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Heat stable mutants of starch biosynthesis enzymes

A technology of thermostability and mutant enzymes, applied in the fields of mutant preparation, biochemical equipment and methods, enzymes, etc.

Inactive Publication Date: 2004-06-09
UNIV OF FLORIDA RES FOUNDATION INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This phenomenon was not observed on AGP activity

Method used

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  • Heat stable mutants of starch biosynthesis enzymes
  • Heat stable mutants of starch biosynthesis enzymes
  • Heat stable mutants of starch biosynthesis enzymes

Examples

Experimental program
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Effect test

Embodiment 1

[0081] Example 1——A test for corn endosperm ADP-glucose pyrophosphorylase containing multiple amino acid mutations

[0082] Expression of ADP-glucose pyrophosphorylase in maize endosperm.Inoculate 10 ml of Luria broth (75 g / mL spectinomycin and 50 g / mL kanamycin prime), and cultured overnight at 37°C with shaking at 220 rpm. These cultures were used to inoculate 250 mL of Luria broth (75 g / mL spectinomycin and 50 g / mL kanamycin). The culture was grown to OD at 37°C with shaking at 220rpm 600 = 0.55. Cultures were induced with 0.2 mM isopropyl-D-thiogalactopyranoside (IPTG) and .02 mg / mL nalidixic acid for 7 hours at room temperature with shaking at 220 rpm. Cells were collected by centrifugation at 3500 rpm for 10 min at 4°C. Resuspend the cell pellet in 800 L of extraction buffer. The above extraction buffer is 50mM HEPES, pH7.5, 5mM MgCl 2 , 5 mM EDTA, 20% sucrose and 30% ammonium sulfate. Add DTT (1mM), 50g / ml lysozyme, 1g / mL pepstatin, 1g / mL leupeptin, 1g / mL antipr...

Embodiment 2

[0103] Example 2 - Combination of thermostable mutations with Rev6

[0104] According to the invention, thermostable mutations may be combined with mutations associated with increased seed weight, such as the Rev6 mutation. The aim is to maintain the desired phosphate insensitivity profile of Rev6 while enhancing stability. Mutants containing Rev6 mutations in combination with thermostabilizing mutations can be constructed and validated as described herein. These "joint" mutants can be transformed into AC70R1-504 with the wild-type small subunit. Increased thermostability can be easily identified by positive glycogen staining on low glucose medium. Rev6 does not stain when grown on this medium. Initially, all combination mutants can be enzymatically screened for maintaining phosphate insensitivity, and then only combinations that maintain phosphate insensitivity are further analyzed.

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Abstract

The present invention relates to novel mutant polynucleotide molecules encoding enzymes with enhanced thermostability. When expressed in plants, these polynucleotides lead to increased yield in plants grown under conditions of heat stress. In one embodiment, the polynucleotide molecule of the present invention encodes maize endosperm ADP glucose pyrophosphorylase (AGP) and soluble starch synthase (SSS) enzyme activities. Also contemplated by the present invention are plants and plant tissues grown to contain or transformed with such mutant polynucleotides and expressing a polypeptide encoded by such polynucleotides. The invention also relates to methods for isolating polynucleotides and polypeptides within the scope of the invention. The present invention also provides methods of increasing yield in plants grown under conditions of heat stress.

Description

[0001] This invention was accomplished under the government support of National Science Foundation Project No. 9316887. The government has certain rights in this invention. [0002] Cross-references to related applications [0003] This application claims the benefit of US Provisional Patent Application Serial No. 60 / 275,768 (filed March 14, 2001). Background of the invention [0004] The sessile nature of plant life is its constant exposure to environmental factors that have both positive and negative effects on plant growth and development. A major obstacle to modern agriculture is unfavorable environmental conditions. An important factor causing significant crop losses is heat stress. Temperature stress has drastically reduced grain yields of many cereal crops such as corn, wheat and barley. Yield losses due to heat stress range from 7 to 35 percent of the world's important cereals. [0005] Many studies have identified possible physiological consequences of heat stres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/00C12N9/12C12N15/29C12N15/54C12N15/82
CPCC12N15/8245C12N9/1241
Inventor C·L·汉娜T·W·格林B·布格尔
Owner UNIV OF FLORIDA RES FOUNDATION INC
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