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Plant RNA (Ribose Nucleic Acid) interference vector for inhibiting lignin from synthesizing, and construction method and application of plant RNA interference vector

An RNA interference and lignin technology, applied in the biological field, can solve problems such as increasing costs and affecting the absorption of nutrients in livestock

Active Publication Date: 2013-06-12
LIAONING UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the excessive deposition of lignin in pastures affects the absorption of nutrients in pastures by livestock; lignin in industrial plants also increases the cost of utilizing plant fibers

Method used

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  • Plant RNA (Ribose Nucleic Acid) interference vector for inhibiting lignin from synthesizing, and construction method and application of plant RNA interference vector
  • Plant RNA (Ribose Nucleic Acid) interference vector for inhibiting lignin from synthesizing, and construction method and application of plant RNA interference vector
  • Plant RNA (Ribose Nucleic Acid) interference vector for inhibiting lignin from synthesizing, and construction method and application of plant RNA interference vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: alfalfa CCoAOMT Cloning of gene fragments

[0034] 1. Extraction of total RNA from alfalfa

[0035] (1) Take 100mg of young alfalfa tissue, add liquid nitrogen to grind; add 1mL RNAiso Plus to the mortar, let stand at room temperature until the sample is completely melted, and continue to grind until the lysate becomes transparent;

[0036] (2) Transfer the homogenate to a centrifuge tube; centrifuge at 12000 r / min, 4°C for 5 min; take the supernatant and transfer it to a new centrifuge tube;

[0037] (3) Add chloroform (1 / 5 volume of RNAiso Plus) to the supernatant, shake for 15 seconds, and wait for the solution to be fully emulsified, then let stand at room temperature for 5 minutes;

[0038] (4) Centrifuge at 12000 r / min, 4°C for 15 minutes; absorb the supernatant and transfer it to another new centrifuge tube;

[0039] (5) Add an equal volume of isopropanol to the supernatant, mix thoroughly, and let stand at room temperature for 10 minutes;

[...

Embodiment 2

[0052] Example 2: Preparation of Plant RNA Interference Carrier Inhibiting Lignin Synthesis

[0053] The forward nucleic acid fragment and the reverse nucleic acid fragment with corresponding restriction sites obtained in Example 1 were respectively connected to the pMD19-T carrier; Asc I and Swa I double-digest the forward nucleic acid fragment connected to the pMD19-T carrier and the plant expression vector pFGC5941, reclaim the target fragment, connect with T4 ligase, and obtain the intermediate vector pFGC5941-CCoAOMT1; Xba I and Sma I double digest the pMD19-T vector containing the reverse nucleic acid fragment and the intermediate vector pFGC5941-CCoAOMT1, recover the target fragment, connect it with T4 ligase, and construct the plant RNA interference vector pFGC5941-CCoAOMTi that inhibits lignin synthesis.

[0054] The double digestion vector pFGC5941-CCoAOMTi was identified, the results are as follows figure 1 and figure 2 As shown, 2 fragments were obt...

Embodiment 3

[0056] Example 3: Transformation of plant RNA interference vector into Agrobacterium tumefaciens.

[0057] 1. Preparation of Agrobacterium Competent Cells

[0058] Pick a single colony of Agrobacterium tumefaciens and inoculate it in 3ml LB liquid medium; 220 r / min, 28°C, shaking culture to OD 600 =0.5; draw 1.5ml of bacterial liquid into a centrifuge tube, ice-bath for 10min; 30s, discard the supernatant; use 100μl 20mM CaCl per tube 2 suspended for transformation.

[0059] 2. Direct Transformation of Plasmids into Agrobacterium

[0060] Add 10ul of plasmid pFGC5941-CCoAOMTi to 50μl of Agrobacterium competent cells, and ice bath for 30 minutes; put it in liquid nitrogen for 1 minute, and then immediately put it in a 37℃ water bath for 5 minutes; take out the centrifuge tube, add 0.5mlLB, 28℃, 220 Shake culture at r / min for 4 hours; take out the bacterial solution and smear it on an LB plate containing kanamycin, and culture it upside down in an incubator at 28°C. Colo...

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Abstract

The invention relates to a plant RNA (Ribose Nucleic Acid) interference vector for inhibiting lignin from synthesizing, and a construction method and application of the plant RNA interference vector. The plant RNA interference vector is that some nucleotide sequences (serving as forward nucleic acid fragments) of alfalfa CCoAOMT as shown in a sequence table SEQ ID No.1 and some nucleotide sequences (serving as reverse nucleic acid fragments) of the alfalfa CCoAOMT as shown in a sequence table SEQ ID No.2 are inserted into a plant expression vector pFGC5941, thus constructing the plant RNA interference vector pFGC5941-CCoAOMTi. The obtained interference vector is used for transforming agrobacterium tumefaciens EHA105 competent cells, so as to obtain agrobacterium engineering bacteria containing the interference vector. The agrobacterium engineering bacteria containing the interference vector is utilized for transforming the plant, and therefore, the plant with low lignin content can be cultivated. By adopting the method, a transgenic plant with normal growth and low lignin content can be gained, and germplasm resources are provided for cultivating low-lignin grass and papermaking plant and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction of a plant interference carrier that inhibits lignin synthesis and its application in cultivating plants with low lignin content. Background technique [0002] Lignin is an important class of macromolecular phenolic polymers in plants. Its quantity is huge. Its content in nature is second only to cellulose and ranks second in organic polymers. It usually accounts for 10% of the dry weight of plants. Around, in trees it can even reach 30%. Lignin plays an important role in the growth and development of terrestrial plants. However, the excessive deposition of lignin in pastures affects the absorption of nutrients in pastures by livestock; lignin in industrial plants also increases the cost of utilizing plant fibers. Therefore, in animal husbandry production and plant fiber utilization industry, it is always desired to obtain new plant strains with low lig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N1/21C12N15/66A01H4/00A01H5/00C12R1/01
Inventor 韩阳王红艳段亚楠卢福荣茹艺李曹娜
Owner LIAONING UNIVERSITY
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