Methods and genetic constructs for modification of lignin composition of corn cobs

A technology of corn cobs and lignin, applied in the field of agricultural biology, can solve problems such as less success

Inactive Publication Date: 2009-08-05
SYNGENTA PARTICIPATIONS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, application of dsRNA inhibition in mammalian systems has been rarely successful

Method used

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  • Methods and genetic constructs for modification of lignin composition of corn cobs
  • Methods and genetic constructs for modification of lignin composition of corn cobs
  • Methods and genetic constructs for modification of lignin composition of corn cobs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0234] Agrobacterium transformation of maize - immature embryos

[0235] Ear of Corn Preparation

[0236] Ears of corn are harvested when the immature embryos are approximately 0.5 to 1.0 mm within the center of the kernel.

[0237] Ears of corn were dehulled and sterilized in a solution of 20% Clorox and 3 drops of Tween / liter solution. Place on an orbital shaker for 20 minutes.

[0238] with sterile ddH 2 O Rinse the ear of corn 3 times.

[0239] The corn kernels are topped off in a sterile environment, the ears are placed on sterile petri dishes and the immature embryos are isolated.

[0240] Preparation of seeding solution for transformation

[0241] • In 100 mL of LP-Lsinf. medium, add 50 μl of acetosyringone (AS) stock solution (40 mg / ml stock solution / mL) so that the final concentration is 100 μM AS.

[0242] • Pipette 4ml (2.5) of the infection medium into a 10ml disposable tube.

[0243] • Prepare Eppendorf centrifuge tubes for collecting the embryos at thi...

Embodiment 2

[0267] T1 CAD results select pSyn12210

[0268] T1 cob samples from a total of 15 transgenic results (10 low-copy, 3 medium-copy and 2 high-copy) contained 65 lines (28 low-copy, 14 medium-copy, 5 high-copy and 18 Blank strain control), which were sent to MSU for NDF (fiber), ADL (lignin), and IVNDFD (in vitro NDF digestibility) analysis. Also included are 3 BM3 congeners and 2 hybrid tests. We compared the differences between individual lines of the same transgene result in the transgene result (transgene vs blank comparison), or the difference between the low copy, medium copy and high copy result with BM3 positive or negative control and hybridization test. Although some of these became the top 16 lines described below, none of them showed a sustained decrease in lignin in subsequent assays. The data are shown in Table 1, and the analysis method used is as described below.

[0269] T1 COMT result selection (pSyn12345)

[0270] T1 cob analysis: A total of 41 transgen...

Embodiment 3

[0407] CAD / COMT double knockout

[0408] Co-expression of dsRNAi constructs of CAD and COMT driven by the OsMADs6 promoter can be achieved in one construct. Using the transformation procedure described in Example 1, transgenic maize bearing such a construct was produced. The sequences of the OsMADs6 promoter and the RNAi constructs are shown herein, and the assays described above were used to determine lignin content, among others. Cobs with reduced lignin content produced in this way can be used with figure 1 Those cobs produced by the plasmids of or 2 were used to the same extent and for the same purpose.

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Abstract

The present invention relates to methods and genetic constructs for the control of expression of enzymes involved in lignin biosynthesis in plants. The method involves the use of double-stranded RNAi to down-regulate or knock out the expression of the CAD and COMT genes. In particular embodiments the method involves the use of cob-specific or cob-preferred promoters for down-regulation of lignin biosynthesis in the cobs of com plants.

Description

[0001] priority claim [0002] This application claims priority based on US Application No. 60 / 665,685, filed March 28, 2005, the disclosure of which is incorporated herein by reference. technical field [0003] The invention relates to the field of agricultural biotechnology. More specifically, the present invention relates to the use of RNAi technology to modify the expression of genes associated with the biosynthesis of lignin in maize (maize) and more specifically in maize cobs. Background technique [0004] Lignin is a complex heterogeneous aromatic polymer that renders membranes impermeable and strengthens the cell walls of certain plant cells. [0005] Lignin is formed by the polymerization of free radicals from monolignols such as paracoumaryl alcohol, coniferyl alcohol and sinapyl alcohol (Higuchi, 1985, in Biosynthesis and degradation of wood components (T. Higuchi, ed.), Academic Press, Orlando, FIa. pp. 141-160). Lignin is formed by the polymerization of at le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/11C12N15/82C12N15/00C12N5/10A01H3/00C07H21/02C07H21/04
CPCY02E50/17C12N15/8218C12N15/8255C12P7/06Y02E50/10
Inventor R·E·卡特J·斯蒂芬斯
Owner SYNGENTA PARTICIPATIONS AG
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