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Paulownia 4-coumaric acid: coenzyme A ligase gene and use thereof

A technology of coumaric acid and ligase, applied in the field of bioengineering, can solve the problems of low bulk density, limited application, low strength and hardness, etc.

Inactive Publication Date: 2009-05-20
河南省绿士达林业新技术研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] But it should be noted that the other side of paulownia material: light wood (low bulk density), low strength and hardness, which seriously limits its application in construction, transportation, home decoration, furniture, papermaking, etc.

Method used

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  • Paulownia 4-coumaric acid: coenzyme A ligase gene and use thereof
  • Paulownia 4-coumaric acid: coenzyme A ligase gene and use thereof
  • Paulownia 4-coumaric acid: coenzyme A ligase gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Cloning and sequencing of Paulownia part 4CL coding sequence:

[0029] 1. Design of degenerate primers: The amino acid homology of plant 4-coumaric acid: Coenzyme A ligase protein was analyzed by GenBanK BLAST system, and its completely homologous part was selected, and 2 pairs of degenerate primers were selected, listed below All PCR primers involved in the present invention have been described.

[0030] (1) Degenerate primers:

[0031] B4F7 5'-CACARCARGTHGAYGGHGAVAAYCC-3'

[0032] B4R7 5'-CCWGCYTCDGTCATBCCRTADCCCTG-3'

[0033] B4F8 5'-CACARCARGTHGAYGGHGAVAAYCC-3'

[0034] B4R8 5'-CKRATGCARATYTCWCCRGSRTGRTT-3'

[0035] (2) 5' RACE primer:

[0036] Outer Primer 5'-catggctacatgctgacagccta-3'

[0037] Inner Primer 5'-cgcggatccacagcctactgatgatcagtcgatg-3'

[0038] 4.5GSP1 5'CAAAACAATCGGCGGCACGAAGGGC3'

[0039] 4.5GSP2 5'AACGGAACAATATCGAATTTCTGCATGATCAGG3'

[0040] (3) 3'RACE Primer 3'RACE:

[0041] Outer Primer 5'-TACCGTCGTTCCACTAGTGATTT-3'

[0042] T...

Embodiment 2

[0080] Example 2: Acquisition of unknown sequence upstream of Paulownia 4CL mRNA 5':

[0081] A preferred solution is to use the 5'-FULL RACE kit (Code No: D315) produced by TaKaRa Company. The first step of this technical system is phosphorylation, the second step is to remove the cap, and the third step is to add a linker. Theoretically speaking, the first step eliminates all those that have lost their "caps", broken in the middle, and incomplete. Incomplete and incomplete mRNA, so that the unknown 5' end of the clone is complete, and the cloned mRNA may be full-length. At the same time, technologies such as adding specific adapters and nested PCR are used to amplify the full-length sequence of the 5' end of cDNA with high sensitivity and high specificity.

[0082] The partial coding sequence of Paulownia 4CL was obtained by RT-PCR with the obtained degenerate primers, and 2 specific nested PCR primers were optimized, as shown in 1 of Example 1 (design of degenerate primers...

Embodiment 3

[0085] Example 3: Acquisition of Unknown Sequence Downstream of Paulownia 4CL mRNA 3':

[0086] A preferred solution is to use 3'-FULL RACE Core Set Ver.20 (Code No: D314) produced by TaKaRa Company. This technology system uses technologies such as Oligad (T) adapters and primers added with specific sequences, so that the 3' end sequence of the cDNA can be amplified more sensitively and specifically through the known partial cDNA sequence.

[0087] The partial coding sequence of Paulownia 4CL was obtained by RT-PCR with the obtained degenerate primers, and 2 specific nested PCR primers were optimized, as shown in 1 of Example 1 (design of degenerate primers).

[0088] Concrete cloning procedures include: a. Extraction of total RNA of Paulownia: the same as 2 in Example 1 (extraction of total RNA of Paulownia); b. Nested PCR reaction: the specific primer GSP1 and 3' RACE Outer Primer [ See 1 in Example 1 (design of degenerate primers)], carry out PCR reaction, and then use the...

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Abstract

The invention discloses a paulownia 4-coumaric-acid: coenzyme A ligase, encoding genes and application thereof. Amino acid residue sequence of the 4-coumaric-acid: coenzyme A ligase is shown in SEQ ID No. of 1. The information can be used for constructing genes of the 4-coumaric-acid: coenzyme A ligase, or antisense genes and RNA disturbing mass of the genes, which can be used for improving plant materials and constructing new materials with higher or lower lignin content.

Description

Technical field: [0001] The present invention relates to a Paulownia 4-coumaric acid: coenzyme A ligase (4-coumarate: coenzymeAligase, 4CL) gene and application thereof, belonging to the field of bioengineering, in particular to Paulowniaceae (Paulowniaceae) Paulownia (Paulownia) white flowers [Paulownia fortunei (Seem.) Hemsl) encoding 4CL gene and its application. Background technique: [0002] 1. Regarding the material improvement of Paulownia and its wood: [0003] Paulownia (Paulownia) is a deciduous tree of the Paulowniaceae (Paulowniaceae) genus Paulownia. It is native to my country. Except for a few species distributed to Vietnam and Laos, other species are unique to my country. [0004] Paulownia is one of the three high-quality, fast-growing timber tree species in the world. It is widely distributed in 23 provinces, municipalities and autonomous regions. In many areas, it is used as the main tree species for tree planting, agricultural paulownia intercropping, an...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A01H1/00
Inventor 裴海朝杨艳坤陈昌民熊治国牛静勇刘志刚叶金山王军军陈占宽
Owner 河南省绿士达林业新技术研究所
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