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Novel application of liriodendron hybrid LhWOX1 gene

A technology of hybridizing Liriodendron tulipifera and genes, applied in genetic engineering, plant genetic improvement, application, etc., can solve problems such as narrowing of leaves and petals, failure of fusion of petals and carpels, etc.

Active Publication Date: 2020-05-08
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In Arabidopsis, although the wox1 single mutant has no obvious visible phenotype, in the wox1 / wox3 double mutant, all organs become narrow stripes; petunia WOX1-like factor MAEWEST (MAW) mutation, petal Inability to fuse with carpels, leaves and petals narrowed

Method used

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  • Novel application of liriodendron hybrid LhWOX1 gene
  • Novel application of liriodendron hybrid LhWOX1 gene
  • Novel application of liriodendron hybrid LhWOX1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Genetic Transformation of Tobacco with LhWOX1 Gene

[0032] (1) Bacterial solution preparation

[0033] Pick a single colony of Agrobacterium containing the target gene (LhWOX1 gene) stored on LB solid medium, inoculate it into 5 mL of LB liquid medium plus corresponding screening antibiotics Kan50mg / L, Str30mg / L, shake at 28°C and 220rpm Cultivate for 24 hours, expand and inoculate into LB liquid medium containing corresponding antibiotics at a ratio of 1:50, and culture with shaking at 28°C and 250rpm for 4-6 hours, then measure the OD value with a UV spectrophotometer (OD value = 0.3-0.4 is enough ). Centrifuge at 4000rpm for 10min, remove the supernatant, and collect the cells. Resuspend the bacteria with a certain volume of MS or 1 / 2MS liquid medium, and dilute the bacteria to OD 600 =0.2, spare.

[0034] (2) Pre-cultivation

[0035] Tissue culture 3-4 week-old tobacco aseptic seedlings, take the size, character, and color of the aseptic seedlings t...

Embodiment 2

[0040] Example 2: Positive Plant Screening of LhWOX1 Gene Transformed Tobacco

[0041] 1. Detection of tobacco plant genomic DNA

[0042] (1) Extraction of tobacco genomic DNA by CTAB method

[0043] 1) Turn on the constant temperature water bath and adjust to 65°C to preheat the lysate CTAB;

[0044] 2) Put the fresh young leaves of the transgenic and control into 2mL centrifuge tubes, add sterile glass beads, and grind the plant material thoroughly with a vibrating ball mill. After the grinding is complete, put the centrifuge tubes on ice, and quickly add CTAB 700μL, shake vigorously to mix;

[0045] 3) Place the material in a 65°C constant temperature mixer for 30 minutes, and shake the centrifuge tube several times during the period;

[0046] 4) After 30 minutes, take out the centrifuge tube and cool it to room temperature, add an equal volume (24:1) of chloroform / isoamyl alcohol, shake vigorously to mix, and let stand for 10 minutes;

[0047] 5) Place the mixed solution...

Embodiment 3

[0093] Example 3: Observation and analysis of LhWOX1 transgenic tobacco T1 phenotype

[0094] Observation and analysis of the leaf phenotype of the T1 generation of transgenic LhWOX1 tobacco, the results are as follows: Figure 4 As shown in middle b-f, it can be seen that the leaves of transgenic LhWOX1 tobacco plants are seriously curled, and the leaves are all curled in the direction of the adaxial plane of the leaves, but the curling degree and shape are different. According to statistics, among the 30 LhWOX1 transgenic tobacco plants transplanted in the greenhouse, 28 plants showed different degrees of leaf curling, while 2 plants did not appear leaf curling. The shape of the leaf tip of the transgenic tobacco leaves with LhWOX1 gene was significantly different from that of the wild-type tobacco leaves. The leaf tips of the transgenic tobacco leaves were sharp-pointed, while the leaf tips of the wild-type leaves were usually short-pointed. The leaf base type of LhWOX1 tr...

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Abstract

The invention discloses a novel application of a liriodendron hybrid LhWOX1 gene, and belongs to the technical field of plant genetic engineering. The novel application of the liriodendron hybrid LhWOX1 gene provided by the invention specifically comprises the following application steps: constructing a vector of the liriodendron hybrid LhWOX1 gene; transforming the constructed vector of the liriodendron hybrid LhWOX1 gene into plants or plant cells; and performing cultivation and screening to obtain transgenic plants with curly leaves. The invention discovers that the liriodendron hybrid LhWOX1 gene can curl the plant leaves for the first time, and the overexpression can cause some plant leaves to be curled and change morphology; and leaf curling is a very useful agronomic trait for somecrops, so that the LhWOX1 gene provided by the present application can be used in preparation of plants having the curled leaves and cultivation of novel plant varieties, and has important applicationvalue.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a new application of the hybrid Liriodendron LhWOX1 gene. Background technique [0002] In the study of Arabidopsis, Petunia, etc., it was found that WOX1 transcription factors were involved in the development of lateral organs in organs. In Arabidopsis, although the wox1 single mutant has no obvious visible phenotype, in the wox1 / wox3 double mutant, all organs become narrow stripes; petunia WOX1-like factor MAEWEST (MAW) mutation, petal Unfused with carpels, leaves and petals narrowed. Studies in Arabidopsis have shown that WOX1 and WOX3 jointly regulate the division activity of organ margin meristem cells, and regulate the developmental patterns of the adaxial and abaxial surfaces of leaves. Tobacco LAM1 gene and Medicago truncatula MtWOX1 gene (STENOFOLIA, STF) are also involved in the regulation of leaf lateral growth and vascular bundle de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/12A01H6/00
CPCC07K14/415C12N15/8261
Inventor 陈金慧李美平龙晓飞施季森胡向阳郝兆东张稼霁成铁龙
Owner NANJING FORESTRY UNIV
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