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Lilium regale wilson LrCCoAOMT gene and application thereof

A lily and gene technology is applied to the LrCCoAOMT gene of Minjiang lily and its application field to achieve the effects of improving yield and quality, short breeding cycle and simple operation

Inactive Publication Date: 2019-06-28
YANGTZE NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no genes with both anti-fungal and anti-lodging effects have been reported in Minjiang lily.

Method used

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  • Lilium regale wilson LrCCoAOMT gene and application thereof
  • Lilium regale wilson LrCCoAOMT gene and application thereof
  • Lilium regale wilson LrCCoAOMT gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Lilium Minjiang LrCCoAOMT full-length gene cloning and sequence analysis

[0038] In the early stage, the research group used Botrytis cinerea to infect Minjiang lily (sprouting stage), and the leaves after 24 hours of treatment were used as materials, and TRIzol was used to TM Plus RNA Purification Kit (12183555, Invitrogen TM ), total RNA was extracted according to the instructions, and DNase I (18047019, Invitrogen TM ) to remove residual traces of DNA, and use a spectrophotometer to measure the concentration of RNA for later use.

[0039] About 2.0 μg of total RNA from leaves of Lilium Minjiang was taken, and the first-strand cDNA was synthesized according to the instructions of PrimeScript II first-strand cDNA synthesis kit (6210A, Takara).

[0040] The PCR amplification system is high-fidelity amplification enzyme Prime STAR HS (R010A, TaKaRa) 0.25 μL, 5×PrimeSTAR Buffer (Mg 2+ Plus) 5 μL, forward primer (LrCCoAOMT-F, 10 μM) 0.5 μL, reverse primer (Lr...

Embodiment 2

[0048] The construction of embodiment 2 plant overexpression vectors

[0049] According to the full-length gene sequence of Lilium Minjiang LrCCoAOMT (SEQ ID NO.1), primers LrCCoAOMT-inf-F and LrCCoAOMT-inf-R were designed, and a seamless cloning (In-fusion) vector linker sequence was introduced into the primers. The TA-ligated positive clone plasmid in Example 1 was used as a template, and LrCCoAOMT-inf-F and LrCCoAOMT-inf-R were used as primers to perform specific amplification of the gene LrCCoAOMT.

[0050] The primer sequences are as follows:

[0051] LrCCoAOMT-inf-F:

[0052]

[0053] LrCCoAOMT-inf-R:

[0054]

[0055] Wherein, the bolded sequence of the forward primer is the Xba I restriction site, and the bolded sequence of the reverse primer is the Sac I restriction site. The linker sequence of the In-fusion cloning vector is underlined.

[0056] The PCR amplification system is high-fidelity enzyme PrimeSTAR HS (R010A, TaKaRa) 0.5μL, 5xPrimeSTARBuffer (Mg 2...

Embodiment 3

[0064] Example 3 Agrobacterium-mediated genetic transformation of Arabidopsis thaliana and screening of transgenic positive lines

[0065] The genetic transformation of Arabidopsis thaliana adopts the flower dipping method (Zhang X., et al., Nat Protoc. 2006, 1:641-646). Agrobacterium carrying pBI121-35S::LrCCoAOMT vector was introduced into Columbia Col type Arabidopsis thaliana. The regenerated seedlings of Arabidopsis thaliana with resistance were screened with kanamycin (100mg / L), the genomic DNA was extracted by CTAB method, and the positive plants were detected by PCR amplification.

[0066] PCR amplification primers are as follows:

[0067] nptII-F:5'-TTGGGTGGAGAGGCTATTCGG-3'

[0068] nptII-R:5'-GCCACAGTCGATGAATCCAG-3'

[0069] The PCR reaction reagents were selected from TaKaRa (RR001A), specifically including: 1 μL of DNA, 10×Buffer (containing 20mM Mg 2+ ) 2 μL, 2.5 mM / L dNTP 2 μL, 10 uM / L forward and reverse primers 0.5 μL each, 5 U / μL Taq DNA polymerase 0.2 μL, 1...

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Abstract

The invention discloses a lilium regale wilson LrCCoAOMT gene and application thereof. The nucleotide sequence of the LrCCoAOMT gene is shown in SEQ ID NO.1, and the amino acid sequence coded by the gene is shown in SEQ ID NO.2. An overexpression vector containing the LrCCoAOMT gene is transferred into arabidopsis thaliana to obtain genetically modified arabidopsis thaliana, compared with a wild plant, the stem of the genetically modified arabidopsis thaliana is thicker and stronger, rosette leaves are larger, and the lignin content is obviously increased. Through functional genomics research,it is further proved that the LrCCoAOMT gene can improve the lodging resistance of the plant and enhance the resistance of plants to pathogenic fungi, especially botryis cinerea and alternaria alternata, the LrCCoAOMT gene plays an important role in breeding work of the plants, and the yield and the quality of crops are easily improved; the LrCCoAOMT gene has an important theoretical and practical significance in improving the plant quality and other key agronomic traits of the crops, and further provides a new means for regulating the resistance by using genetic engineering.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a Minjiang Lily LrCCoAOMT gene and its application. Background technique [0002] Lodging is a ubiquitous problem in crop production and has become one of the important limiting factors for high yield, stable yield and high quality. After the crops fall, the normal distribution order of the leaves in space is disrupted, the plant population structure is destroyed, and the photosynthetic efficiency of the leaves is reduced sharply. Nutrients, the yield is greatly reduced, which increases the difficulty of harvesting crops, which will greatly affect the yield and quality of crop harvesting, and also greatly reduce the ornamental value of plants. Therefore, preventing and improving crop lodging is of great significance to ensure high, stable, high-quality and ornamental crops. [0003] Plant fungal diseases have always been a very difficult problem in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/82A01H5/00A01H6/20A01H6/82A01H6/56
Inventor 符勇耀杨利平杨韦朱艺勇徐文姬
Owner YANGTZE NORMAL UNIVERSITY
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