Rice Waxy Gene Research Articles

A straightforward plant prime editing system enabled highly efficient precise editing of rice Waxy gene

CRISPR Cas9-mediated genome editing is highly efficient at targeted site-specific gene knock-out through NHEJ (Non-Homology End Joining), but ineffective for specific DNA integration through HDR (Homology Directed Repair) for precise gene editing. Base editors can make limited base substitutions but only within restricted small windows of the protospacer. Prime editing has been applied in plants with various degrees of success. However, several questions such as low and inconsistent editing efficiencies across different target sites need to be addressed. We compared two prime editing approaches PE3 and PE2 at two neighboring target sites within rice Waxy gene to partially address those questions. A straightforward PE2 plant prime editing system retrofitted from a regular CRISPR-Cas9 editing system can deliver highly efficient up to 66.7% precise gene editing. Various forms of precise editing including base substitutions, small deletions and insertions can be accurately achieved. The secondary structure variations of different pegRNAs may be the primary reason for inconsistent editing across different target sites and should be the optimization focus to further improve plant prime editing.

Wxlv, the Ancestral Allele of Rice Waxy Gene

In rice grains, the Waxy (Wx) gene is responsible for the synthesis of amylose, the most important determinant for eating and cooking quality. The effects of several Wx alleles on amylose content and the taste of cooked rice have been elucidated. However, the relationship between artificial selection and the evolution of various Wx alleles as well as their distribution remain unclear. Here we report the identification of an ancestral allele, Wxlv, which dramatically affects the mouthfeel of rice grains by modulating the size of amylose molecules. We demonstrated that Wxlv originated directly from wild rice, and the three major Wx alleles in cultivated rice (Wxb, Wxa, and Wxin) differentiated after the substitution of one base pair at the functional sites. These data indicate that the Wxlv allele played an important role in artificial selection and domestication. The findings also shed light on the evolution of various Wx alleles, which have greatly contributed to improving the eating and cooking quality of rice.

<i>In silico</i> promoter analysis reveals rice <i>Wx</i> promoter could be a TATA-less promoter with a putative pyrimidine rich region

Rice waxy gene is one of the major genes, which determines the amylose content of a rice grain. The expression of this gene is regulated by various stimuli such as temperature, light, drought and diseases. Promoter region of a gene is responsible for changing the level of expression with the help of transcription factors and their binding sites, cis-acting regulatory elements found in the promoter region ranging from 2000 bp upstream and 500 bp downstream relative to the transcription start site. Present study investigated the putative transcription factor binding sites in the promoter region and possible expression regulation stimuli using five Sri Lankan rice varieties, two glutinous rice varieties, Arabidopsis thaliana and potato. The promoter regions of the plants were extracted either after mapping selected varieties from rice 3000 genome project or from promoter databases. Signal scan was carried out to capture putative signal sequences, and kappa incidence of coincidence vs guanine cytosine content, guanine cytosine skew, and multiple sequence alignment were also performed. The results revealed that rice Wx promoter could be a TATA-less promoter with a putative pyrimidine patch, and categorized under ATCG middle class of promoters. The possible transcription regulation stimuli of waxy gene include dehydration, light, temperature, and hormones. There were signal sequences related to mesophyll, root nodule, seed and root specific regulation of expression.

A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice.

We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing.

Development of PCR-based CNP marker of rice Waxy gene with confronting two-pair primers

Development of PCR-based CNP marker of rice Waxy gene with confronting two-pair primers

Modulation of amylose content by structure-based modification of OsGBSS1 activity in rice (Oryza sativa L.).

The rice Waxy (Wx) gene encodes granule-bound starch synthase 1 (EC 2.4.1.242), OsGBSS1, which is responsible for amylose synthesis in rice seed endosperm. In this study, we determined the functional contribution of eight amino acids on the activity of OsGBSS1 by introducing site-directed mutated Wx gene constructs into the wx mutant glutinous rice. The eight amino acid residues are suspected to play roles in OsGBSS1 structure maintenance or function based on homologous enzyme sequence alignment and homology modelling. Both OsGBSS1 activity and amylose content were analysed in homozygous transgenic lines carrying the mutated OsGBSS1 (Wx) genes. Our results indicate that mutations at diverse sites in OsGBSS1 reduces its activity by affecting its starch-binding capacity, its ADP-glucose-binding capability or its protein stability. Our results shed new light on the structural basis of OsGBSS1 activity and the mechanisms of OsGBSS1 activity on amylose synthesis in vivo. This study also demonstrates that it is feasible to finely modulate amylose content in rice grains by modifying the OsGBSS1 activity.

Developing and Validating the Functional Marker of Rice Waxy Gene, M-Wx

Developing and Validating the Functional Marker of Rice Waxy Gene, M-Wx

Developing and Validating the Functional Marker of Rice Waxy Gene, M-Wx

It would be an important significance of developing molecular markers that could effectively track rice amylose content in the breeding program of high-quality rice varieties. In this study the dominant functional marker M-Wx was developed based on the SNP difference that can cause Wx gene function changes. The effectiveness of the functional marker M-Wx was validated by detecting the rice genotypes of 72 parents and amylose contents. The results showed that 228 bp and 425 bp bands were detected in 43 of 72 parents, genotype assigning to TT, which the amylose content were less than 15.6%, whereas the remaining 29 parents only appeared a 425 bp control band, genotype assigning to GG, which the amylose content were much more than 20.1%. The results of this study indicated the marker M-Wx should accurately detect the level of the amylose content in rice, which and can be applied to improve varieties and enhance rice quality in marker-assisted selection.

Inheritance of flour paste viscosity is associated with a rice Waxy gene exon 10 SNP marker

Apparent amylose content is a key element for characterizing a rice (Oryza sativa L.) cultivar for cooking quality. However, cultivars with similar apparent amylose content can have widely varying quality attributes, including major parameters of flour paste viscosity. It has been postulated that the presence of a rice Waxy gene single nucleotide polymorphism (SNP) marker is associated with elevated Rapid Visco Analyser (RVA) properties in specific high amylose rice cultivars. A mapping population derived from a cross between two varieties, Cocodrie and Dixiebelle, having similar high apparent amylose contents, but with different paste viscosity properties and Waxy gene markers was analyzed for the genetic segregation of various pasting properties, measured with RVA instrumentation. Marker inheritance analyses revealed that the Waxy exon 10 SNP marker was associated with the proportion of soluble to insoluble apparent amylose and most RVA pasting measurements. Waxy gene markers can be used to efficiently improve the selection of rice with desirable characteristics, particularly for superior parboiling and canning quality.

Development of three allele-specific codominant rice Waxy gene PCR markers suitable for marker-assisted selection of amylose content and paste viscosity

Four Waxy haplotypes, previously identified as each having a different combination of three single nucleotide polymorphisms (SNPs) in the Waxy gene, were highly correlated with apparent amylose content and pasting properties, which are important grain quality traits for predicting cooked rice (Oryza sativa L.) texture and processing properties (Chen et al. in J Cereal Sci 47:536–545, 2008a; Chen et al. in J Cereal Sci 48:781–788, 2008b). Three allele-specific PCR markers were developed to genotype the three aforementioned functional SNPs in a single PCR amplification. Each marker contained two allele-specific primers and one common primer. For each marker, the two allele-specific primers differed by one base at the 3′-end to provide discrimination of SNP alleles, and were labeled with unique fluorescence probes. An additional mismatched base, the third base from the 3′-end, was inserted in some allele-specific primers to increase selectivity. The amplification step of the PCR thermal cycling program was initially set for 20× touch-down cycles with the annealing temperature of the first cycle approximately 6°C above the thermal melting temperature of all three primers at a touch-down rate of −0.3°C per cycle, and followed by 25× regular thermal cycles with the annealing temperature at their thermal melting temperature. The allelic genotypes for each SNP were distinguished from each other by both their differential primer-allele fluorescences and their amplification product lengths. The simplicity of these assays makes it easy to utilize these markers as part of a marker-assisted selection strategy in rice breeding programs selecting for these important grain quality traits.

Introduction of antisensewaxy gene into main parent lines ofindica hybrid rice

Amylose content in rice endosperm is one of the key determinants of rice eating and cooking quality, and the poor quality ofindica hybrid rice is closely related to the high amylose level in rice grains. In order to improve the grain quality of theindica hybrid rice by genetic engineering, an antisense fragment of ricewaxy gene, driven by the 5′-franking sequences of the ricewaxy gene, was successfully introduced into three major parent lines ofindica hybrid rice, all contain a high amylose level in the grains, viaAgrobacterium, and more than 100 hygromycinresistant plants were regenerated. The analysis of PCR amplification and Southern blots indicated that the T-DNA containing the antisensewaxy gene had been integrated into the genome of transgenic rice plants. Most of the primary transgenic rice plants grew normally, and the mature seeds from these transgenic plants were performed for analysis of the amylose content. The results showed that the amylose content in the endosperm of some grains was reduced and the lowest reached 7.02% in one homozygous transgenic line, 72.4% lower than that of the wild type. The influence of the altered amylose content on the gelatinization temperature and gel consistency was also observed in several homozygous transgenic rice plants.

Interaction of rice bZIP protein REB with the 5?- upstream re-gion of both rice sbe1 gene and waxy gene

Rice starch branching enzyme 1 (SBE1) and granule-bound starch synthase (GBSS) catalyzed the biosynthesis of amylopectin and amylose in rice developing endosperm respectively, and the genes encoding these two enzymes, sbe1 and waxy, are mainly expressed in the endosperm. Within the 5′-upstream region of rice sbe1 gene, we identified a 53 bp fragment C53 which could interact with the nuclear proteins extracted from rice endosperm. We also found that fragment Ha-2 in the 5′-upstream region of rice waxy gene could compete with C53 for its interaction. Further experiments demonstrated that rice bZIP protein REB could interact with two ACGT elements (G-box and Hex) in the fragment C53 as well as three ACGT elements (WG1, WG2, and WG3) in the fragment Ha-2. The WG1 element could compete well with Hex element for its interaction with REB. These results suggested that the expression of sbe1 and waxy gene in rice developing endosperm may be coordinately regulated by REB-like bZIP transcriptional factors.

Evaluation of expression cassettes in developing rice endosperm using a transient expression assay

In order to evaluate rapidly the potential of various expression cassettes for transgene expression in rice grain, a transient expression assay was developed using rice immature endosperms. The promoters of several seed storage protein genes including the rice glutelins ( Gt3, GluB-1, GluB-2), rice prolamins ( PG5a, RP6), rice α-globulin ( Glb) and wheat glutenin ( Bx7), were examined for their expression levels in rice immature endosperms isolated from the caryopses at 7–9 days after pollination. In this promoter comparison, the wheat Bx7 promoter showed the highest transcription activity, followed by the promoters of the Glb, GluB-2, and Gt3 genes. Based on this result, further studies were conducted using the Glb and Bx7 promoters. We examined the effects of the addition of a second promoter fragment, the alfalfa mosaic virus (AMV) 5′untranslated leader sequence (UTL), and various introns and terminators from different genes. Although the addition of a second promoter fragment and the AMV 5′UTL had no effect on GUS expression levels, the insertion of the rice waxy first intron increased GUS expression of the Glb and Bx7 constructs 3 and 6-fold, respectively. The maize adh1 first intron also showed a 2–3-fold increase in the GUS expression levels of both the Glb and Bx7 reporter gene constructs. A comparison of terminator sequences revealed the nopaline synthase gene terminator from Agrobacterium and the rice waxy gene terminator were the most effective in contributing to elevated expression levels.

An Improved Method for Using a Microsatellite in the Rice Waxy Gene to Determine Amylose Class

ABSTRACTRice (Oryza sativa L.) breeders must evaluate progeny across multiple years and locations in part due to environmental effects on amylose content, the primary constituent that influences rice end‐use quality. A microsatellite correlated with the various classes of apparent amylose content in rice has been used to decrease the development time for the U.S. cultivars Cadet and Jacinto by several years. The objective of this project was to develop a relatively inexpensive method for assaying this microsatellite that is suitable for screening large numbers of progeny and to evaluate this method by analyzing a diverse set of breeding lines and cultivars. Rapid multiple‐kernel (brown and milled), single kernel, and leaf tissue alkali DNA extraction procedures were developed. Enhanced resolution of allele classes and separation speed was achieved by electrophoresing polymerase chain reaction (PCR) amplification products encompassing the waxy microsatellite in a polyacrylamide and Spreadex gel matrix using a triple‐wide mini electrophoresis unit. For germ plasm characterization, allele scoring accuracy and speed were improved by loading standards, consisting of three microsatellite classes in a single lane, several times across the gel. The microsatellite explained 88% of the variation in the apparent amylose content of 198 nonwaxy U.S. cultivars and breeding lines of diverse parentage, grown in four locations. The utility of this method was demonstrated by one technician analyzing a breeding population of 142 progeny in 1.5 days using relatively inexpensive laboratory equipment.

Use of alternate splice sites in granule-bound starch synthase mRNA from low-amylose rice varieties.

The rice Waxy gene encodes a granule-bound starch synthase (GBSS) necessary for the synthesis of amylose in endosperm tissue. We have previously shown that a CT microsatellite near the transcriptional start site of the GBSS gene can distinguish 7 alleles that accounted for more than 80% of the variation in apparent amylose content in an extended pedigree of 89 US rice cultivars (Oryza sativa L.). Furthermore, all the cultivars with 18% or less amylose were shown to have the sequence AGTTATA at the putative leader intron 5' splice site, while all cultivars with a higher proportion of amylose had AGGTATA. Here we demonstrate that this single-base mutation reduces the efficiency of GBSS pre-mRNA processing and results in alternate splicing at three cryptic sites. The predominant 5' splice site in CT18 low-amylose varieties is 93 bp upstream of the splice site used in intermediate and high amylose varieties and is immediately 5' to the CT microsatellite that we previously demonstrated to be tightly correlated with amylose content. Use of the leader intron 5' splice site at either -93 or -1 in conjunction with the predominant 3' splice site results in formation of a small open reading frame 38 bp upstream of the normal ATG and out of frame with it. This open reading frame is not produced when any of the 5' leader intron splice sites are used in conjunction with an alternate 3' splice site five bases further downstream which was observed in all rice varieties tested.

Effects of the first intron of rice Waxy gene on the expression of foreign genes in rice and tobacco protoplasts

The rice Waxy ( Wx) gene codes for the starch granule-bound starch synthase, which is responsible for the synthesis of amylose in the developing endosperm and pollen. Our previous works have located the transcriptional starting site of the rice Wx gene at 1288 bp upstream of the translation initial codon and identified a 1126 bp intron (intron 1) in the 5′-untranslated region. Here we report the influence of the 5′-untranslated region including intron 1 and the area around the translational initial codon of Wx gene on the expression of GUS gene in several chimeric constructs in rice and tobacco protoplasts. The GUS activity in rice and tobacco protoplasts transformed with the chimeric gene, pWG3.4 plasmid, was much higher than that of a construct with 1.3-kb 5′ untranslated region deleted. When Wx intron 1 was inserted into the site between the CaMV 35S promoter and GUS coding sequence, the GUS activity was increased about 15-fold in rice protoplasts, but with practically no effect in tobacco mesophyllous protoplasts. Sequence analyses of RNAs transcribed from pWG3.4 plasmid DNA in protoplasts showed that the intron 1 of the Wx gene was spliced accurately both in rice protoplasts and in tobacco mesophyllous protoplasts.

Structure, Organization, and Chromosomal Location of the Gene Encoding a Form of Rice Soluble Starch Synthase

A rice (Oryza sativa L.) genomic clone encoding the gene for a form of soluble starch synthase (SSS1) and its 5'- and 3'-flanking regions has been isolated and sequenced. The SSS1 gene contained 15 exons interrupted by 14 introns. The exon/intron organization of the SSS1 gene was divergent from that of the rice Waxy gene coding for granule-bound starch synthase, thus suggesting that the SSS1 and granule-bound starch synthase genes have evolved from an ancestral gene in a different way or that the two genes are products of different ancestral genes that have converged during evolution. However, these two genes were closely located to each other on rice chromosome 6 at an approximate map distance of 5 centimorgans. The nucleotide sequence of the 5'-end region of the gene is unique because of the presence of some repetitive sequences.

澱粉生合成の分子機構 イネｗａｘｙ遺伝子の発現制御機構

澱粉生合成の分子機構 イネｗａｘｙ遺伝子の発現制御機構

Antisense regulation of the rice waxy gene expression using a PCR-amplified fragment of the rice genome reduces the amylose content in grain starch

The waxy gene encodes a granule-bound starch synthase. A 1.0-kb portion of the sequence of the rice waxy gene, which includes the region between exon 4 and exon 9, was inserted in an antisense orientation between the 35 S promoter and the GUS gene of pBI221. The resultant plasmid, pWXA23, was introduced into rice protoplasts by electroporation. GUS activity was clearly detected in derived callus lines, suggesting that the antisense component of the fusion gene was also expressed. Transgenic rice plants were regenerated from these callus lines and their GUS activity was confirmed. Some of the rice seeds from these transformants showed a significant reduction in the amylose content of grain starch, even though they had become polyploid. These results suggest that even when intron sequences are included, antisense constructs can bring about a reduced level of expression of a target gene. The utility of GUS, included as a reporter gene, for the simple detection of expression of an antisense gene, was apparent from these results.

Nucleotide sequence of a long cDNA from the rice waxy gene.

Nucleotide sequence of a long cDNA from the rice waxy gene.