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Guayule with increased rubber production and yield

a technology of guayule and rubber, applied in the field of altered guayule plants, can solve problems such as allergic reactions, and achieve the effects of increasing the amount of rubber, and reducing the altered paaos' functionality

Inactive Publication Date: 2018-11-15
UNITED STATES OF AMERICA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention relates to a method for producing guayule plants or seeds with increased rubber production compared to non-altered guayule plants or seeds. The method involves mutating the guayule plant's Allene oxide synthase (PaAos) gene to reduce its functionality and cause the guayule plant to produce more rubber. The mutation can occur through various methods such as changing the codon sequence, altering the translation initiation codon, changing the ribosome binding site sequence, or inserting or deleting DNA. The resulting guayule plants or seeds with the mutated PaAos gene produce more rubber compared to non-altered guayule plants or seeds. The invention also includes identifying high rubber producing guayule plants or seeds by detecting the presence of specific amino acids in PaAos.

Problems solved by technology

The Hevea tree is the main source of natural rubber but concerns exist as it is limited geographically to tropical climates, mainly in Southeast Asia, is susceptible to diseases, and produces rubber that causes allergic reactions.

Method used

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  • Guayule with increased rubber production and yield
  • Guayule with increased rubber production and yield
  • Guayule with increased rubber production and yield

Examples

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Effect test

example 1

ion of Genetically Altered Guayule

[0064]To better understand the role of PaAos in rubber synthesis, genetically altered P. argentatum plants are generated in which either PaAos is over-expressed or PaAos is silenced by RNAi. The various plasmids used to achieve the overexpression or silencing of PaAos in guayule are shown in FIG. 1. To generate these plasmids, the guayule Aos (PaAos) is amplified by PCR using genomic DNA as a template. The primers used to amplify PaAos are designed from the cDNA PaAos sequence published in NCBI database (GeneBank accession no. X78166.2) and have the following sequences: forward primer 5′-cttaagaggtggtATGGACCCATCGTCTAAACCC-3′ (SEQ ID NO: 1) and reverse primer 5′-ggatccTCATATACTAGCTCTCTTCAGGG-3′ (SEQ ID NO: 2). The nucleotides in lower case and underlined in the forward primer are the recognition nucleotides for restriction enzyme AflII; the nucleotides in lower case and underlined in the reverse primer are the recognition nucleotides for restriction ...

example 2

tion of RNA Expression Levels in Genetically Altered Plants

[0068]Guayule containing intact PaAos (non-altered; G7-11) and genetically altered guayule containing one of the plasmids (pND6, pND6-Aos, or pND6-AosiL) are further screened to determine the RNA level (see Table 3). Leaves from the various genetically modified plants (which are grown in tissue culture) are collected and snap-frozen in liquid nitrogen for RNA extraction. RNA is extracted using TRIzol® according to manufacturer's recommended protocol (Ambion, Pittsburgh, Pa.). RNA concentration is quantified with the NanoDrop ND1000 (ThermoScientific, Wilminton, Del.). RNA cleanup is performed using the RNeasy MinElute Cleanup kit according to manufacturer's recommended protocol (Qiagen Inc., Valencia, Calif.). The RNA is eluted with 30-50 μL of RNase-free water along with on-column DNase1 treatment.

[0069]Using the RNA isolated from the leaves of the genetically altered plants, cDNA is generated using iScript cDNA synthesis k...

example 3

antification in Tissue

[0071]Rooted plantlets (genetically altered, empty vector transformed (pND6 without PaAos), and wild-type control) from transferred shoot tips are grown on half-strength MS medium (PhytoTechnology Laboratories, Overland Park, Kans.) in Magenta boxes (Caisson Labs, Smithfield, Utah) for 6 weeks. The top part of the plantlets are separated from the medium and lyophilized for 48 hours. The dried tissues are placed in a 50 mL stainless steel grinding jar containing grinding ball, frozen in liquid nitrogen for 5 minutes and finely ground using the Retsch mixer mill MM 400 at a frequency of 30 / second for 1 minute (Verder Scientific Inc., Newtown, Pa.). Three hundred milligrams (0.3 g) of pulverized tissues are partitioned with Ottawa sand (Fisher Scientific, Fair Lawn, N.J.) and loaded into 11 mL stainless steel extraction cells (Dionex, Sunnyvale, Calif.). Three sequential extractions are performed using the Accelerated Solvent Extractor (ASE 2000; Dionex, Sunnyvale...

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Abstract

A reduction in the amount of functional PaAos in guayule results in the production of increased amounts rubber compared to the amount of rubber produced by wild-type guayule having a non-reduced amount of functional PaAos. Further, the guayule with reduced amount of functional PaAos are larger than wild-type guayule and thus have larger rubber yield per acre than wild-type guayule. Reduction of the amount of functional PaAos in guayule can be caused by genetic alterations in PaAos. Guayule having PaAos with a specific amino acid sequence produces more rubber than guayule with PaAos having a different amino acid sequence. Thus, one can use the sequence differences as a biomarker for selecting high rubber producing guayule plants.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This patent application claims priority to U.S. Patent Application 62 / 504,762 filed on May 11, 2017, contents of which are expressly incorporated by reference herein.BACKGROUND OF THE INVENTIONSequence Listing[0002]The Sequence Listing submitted via EFS-Web as ASCII compliant text file format (.txt) filed on May 10, 2018, named “SequenceListing_ST25”, (created on May 9, 2018, 24 KB), is incorporated herein by reference. This Sequence Listing serves as paper copy of the Sequence Listing required by 37 C.F.R. § 1.821(c) and the Sequence Listing in computer-readable form (CRF) required by 37 C.F.R. § 1.821(e). A statement under 37 C.F.R. § 1.821(f) is not necessary.Field of the Invention[0003]This invention relates to altered guayule plants that grow larger and produce more rubber than non-altered guayule plants, when grown under the same conditions. The altered guayule contain the cDNA sequence of Parthenium argentatum Allene oxide synthase...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/82
CPCC12N15/8261C12N15/8251C12Y402/01092C08L7/02C12N15/8218C12N15/8243C12N9/88Y02A40/146C12N15/1137C12N2310/14C07K14/415C12N2310/11
Inventor MCMAHAN, COLLEEN M.PLACIDO, DANTEDIERIG, DAVIDCRUZ, VON MARK
Owner UNITED STATES OF AMERICA
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