WO2015004638A2 - Mutations des gènes de biosynthèse de l'amidon à l'origine d'une forte expression de fibres et d'amidon résistant dans l'endosperme du riz - Google Patents

Mutations des gènes de biosynthèse de l'amidon à l'origine d'une forte expression de fibres et d'amidon résistant dans l'endosperme du riz Download PDF

Info

Publication number
WO2015004638A2
WO2015004638A2 PCT/IB2014/063040 IB2014063040W WO2015004638A2 WO 2015004638 A2 WO2015004638 A2 WO 2015004638A2 IB 2014063040 W IB2014063040 W IB 2014063040W WO 2015004638 A2 WO2015004638 A2 WO 2015004638A2
Authority
WO
WIPO (PCT)
Prior art keywords
starch
rice
endosperm
mutations
amylopectin
Prior art date
Application number
PCT/IB2014/063040
Other languages
English (en)
Other versions
WO2015004638A3 (fr
Inventor
Bhikane Shankarrao NANDKISHOR
Original Assignee
Texcity Bio-Sciences Pvt. Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Texcity Bio-Sciences Pvt. Ltd filed Critical Texcity Bio-Sciences Pvt. Ltd
Publication of WO2015004638A2 publication Critical patent/WO2015004638A2/fr
Publication of WO2015004638A3 publication Critical patent/WO2015004638A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/10—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits
    • A01H1/101—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine or caffeine
    • A01H1/102—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine or caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4636—Oryza sp. [rice]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)

Definitions

  • the present invention relates to an endosperm of rice with increased dietary fiber and resistant starch expression. More particularly, the invention relates to five mutations in starch synthase genes of rice leading to increased dietary fiber and resistant starch expression by modifying the amylopectin structure of starch which reduces the glycemic index.
  • Rapid urbanization and industrialization have produced advancement on the social and economic front in many countries that has resulted into dramatic lifestyle changes leading to lifestyle related diseases.
  • the transition from a traditional to modern lifestyle consumption of diets rich in fat and calories combined with a high level of mental stress has compounded the problem further.
  • more people are succumbed to various diseases owing to high lifestyle that includes eating pattern.
  • Trend of eating food in restaurants and fast food joints is increasing day by day, making people more prone to diseases that develop due to unhealthy eating habits.
  • One of such common diseases includes diabetes that happens due to increased blood sugar level in a person's body.
  • Diabetes mellitus generally known as Diabetes, is the most common endocrine disorder in both developing and developed countries. Diabetes is a chronic disease, which occurs when the pancreas fails to produce enough insulin, or when the body is not able to effectively use the insulin it produces. This leads to an increased concentration of glucose in the blood (hyperglycemia).
  • Type 1 diabetes previously known as insulin-dependent or childhood-onset diabetes
  • Type 2 diabetes previously called non-insulin-dependent or adult-onset diabetes
  • Another type of diabetes termed as gestational diabetes, is hyperglycemia which is first recognized during pregnancy.
  • the Glycemic index is a ranking of carbohydrates based on their immediate effect on blood glucose (blood sugar) levels. Foods that raise blood sugar content quickly, have high GI values. Conversely, foods that raise blood sugar content slowly have low GI values. As a result, the GI can be a useful indicator of starch digestion of food-based products. World health organization define GI as the incremental area under the blood glucose response curve of a 50 g available carbohydrate portion of a test food, expressed as a percent of the response to the same amount of carbohydrate from a standard food consumed by the same subject.
  • the GI consists of a scale from 1 to 100, indicating the rate at which 50 grams of carbohydrate in a particular food is absorbed into the bloodstream as blood-sugar. Glucose itself is used as the main reference point and is rated 100.
  • the GI values of foods are grouped into low GI ( ⁇ 55), medium (55-70), and high (>70) (Miller et al, 1992).
  • carbohydrates that break down quickly have high GI (giving fast and high glucose responses).
  • Lowering postprandial blood glucose by consuming low GI foods) has positive health outcomes for both healthy subjects and patients with insulin resistance.
  • the high starch content and low non-starch polysaccharide content of polished rice means that rice typically gives a high Glycemic response and contain low levels of dietary fiber and resistant starch.
  • Jenkins et al. (1981) reported a GI of 83 for white rice.
  • the viable solution will be to increase the fraction of dietary fiber and resistant starch (RS) within the endosperm of rice grains.
  • Dietary fiber and RS can elicit three major effects when included in the diet that is dilution of dietary metabolizable energy, a bulking effect, and fermentation to short-chain fatty acids and increase in expression of Peptide YY (PYY) and glucagon-like peptide (GLP)-l in the gut.
  • PYY Peptide YY
  • GLP glucagon-like peptide
  • DF and RS also encourage the growth of beneficial bacteria in the large intestine and lower the pH of the intestinal contents, all of which may reduce the development of colon cancer.
  • breeding rice with high DF and RS is of particular interest, as it will be easy to incorporate into the dietary-prevention strategy of diabetes.
  • High DF and RS cereals are useful for various applications but the genetic and molecular basis of these desirable traits are not fully understood yet.
  • RNA silencing techniques have been deployed in order to down- regulate the expression of starch branching enzyme lib (SBEIIb) to produce rice lines exhibiting the amylose extender (ae) phenotype with the aim of increasing the levels of RS and attenuating glycemic response.
  • SBEIIb starch branching enzyme lib
  • the RNA silencing techniques as per the current genetically modified organism regulatory norms cannot be applied to food crops like rice.
  • All the current strategies of enhancing the resistant starch in rice endosperm target the increase in amylose content, which has the drawback of severely affecting the cooking quality of rice grain. As rice is the only cereal cooked and eaten, augmenting the amylose content beyond 28% will lead to hardness in cooking of rice grains.
  • the present invention describes identification of the induced mutations in different starch synthase genes of rice.
  • the mutation is associated with down-regulation of these genes and subsequently expression of enzymes encoded by these genes.
  • the mutation targets the structural modification of amylopectin instead of increasing the amylose content, which does not offset the cooking quality of the rice grains and provides additional dietary fiber and resistant starch contents in the endosperm.
  • the present invention also discloses at increasing the fraction of dietary fiber and resistant starch (RS) within the endosperm of rice grains.
  • the increased dietary fiber and resistant starch is associated with decreased glycemic index in rice.
  • the wild type ADT 43 is hybridized with a mutant Tx2.
  • the first generation hybridization resulted in 27 true Fl plants, which were tagged based on morphological traits and selfed for the second generation.
  • the second hybridization resulted in generation of 540 F2 plants. Out of this, 384 F2 plants were randomly selected for Single Nucleotide Polymorphism (SNP) genotyping.
  • SNP Single Nucleotide Polymorphism
  • the Oryza sativa primers for the genes coding the enzymes Starch Synthase I (SSI), Starch Synthase Ila (SSIIa), Starch Synthase Ilia (SSIIIa), Granule Bound Starch Synthase (GBSSI), Starch Branching enzyme la (SBEIa), Starch Branching enzyme lib (SBEIIb) were manually designed using Gene Runner version 3.05.
  • the PGR is carried out with initial denaturation at 95°C for 10 min followed by 40 cycles at 95°C for 30s, 60°C for lmin, 72 °C for 1 min.
  • the results showed that there were no recombinants found for all the four SSIIIa alleles indicating that the gene is present in a recombination cold spot that does not allow free crossovers.
  • the invention also describes the effect of combined mutations of SSIIa and SSIIIa genes in the modification of amylopectin structure.
  • the endosperm of the Tx2 mutant showed the round shaped amyloplasts, which are relatively loosely packed into endosperm cell.
  • the genes coding the enzymes SSIIa and SSIIIa showed reduced fold change in qPCR studies of the mRNA isolated from the developing grains of the mutants and wild type suggesting down regulation is resulting from higher functionality of the mutation in suppressing these genes.
  • the invention may be employed to enhance the dietary fiber and resistant starch (RS) levels in the endosperm of any rice variety of interest through marker assisted breeding strategies.
  • RS dietary fiber and resistant starch
  • Such an enhancement of dietary fiber and resistant starch results in the development of low Glycemic Index rice varieties, which are of great demand for diabetic population of the country.
  • the fiber content of the mutant rice endosperm was found to be 8.20% and showed low glycemic index with a value of 51+5.2.
  • FIG 1 shows a table depicting resistant starch content in the seeds of the rice lines mutant Tx2 and wild type ADT 43, in accordance to one or more embodiments of the invention.
  • FIG 2 shows a table depicting the details of candidate genes explored for sequence variations, in accordance to one or more embodiments of the invention.
  • FIG 3 shows a table depicting primers and their fragments (PCR amplicon) size coverage, in accordance to one or more embodiments of the invention.
  • FIG 4 shows a PCR amplification of the seven candidate genes, in accordance to one or more embodiments of the invention.
  • FIG 5 shows position of mutations identified with respect to SSIIa (A) and gene SSIIIa (B), in accordance to one or more embodiments of the invention.
  • FIG 6 shows a table depicting the list of mutations identified in the key candidate genes of mutant Tx2, which are likely to increase the dietary fiber and resistant starch content in the endosperm, in accordance to one or more embodiments of the invention.
  • FIG 7 shows the sequence length and the size of the primers involved in starch biosynthesis, in accordance to one or more embodiments of the invention.
  • FIG 8 shows the delta ct values for the starch synthesis genes after qPCR with average ct values of target gene and average ct of UBQ5 gene, in accordance to one or more embodiments of the invention.
  • FIG 9 shows the fold change values (DDCt) values for the starch synthesis genes, in accordance to one or more embodiments of the invention.
  • FIG 10 shows the schematic representation of hybridization of wild type ADT 43 and mutant Tx2, in accordance to one or more embodiments of the invention.
  • FIG 11 shows the results of genetic analysis, in accordance to one or more embodiments of the invention.
  • FIG 12 shows the results of scanning electron microscope, in accordance to one or more embodiments of the invention.
  • FIG 13 depicts a table for the results of particle size of starch granules with percentage volume distribution, in accordance to one or more embodiments of the invention.
  • FIG 14 illustrates the graph showing the distribution of particle size of starch granules, in accordance to one or more embodiments of the invention.
  • FIG 15 shows the microscopic view of the starch granules from wild type ADT 43 and mutant Tx2, in accordance to one or more embodiments of the invention.
  • FIG 16 shows the values of fiber content and resistant starch of mutant Tx2, in accordance to one or more embodiments of the invention.
  • Resistant starch means portion of the starch, which is not broken down by human enzymes in the small intestine. It enters the large intestine where it is partially or wholly fermented, as context requires.
  • DNA sequencing means the process of determining the precise order of nucleotides within a DNA molecule, as context requires.
  • PCR means polymerase chain reaction (PCR), which is a biochemical technology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence, as context requires.
  • the term “Mutation”, means a permanent change in the DNA sequence of a gene that can alter the amino acid sequence of the protein encoded by the gene, as context requires.
  • “Expressed sequence tags”, means a short sub-sequence of a cDNA sequence. These tags may be used to identify gene transcripts, and are instrumental in gene discovery and gene sequence determination, as the context requires.
  • Quantitative polymerase chain reaction The term a laboratory technique based on the polymerase chain reaction, which is used to amplify and simultaneously quantify a targeted DNA molecule, as the context requires.
  • Glycemic index we mean a numerical scale used to indicate how fast and how high a particular food can raise the blood glucose (blood sugar) level, as the context requires.
  • Hybridization we mean a process of interbreeding between individuals of different species or genetically divergent individuals from different species, as context requires.
  • the present invention overcomes the drawback of the existing state of the art technologies by exhibiting mutations in the key target genes that are responsible for starch biosynthesis and modifies the amylopectin structure leading to increased dietary fiber (DF) and resistant starch (RS) content in rice.
  • the above methodology is successful in achieving the desired increased level of dietary fiber and resistant starch content in rice without increasing the amylose content of starch, which may destroy the cooking quality of rice by making it hard after cooking.
  • Plant materials used for selection of suitable mutants were Txl and Tx2 rice lines with high resistant starch content (> 7 %). These rice lines were isolated after screening of more than 1200 rice mutant lines generated from the variety ADT 43 through a mutagenic treatment with ethyl methane sulfonate. Txl and Tx2 lines were tested for their stability of resistant starch expression over locations and seasons consistently. Txl was relatively less stable for resistant starch expression hence eliminated from further characterization. Tx2 was selected for further characterization.
  • FIG 1 shows a table depicting resistant starch content in the seeds of the rice lines including mutant Tx2 and wild type ADT 43, in accordance to one or more embodiments of the invention.
  • the resistant starch content of the rice lines were estimated using the megazyme RS estimation kit.
  • Tx2 was isolated through biochemical screening of a large number of mutant lines for RS content in the endosperm and showed high content of resistant starch.
  • the highest RS expressing and stable line Tx2 was used for the identification of DNA sequence variants (mutations) leading to high RS and DF expression.
  • FIG 2 shows a table depicting the details of candidate genes explored for sequence variations, in accordance to one or more embodiments of the invention.
  • the Expressed Sequenced Tag (ES s) sequences for six candidate genes governing the biosynthesis of starch in rice endosperm along with the amino acid sequences were retrieved from the gene bank database of NCBI. These EST sequences analyzed through BLAST in comparison with the rice whole genome sequence available in NCBI.
  • FIG 3 shows a table depicting primers and their fragments (PCR amplicon) size coverage, in accordance to one or more embodiments of the invention Forward primers and reverse primers were designed for all the six candidate genes using the software Primer 3. The table also shows the length of the primers.
  • FIG 4 shows a PCR amplification of the seven candidate genes, in accordance to one or more embodiments of the invention. Mutations in these genes were identified through DNA sequencing of the PCR amplicons of the seven key candidate genes that are involved in the starch biosynthesis. Genomic DNA was isolated from ADT43 control and high RS expressing line Tx2 using the DNA isolation kit. Long Amp Taq DNA polymerase (Cat#M0323) was used for the PCR amplification of the genes. Next Generation Sequencing platform Ion Torrent was used for sequencing with the output of 1GB data on a 216 chip adopting suitable bar coding strategies. The shot gun sequences of ⁇ 100 base lengths were aligned over control sequence from ADT 43 and the mutations were confirmed using bioinformatics pipelines. Many mutations were identified as SNP changes between the wild and mutant.
  • FIG 5 shows position of mutations identified with respect to SSIIa and gene SSIIIa, in accordance to one or more embodiments of the invention.
  • One mutation position C978G with respect to the exon position in SSIIa is furnished in FIG 5A and four mutation positions namely C1615T, C1834T, T3559A and C10761T with respect to the exon positions in SSIIIa are furnished in FIG 5B.
  • FIG 6 shows a table depicting the list of mutations identified in the key candidate genes of mutant Tx2, which are likely to increase the dietary fiber and resistant starch content in the endosperm, in accordance to one or more embodiments of the invention.
  • the five mutations were identified from a high resistant starch expressing mutant line Tx2. Exon mutations were taken into account for functional analysis with PARSESNP, an online bioinformatics tool to know whether these mutations are capable of causing amino acid changes or not. Furthermore, PARSESNP analysis also gives details about introduction and disappearance of restriction sites.
  • SIFT Session Intolerant from Tolerant.
  • SIFT is a sequence homology-based tool that identifies intolerant from tolerant amino acid substitutions and predicts whether an amino acid substitution in a protein will have a phenotypic effect.
  • SIFT is based on the concept that protein evolution is correlated with protein function. The amino acid positions important for protein function will be conserved in an alignment of the protein family, whereas unimportant positions will appear diverse in an alignment.
  • the SIFT scores for all the five mutations of SSIIa and SSIIIa genes showed zero indicating that all these mutations are capable of altering an amino acid in the protein and these changes in the protein are intolerable and will either lead to disruption or down regulation of the concerned enzymes.
  • the mutations performed in the candidate genes are capable of increasing the resistant starch and dietary fiber in the endosperm to a level more than 8 %. All cultivated rice varieties normally has a level of ⁇ 2-3 % of resistant starch in their endosperm thus this mutation has the capability to enhance resistant starch by two to three times as compared to other rice varieties.
  • FIG 7 shows the sequence length and the size of the primers involved in starch biosynthesis, in accordance to one or more embodiments of the invention.
  • the starch synthase genes are subjected to qPCR amplification.
  • the Oryza sativa primers SSI, SSIIa, SSIIIa, Granule Bound Starch Synthase (GBSSI), Starch Branching enzyme la (BEIa), and SBEIIb and reference 18S rRNA and UBQ5 were manually designed using Gene Runner version 3.05.
  • PCR amplification curves were monitored for 40 cycles. Melting curves were analyzed for single product formation. Amplicons were run on the Bioanalyzer 2100 to confirm the expected sizes of the genes.
  • Relative quantification by qPCR was done using Brilliant II SYBR Green qPCR Master mix (Lot # 1105284). Each sample was run in duplicates for each gene using 50ng input per reaction. The experiment was conducted using Stratagene Mx3005P (Agilent technologies) platform. The relative expression levels of the genes were determined after normalizing with 18s rRNA and UBQ5 as the reference genes by using Delta Ct method. The PCR is carried out with initial denaturation at 95 °C for 10 min followed by 40 cycles at 95 °C for 30 s, 60°C for 1 min, 72°C for 1 min. A melt curve was also performed after the assay to check for specificity of the reaction. The down regulation of the concerned enzymes is clearly evident in the quantitative real time polymerase chain reaction (qPCR) study conducted from the RNA isolated from the endosperm of the wild type ADT 43 and mutant Tx2.
  • qPCR quantitative real time polymerase chain reaction
  • FIG 8 shows the delta ct values for the starch synthesis genes after qPCR with respect to ct of target sample with ct of control, in accordance to one or more embodiments of the invention.
  • the delta ct values are the difference of threshold cycles ct of the starch synthesis genes with ct of the control gene i.e. UBQ 5, which are determined to analyze the activity of the mutation.
  • the delta ct values for all the starch synthesis genes like SSI, SSIIa, SSIIIa, GBSS I, SBE la and SBE lib are increased in the Tx2 mutant compared to ADT 43 wild type.
  • the increased delta ct values indicate that the enzymes of starch biosynthesis are down regulated in the mutant Tx2 compared to the wild type ADT 43.
  • FIG 9 shows the fold change values (DDct) values for the starch synthesis genes, in accordance to one or more embodiments of the invention.
  • the lower reduction of fold change values in other genes including SSI, GBSS I, BE I, and SBE II B indicates that mechanisms like feedback inhibition of the enzymes has occurred in these enzymes.
  • FIG 10 shows the schematic representation of hybridization of wild type ADT 43 and mutant Tx2, in accordance to one or more embodiments of the invention.
  • the wild type ADT 43 is hybridized with the mutant Tx2.
  • the first generation hybridization resulted in 27 true Fl plants, which were tagged based on morphological traits and selfed for the second generation.
  • This resulted in generation of 27 F2 families constituted by 20 plants each accounting to a population of 540 F2 plants.
  • 384 F2 plants were randomly selected for Sequence Nucleotide Polymorphism (SNP) genotyping.
  • SNP Sequence Nucleotide Polymorphism
  • FIG 11 shows the results of genetic analysis, in accordance to one or more embodiments of the invention.
  • the genetic analysis depicts the association of wild type and mutant alleles with various starch fractions released from a amylase digestion.
  • the matured grains from 384 F2 plants were harvested, milled and grinded and subjected to biochemical analysis for determination of amylase digestion and estimating starch digestion fractions such as Rapidly Digestible Starch (RDS), Slowly Digestible Starch (SDS) and Resistant Starch (RS).
  • RDS Rapidly Digestible Starch
  • SDS Slowly Digestible Starch
  • RS Resistant Starch
  • the double mutant combination is uniquely responsible for reduced RDS level thereby lowering GI in rice.
  • the present invention is a unique attempt to study the effect of combined mutations of these two key genes in the modification of amylopectin structure. These two genes are found to be the key enzymes involved in the amylopectin chain elongation in rice endosperm hence mutations in these genes will down regulate these key enzymes resulting in short chain amylopectin with more proportion of 1-6 linkages. As 1-6 linkages are not prone for a amylase digestion, this result in slower release of glucose finally resulting in low GI.
  • FIG 12 shows the results of scanning electron microscope, in accordance to one or more embodiments of the invention. The scanning electron microscopic studies of the cross section of the rice seed of wild type ADT 43 and mutant Tx2 were carried out to test the effects of SSIIa and SSIIIa activity on the granular structure of starch in the endosperm.
  • the endosperm starch granules formed homogenous polygonal granules with sharp edges and the granules were tightly packed into the amyloplasts, which were very abundant in the endosperm cells as shown in Fig 12A.
  • the amyloplasts of the mutant Tx2 were round shaped and relatively loosely packed into the endosperm cell as shown in Fig 12B.
  • FIG 13 depicts a table for the results of particle size of starch granules with percentage volume distribution, in accordance to one or more embodiments of the invention.
  • the particle size of the starch granules determines resistant starch and dietary fiber contents in natural and cooked foods.
  • the particle size of the starch granules of wild type ADT43 and mutant Tx2 are determined in the present invention, lg of finely ground rice flour is mixed with 5ml of 0.01M sodium hydroxide and ⁇ of 1% protease overnight at 37°C with moderate shaking.
  • the samples are neutralized with 1M hydrochloric acid and centrifuged at 3000g.
  • the precipitate is suspended with water, layered over 80% (w/v) caesium chloride solution and centrifuged at 13000g for 20 minutes. After centrifugation, again the pellet is suspended with water and filtered through ⁇ pore size nylon filter. Supernatant is discarded and dark tailing layer is removed. The starch pellet is washed thrice with water, followed by acetone and finally overnight air dried in the fume hood. The dried starch pellet is subjected to particle size analysis using laser diffraction particle size analyzer. The table shows the results of percentage volume proportion in wild type ADT 43 and mutant Tx2 with reference to particle size of the starch granule.
  • FIG 14 illustrates the graph showing the distribution of particle size of starch granules, in accordance to one or more embodiments of the invention.
  • the graph represents the association of the particle size of the starch granules with percentage volume proportion in wild type ADT 43 and mutant Tx2. It interprets that the mutant Tx2 showed higher dispersion of particle size than the wild type ADT 43.
  • FIG 15 shows the microscopic view of the starch granules from wild type ADT 43 and mutant Tx2, in accordance to one or more embodiments of the invention.
  • the starch granules isolated from wild type ADT 43 showed higher dispersion of particles and smaller particles of less than 10 micron occupied more volume proportion.
  • the starch granules from Tx2 showed reduced dispersion of particles and particles with 10-20 mcirons size occupied more than 90 % volume.
  • the particle size in the Tx2 mutant is more homogenous with a shift in dispersion towards higher size as compared to wild type ADT 43.
  • Fig 16 shows the fiber content and the glycemic index of the mutant Tx2.
  • the fiber content was found to be 8.20%.
  • the rice is also subjected to glycemic analysis to determine the glycemic index.
  • the rice showed the low glycemic index 51.2+5.2. This value falls under the low glycemic category.
  • the mutation in starch synthase genes of rice is associated with increased dietary fiber and resistant starch. This is also associated with modified amylopectin structure of starch, which reduces the glycemic index.
  • the rice showed increased fiber content of 8.20% and low glycemic index with a value of 51.2+5.2, which is of high demand in the market.
  • the induced mutation is also effective in altering functional mutation in SSIIa gene, which gains significance with respect to manipulation of amylopectin chain length in indica varieties cultivated in India.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Physiology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Molecular Biology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne les mutations touchant les gènes de l'amidon synthétase du riz mutant Tx2 qui sont associées à des teneurs en fibres alimentaires et en amidon résistant améliorées dans l'endosperme. Les teneurs accrues en fibres alimentaires et en amidon résistant chez le riz ont pour résultat un indice glycémique bas, d'une valeur de 51,2 + 5,2, ce qui fait que ces variétés de riz sont très recherchées par les diabétiques. Les mutations touchant les gènes de l'amidon synthétase ciblent la modification structurelle de l'amylopectine plutôt qu'elles n'entraînent une augmentation de la teneur en amylose, et ce, sans affecter les caractéristiques de cuisson des grains de riz. La mutation touchant le gène SSIIa est également associée à un moindre allongement de chaîne dans la forme L de l'amylopectine si bien que la longueur de chaîne de l'amylopectine peut être modifiée dans les variétés de riz Indica.
PCT/IB2014/063040 2013-07-12 2014-07-11 Mutations des gènes de biosynthèse de l'amidon à l'origine d'une forte expression de fibres et d'amidon résistant dans l'endosperme du riz WO2015004638A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN3126/CHE/2013 2013-07-12
IN3126CH2013 2013-07-12

Publications (2)

Publication Number Publication Date
WO2015004638A2 true WO2015004638A2 (fr) 2015-01-15
WO2015004638A3 WO2015004638A3 (fr) 2015-05-21

Family

ID=52280680

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2014/063040 WO2015004638A2 (fr) 2013-07-12 2014-07-11 Mutations des gènes de biosynthèse de l'amidon à l'origine d'une forte expression de fibres et d'amidon résistant dans l'endosperme du riz

Country Status (1)

Country Link
WO (1) WO2015004638A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015004638A3 (fr) * 2013-07-12 2015-05-21 Texcity Bio-Sciences Pvt. Ltd Mutations des gènes de biosynthèse de l'amidon à l'origine d'une forte expression de fibres et d'amidon résistant dans l'endosperme du riz
WO2017141128A1 (fr) * 2016-02-15 2017-08-24 Udaya Agro Farm Procédé d'augmentation de l'amidon résistant et des fibres alimentaires dans le riz
CN108949861A (zh) * 2018-08-13 2018-12-07 江南大学 一种制备慢消化糊精的方法
CN109662307A (zh) * 2018-12-29 2019-04-23 上海市农业科学院 一种富含慢消化淀粉的复合青稞粉及其制备方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6585930B2 (en) * 2001-04-25 2003-07-01 Extrude Hone Corporation Method for article fabrication using carbohydrate binder
CN1886507B (zh) * 2003-10-27 2010-12-29 联邦科技产业研究组织 淀粉中具有增加了直链淀粉含量的水稻及水稻制品
AR064557A1 (es) * 2006-12-29 2009-04-08 Bayer Cropscience Ag Almidon de maiz y harinas y alimentos de maiz que comprenden este almidon de maiz
NZ597971A (en) * 2009-07-30 2013-09-27 Commw Scient Ind Res Org Barley and uses thereof
WO2015004638A2 (fr) * 2013-07-12 2015-01-15 Texcity Bio-Sciences Pvt. Ltd Mutations des gènes de biosynthèse de l'amidon à l'origine d'une forte expression de fibres et d'amidon résistant dans l'endosperme du riz

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015004638A3 (fr) * 2013-07-12 2015-05-21 Texcity Bio-Sciences Pvt. Ltd Mutations des gènes de biosynthèse de l'amidon à l'origine d'une forte expression de fibres et d'amidon résistant dans l'endosperme du riz
WO2017141128A1 (fr) * 2016-02-15 2017-08-24 Udaya Agro Farm Procédé d'augmentation de l'amidon résistant et des fibres alimentaires dans le riz
US20190071687A1 (en) * 2016-02-15 2019-03-07 Udaya Agro Farm A method for increasing resistant starch and dietary fiber in rice
CN109689874A (zh) * 2016-02-15 2019-04-26 乌达亚农业农场 用于增加水稻中的抗性淀粉和膳食纤维的方法
EP3430144A4 (fr) * 2016-02-15 2019-12-04 Udaya Agro Farm Procédé d'augmentation de l'amidon résistant et des fibres alimentaires dans le riz
CN108949861A (zh) * 2018-08-13 2018-12-07 江南大学 一种制备慢消化糊精的方法
CN109662307A (zh) * 2018-12-29 2019-04-23 上海市农业科学院 一种富含慢消化淀粉的复合青稞粉及其制备方法
CN109662307B (zh) * 2018-12-29 2021-12-21 上海市农业科学院 一种富含慢消化淀粉的复合青稞粉及其制备方法

Also Published As

Publication number Publication date
WO2015004638A3 (fr) 2015-05-21

Similar Documents

Publication Publication Date Title
Fitzgerald et al. Identification of a major genetic determinant of glycaemic index in rice
Bao et al. Genome‐wide association study of the resistant starch content in rice grains
CN106086028B (zh) 一种通过基因组编辑提高水稻抗性淀粉含量的方法及其专用sgRNA
Fitzgerald et al. Not just a grain of rice: the quest for quality
Yang et al. A putative gene sbe3-rs for resistant starch mutated from SBE3 for starch branching enzyme in rice (Oryza sativa L.)
Parween et al. Balancing the double‐edged sword effect of increased resistant starch content and its impact on rice texture: its genetics and molecular physiological mechanisms
Gurunathan et al. Single nucleotide polymorphisms in starch biosynthetic genes associated with increased resistant starch concentration in rice mutant
Shu et al. The influences of chain length of amylopectin on resistant starch in rice (Oryza sativa L.)
Raja et al. EcoTILLING by sequencing reveals polymorphisms in genes encoding starch synthases that are associated with low glycemic response in rice
WO2015004638A2 (fr) Mutations des gènes de biosynthèse de l'amidon à l'origine d'une forte expression de fibres et d'amidon résistant dans l'endosperme du riz
US11785907B2 (en) Wheat lines, plants, and grain with increased beta-glucan content
Naseer et al. Variability in waxy (Wx) allele, in-vitro starch digestibility, glycemic response and textural behaviour of popular Northern Himalayan rice varieties
Qi et al. Ae1/Sbe1 maize-derived high amylose improves gut barrier function and ameliorates type II diabetes in high-fat diet-fed mice by increasing Akkermansia
Karaman et al. Development of EMS-induced Mutagenized Groundnut Population and Discovery of Point Mutations in the ahFAD2 and Ara h 1 Genes by TILLING
Kumar et al. Low light stress influences resistant starch content and glycemic index of rice (O. sativa L)
Dwiningsih et al. Glycemic index of diverse rice genotypes and rice products associated with health and diseases
CN107920490A (zh) 具有降低的脂氧合酶活性的小麦
WO2018092155A1 (fr) Variété de riz non ogm riche en amidon résistant et en fibres alimentaires
JP2019509035A (ja) イネにおける難消化性デンプンおよび食物繊維を増加させる方法
Mudge et al. Genomic approaches for improving grain quality of sorghum
Zafeiriou A forward genetic approach to identify new loci controlling wheat starch digestibility
Dhanalakshmi et al. Breeding Low Glycemic Index Rice (Oryza sativa L.) Cultivars: Progress, Benefits, and Challenges
John et al. Type‐2 Diabetes and Identification of Major Genetic Determinants of Glycemic Index in Rice–A Review
Singh et al. Exploring the glycaemic impact and culinary qualities of rice through Genome-Wide Association Studies on starch composition and viscosity profiles
Kumar et al. Cooking quality of rice is a potential predictor to identify low starch digestibility rice

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14822298

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14822298

Country of ref document: EP

Kind code of ref document: A2