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Wheat cinnamyl alcohol desaturase, encoding gene and uses thereof

A cinnamyl alcohol dehydrogenase and gene technology, applied in the application field of cultivating lodging-resistant plants, can solve the problems of yield loss and huge loss

Inactive Publication Date: 2009-08-19
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In production practice, lodging caused by insufficient mechanical strength of wheat stalks can lead to yield loss of more than 20%
In addition, the losses caused by the invasion of diseases and insect pests are also very huge.

Method used

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  • Wheat cinnamyl alcohol desaturase, encoding gene and uses thereof
  • Wheat cinnamyl alcohol desaturase, encoding gene and uses thereof
  • Wheat cinnamyl alcohol desaturase, encoding gene and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Isolation and sequence analysis of cinnamyl alcohol dehydrogenase gene probe in wheat

[0049] Wheat (Triticum aestivumL, variety H4564, Handan Seed Company, Hebei Province) was planted in a greenhouse, watered and fertilized normally, until it grew to 2-3 nodes, and roots, stems, and leaf tissues were collected and tested with TRI reagent ( Molecular Research Center, Inc, Cincinnati, USA) extract total RNA, use PolyAT mRNA Isolation kit (Promega company, Madison, USA) separates Poly(A) + RNA.

[0050] The conditions for reverse transcription to synthesize the first strand of cDNA are: Poly(A) + RNA 1ug, 5μmol / L Primer 5'-GACTCGAGTCGACATCGA(T) 17 -3', 0.5mmol / L dNTP, 50 units of RNasin, incubate at 65°C for 10 minutes, after cooling on ice, add 200U SuperScript TM II RNase H - -Reverse Transcriptase (Gibco Company, Grand Island, NY, USA), incubated at 42°C for 1 hour, added EDTA to a final concentration of 10 mM, and incubated at 95°C for 10 minutes, ...

Embodiment 2

[0054] Example 2. Screening and sequence analysis of wheat cinnamyl alcohol dehydrogenase gene

[0055] With Wheat Stem Poly(A) + Using RNA as a template, cDNA was synthesized with a ZAP vector (Stratagene, La Jolla, CA, USA), and packaged in vitro to construct a cDNA library of wheat stems. Take 50ng probe DNA, add 50uCi 32 P-dCTP was labeled, incubated at 37°C for 1 hour, and EDTA was added to a final concentration of 10mM to terminate the reaction. After the probe passed through the Sephadex G-50 column, it was boiled at 100° C. for 10 minutes, and immediately cooled on ice for hybridization.

[0056] Take 50000pfu to spread the plate, and transfer it to the nitrocellulose membrane, denature in 0.5M NaOH plus 1.5MNaCl for 2 minutes, 1.5M NaCl plus 0.5M Tris-HCl (pH8.0) for 5 minutes, 0.2MTris-HCl (pH7.5) rinsed with 2x SSC for 30 seconds, sandwiched the membrane between two layers of Whatman filter paper, baked in vacuum at 80°C for 2 hours, then added hybridization solu...

Embodiment 3

[0059] Embodiment 3, the translation of wheat cinnamyl alcohol dehydrogenase gene coded protein

[0060] Artificially synthesize a pair of primers and introduce EcoRI and NotI restriction sites respectively. The primers are as follows:

[0061] 5'-Primer: 5'-CGGAATTCATGGGCAGCGTCGACGCCTC-3',

[0062] 3'-Primer: 5'-ATAAGAATGCGGCCGCTCAGGCGGCGTCCTCGATG-3'.

[0063] With the p1Ta-CAD1 described in Example 2 as template, carry out PCR amplification, amplification condition: 10ng DNA, 1 μ mol / L primer, 0.4mmol / L dNTP, 2.5U Taq DNA polymerase (Gibco company, Grand Island, NY , USA). Denaturation at 95°C for 5 minutes, followed by 30 cycles (1 minute at 95°C, 1 minute at 55°C, 1.5 minutes at 72°C), and a final extension at 72°C for 10 minutes.

[0064] PCR products were subjected to 1.0% agarose gel electrophoresis, using DNA Isolation Kit (Gibco Company, Grand Island, NY, USA) was used to purify the electrophoresis product, and after double digestion with EcoRI and NotI, the cor...

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Abstract

The invention discloses a cinnamyl alcohol dehydrogenase of wheat and an encoding gene and application thereof; the cinnamyl alcohol dehydrogenase is the protein of the following a) or b): a) the protein composed of the amino acid sequences represented by the sequences 2 in the sequence table; b) the protein derived from a) and related to the cinnamyl alcohol dehydrogenase function by substituting and / or deleting and / or adding one or more amine acid in the amino acid sequences represented by the sequences 2 in the sequence table. The invention further provides the encoding gene of the cinnamyl alcohol dehydrogenase, and application of the encoding gene in culturing lodging-resistant plants. The encoding gene is introduced in the plants; and the transformed plant hosts can be wheat. The protein in the invention utilizes coniferyl aldehyde as base materials and has enzyme activities of 94.49 nmol / sec / mg, showing that the protein can control the lignin synthesis of the wheat. Northern hybridization is carried out on the Ta-CADI genes of the stems of easily-lodging and lodging-resistant wheat, which shows that the Ta-CADI genes not only control the lignin synthesis of the wheat, but also are closely related to the lodging-resistant characteristics of the stems of the wheat.

Description

technical field [0001] The invention relates to a cinnamyl alcohol dehydrogenase and its coding gene and application, in particular to a cinnamyl alcohol dehydrogenase from wheat and its coding gene and its application in cultivating lodging-resistant plants. Background technique [0002] In vascular plants including ferns, gymnosperms, and angiosperms, the appearance of lignin plays an important role in the evolution of plants to land, allowing plants to adapt to drier external environments and develop into larger spaces , thus forming a more diverse plant group (Peter, G. and Neale, D., 2004, Current Opinion in Plant Biology 7: 737-742). The synthesis of lignin also plays an important role in the growth and development of plants. Lignin is mainly deposited on the cell walls of plant vessels, rays, thick-walled cells, etc., which can increase the mechanical strength of the cell wall and reduce its permeability. Therefore, the synthesis of lignin enhances the mechanical sup...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/63C12N5/10C12N1/00C12N15/82A01H5/00C12N15/11
Inventor 马庆虎
Owner INST OF BOTANY CHINESE ACAD OF SCI
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