and floury genes that have been cloned regulate zein OAA, also showed correlated changes. The mutation might
synthesis by affecting regulatory genes or structural genes act through a-KG and OAA, affecting the biosynthesis of
for storage proteins such as o2, fl2, De-B30, and Mc (Schmidt several amino acids. Changes in the levels of these amino
et al. 1987; Gillikin et al. 1997; Kim et al. 2004, 2006; Gibbon acids might lead to translational suppression of zeins, which
and Larkins, 2005). Motto et al. (1996) considered that other are highly biased for certain amino acids, such as Gln and
regulatory mechanisms, such as amino acid supply, could Lys. Thus, O7 may represent a new regulatory mechanism
affect zeins gene expression. Tonelli et al. (1986) predicted unlike those of known opaque mutants. Because the exact
that the starchy endosperm phenotype of proline1 (pro1) is biochemical function of O7 as an AAE3 protein remains un-
associated with a defect in amino acid biosynthesis. However, known, the details of such a regulatory mechanism remain
because pro1 has not been cloned yet, this prediction has not to be explored.
been validated. The Mto140 gene was found to encode arro- Phylogenetic analysis indicates that O7 orthologs in other
gate dehydrogenase, which provided more direct evidence major cereal crops are monophyletic. In other words, this
The Cloning and Analysis of Maize Opaque7 1293gene was present in the common ancestor of the major ce- Black, P. N., Q. Zhang, J. D. Weimar, and C. C. Dirusso,
real crops. The O7 gene might have a conserved function 1997 Mutational analysis of a fatty acyl-coenzyme a synthetase
signature motif identifies seven amino acid residues that modu-
among cereal crops. Synteny analysis showed that the
late fatty acid substrate specificity. J. Biol. Chem. 272: 4896–4903.
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Acknowledgments Coleman, C. E., M. A. Lopes, J. W. Gillikin, R. S. Boston, and B. A.
Larkins, 1995 A defective signal peptide in the maize high-
We thank Frances Burr at Brookhaven National Laboratory lysine mutant floury 2. Proc. Natl. Acad. Sci. USA 92: 6828–6831.
for his providing maize genetic stocks. We thank Weibin Damerval, C., and D. De Vienne, 1993 Quantification of domi-
Song at the China Agricultural University and Chunhua nance for proteins pleiotropically affected by opaque-2 in maize.
Yang at the Institute of Genetics and Developmental Biology, Heredity 70: 38–51.
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Chinese Academy of Sciences, for their technical support
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We thank Jian Zhu at Tongji University for SEM technical Dombrink-Kurtzman, M. A., and J. A. Bietz, 1993 Zein composition
assistance. We thank Liangliang Zhou, Jianpin Xu, Dianbin in hard and soft endosperm of maize. Cereal Chem. 70: 105–108.
Lin, Xiaowei Zhang, Zhihong Ren, and Lihua Wang at Feng, L. N., J. Zhu, G. Wang, Y. P. Tang, H. J. Chen et al.,
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Shanghai University for their technical assistance. We thank
sional patterns and dramatic expressional divergence of maize
the Kansas Lipidomics Research Center for lipid analysis. a-zein super gene family. Plant Mol. Biol. 69: 649–659.
This work was supported by the Ministry of Science and Frame, B. R., H. Shou, R. K. Chikwamba, Z. Zhang, C. Xiang et al.,
Technology of China (2006AA10A107, 2006AA10Z148, and 2002 Agrobacterium tumefaciens-mediated transformation of
2009CB118400) and the Natural Sciences Foundation of maize embryos using a standard binary vector system. Plant
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Gibbon, B. C., X. Wang, and B. A. Larkins, 2003 Alteredstarch-
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Gibbon, B. C., and B. A. Larkins, 2005 Molecular genetic ap-
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