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Methods for increasing starch content in plant cobs

一种淀粉含量、穗轴的技术，应用在植物分子生物学领域，能够解决效率低等问题

Inactive Publication Date: 2012-04-04
SYNGENTA PARTICIPATIONS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another limitation may be the inefficiency of the method of extracting sugars from cobs due to the dense structure of lignocellulose

Method used

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  • Methods for increasing starch content in plant cobs
  • Methods for increasing starch content in plant cobs
  • Methods for increasing starch content in plant cobs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] Generation of transgenic maize plants

[0132] Eight RNAi cassettes were constructed for plant expression. These cassettes are designed to downregulate the enzymes alpha-amylase, beta-amylase, dextran water dikinase (R1), phosphodextran water dikinase (PWD), and chloroplast alpha-amylase (AMY3) in maize cob tissue . The maize cDNA sequences for α-amylase (Genbank Accession No. L25805), β-amylase (Genbank Accession No. Z25871 ) and α-glucan water dikinase (R1 , Genbank Accession No. CD973834) were obtained from NCBI. The maize orthologue of Arabidopsis chloroplast phosphoglucan water dikinase (GenBank accession number AJ635427) was generated based on sequence homology to the maize genomic sequence. Likewise, a maize orthologue of Arabidopsis Amy3 (GenBank Accession No. NM105651 ) was generated based on sequence homology to the maize genomic sequence. RNAi fragment from the 3' end of the coding region of each gene (α-amylase of 495 bp, SEQ ID NO: 1; β-amylase of 500 b...

Embodiment 2

[0145] Sample collection and pre-screening of transgenic maize events.

[0146] 10 days after pollination from mature T 0 Cob samples were collected from corn plants. Then, fresh cobs were frozen at -80°C. Afterwards, the seeds were removed from the frozen cobs using a paint scraper. Freezing the cobs allows for better removal of corn seeds for cob analysis. After removing the cob seeds, each cob was then sliced ​​into slices less than 1 / 4 inch thick. Two sections (one from the middle and one from the end) were used for Lugol's iodine staining. The method as described in Example 1 can be used by using T transformed with an empty binary vector without RNAi cassette 0 plants to generate control plants.

[0147] Use Lugol's staining solution to perform T 0 Prescreening for starch accumulation in maize events. Lugol's solution selectively stains starch from dark blue to black and can be visualized under a microscope. The degree of staining relative to the null control a...

Embodiment 3

[0164] Starch and Sugar Evaluation in Corn Cob Tissue Samples

[0165] The following assay protocol was used to assess the amount of sugar and total starch by dry weight in cob samples. This method employs the Megazyme Total Starch Assay (MEGAZYME, Wicklow, Ireland) (AOAC Method 996.11 and AACC Method 76.13), which involves complete digestion of sample starch into free D-glucose by alpha-amylase and amyloglucosidase hydrolysis , followed by a glucose oxidase-peroxidase reaction and a colorimetric measurement of free D-glucose released from the sample. The amount of starch in the sample can be calculated via a simple conversion of the measured amount of D-glucose released. The sugars were extracted with water and the amount analyzed by HPAEC (High Performance Anion Exchange Chromatography).

[0166] Cob sample preparation

[0167]Fresh cobs were frozen at -80°C, photographs were taken, and a paint scraper was used to remove the seeds. Then, slice each cob into slices le...

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Abstract

Methods and compositions for increasing the starch content in green tissues of a plant are provided. The method comprises down-regulating the activity of starch degradation enzymes in a plant. The resulting transgenic plants of the invention have increased starch content in green tissues and exhibit a starch excess phenotype. In one embodiment the method involves manipulating a monocot plant to down-regulate the activity of a starch degradation enzyme. The plants are useful for improving the yield of free sugars from plant biomass and increase dried green tissue storage stability.

Description

field of invention [0001] The present invention relates to plant molecular biology, and in particular to methods and compositions for increasing starch accumulation in plant cobs and the use of these plant tissues in commercial applications. Background of the invention [0002] Plant biomass is composed of sugars and represents the largest source of renewable hydrocarbons on Earth. Unlike other renewable energy sources, biomass can be converted directly into liquid fuels. The two most common types of biofuels are ethanol (ethyl alcohol) and biodiesel. Ethanol is an alcohol that can be produced by fermenting any biomass high in carbohydrates (starch, sugar, or cellulose). Once fermentable sugars are obtained from the biomass material, these sugars can be fermented to produce ethanol via a process similar to brewing beer. However, this vast resource is underutilized due to the fact that sugars are locked in complex polymers, often collectively referred to as lignocellulose....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87A01H5/00
CPCC12N15/8245C12N9/00Y02E50/17Y02E50/10
Inventor J.斯蒂芬斯
Owner SYNGENTA PARTICIPATIONS AG
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