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Lilium regale Lr4CL-1 gene and application thereof

A technology of lr4cl-1 and gene, applied in Minjiang lily Lr4CL-1 gene and its application fields, can solve the problems of blank biological function and mechanism of action, improve plant fungal disease resistance, shorten breeding cycle, and reduce environmental pollution Effect

Inactive Publication Date: 2019-05-28
YANGTZE NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the 4CL gene of Lily of Minjiang River, and there are gaps in its biological function and mechanism of action.

Method used

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  • Lilium regale Lr4CL-1 gene and application thereof
  • Lilium regale Lr4CL-1 gene and application thereof
  • Lilium regale Lr4CL-1 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Lilium Minjiang Lr4CL-1 full-length gene cloning and sequence analysis

[0028] The genome sequencing of Minjiang lily (Lilium regale Wilson) has not yet been completed. The research group used Botrytis cinerea to infect Minjiang lily (sprouting stage) in the early stage, and used the leaves after 24 hours of treatment as materials, using TRIzol 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.

[0029] 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).

[0030] PCR amplification system: 2xfast pfu master Max 10ul, forward primer (Lr4CL-1-F, 10μM) 1μL, reverse primer (Lr4CL-1-R, 10μM) 1μL, templ...

Embodiment 2

[0038] The construction of embodiment 2 plant overexpression vectors

[0039] (1) Construction of pART-CAM::Lr4CL-1 overexpression vector

[0040] According to the full-length gene sequence of Lilium Minjiang Lr4CL-1 (SEQ ID NO.1), primers Lr4CL-1-inf-F and Lr4CL-1-inf-R were designed, and the seamless cloning (In-fusion) vector linker sequence was introduced into the primers . Using the TA-ligated positive clone plasmid in Example 1 as a template, Lr4CL-1-inf-F and Lr4CL-1-inf-R were used as primers to perform specific amplification of the gene Lr4CL-1.

[0041] The primer sequences are as follows:

[0042] Lr4CL-1-inf-F:5'- GGAGAGGACACGCTCGAG ATGGGCTCCATCTCTCCG-3'

[0043] Lr4CL-1-inf-R:5'- TTAAAGCAGGACTCTAGA TCACTGCTGTCGACCGTT-3'

[0044] Among them, the underline is the linker sequence of the In-fusion cloning vector.

[0045] PCR amplification system: 2xfast pfu master Max 10ul, forward primer (Lr4CL-1-F, 10μM) 1μL, reverse primer (Lr4CL-1-R, 10μM) 1μL, template ...

Embodiment 3

[0053] Example 3 Agrobacterium-mediated transformation of tobacco and screening of transgenic positive lines

[0054] Rinse the seeds of tobacco (Nicotiana tabacum cv.Petit Havana SR1) with sterile water, sterilize with 75% ethanol for 1 min, sterilize with sodium hypochlorite solution (2% available chlorine) for 15 min, rinse with sterile water 3-5 times, and absorb them with sterile filter paper. Moisture on the surface of dry seeds, inoculated on LS medium (PH5.8).

[0055] Glycerol bacteria were streaked on YEB solid medium (50 μg / mL streptomycin + 20 μg / mL rifampicin), and after two days of culture at 28 ° C, single clones were picked and inoculated into 5 ml of YEB liquid medium (50 μg / mL streptomycin). Mycin+20μg / mL rifampicin), 28°C, 220rpm constant temperature shaking culture overnight to OD 600 At about 0.6, centrifuge at 5000rpm for 10min to collect the bacteria, and resuspend the collected bacteria to OD with MS liquid medium 600 = 0.4.

[0056] After 1 to 2 mon...

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Abstract

The invention discloses a lilium regale Lr4CL-1 gene and application. A nucleotide sequence of the Lr4CL-1 gene is as shown in SEQ ID NO.1, an amino acid sequence coded by the Lr4CL-1 gene is as shownin SEQ ID NO.2, an overexpression vector containing the Lr4CL-1 gene is transferred into tobacco, an obtained transgenic tobacco plant has obvious inhibiting effects on pathogenic bacteria of botrytis cinerea, alternaria alternata, anthracnose and the like, and meanwhile, the fact that leaves of the transgenic plant is more dark-green and slightly droop is found. According to the lilium regale Lr4CL-1 gene and the application, through studies of functional genomics, the fact that the Lr4CL-1 gene has double functions of improving fungus resistance of plants and changing the leaf characters isproven, and theory and technique support is provided for using the gene for conducting plant molecular heredity improvement in future, so that the lilium regale Lr4CL-1 gene has potential applicationvalues.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a Minjiang lily Lr4CL-1 gene and its application. Background technique [0002] In agricultural production, plant diseases, especially fungal diseases, have always been an important factor limiting the improvement of crop yields. At present, chemical pesticides are mainly used as the main means of disease control, but long-term use of chemical pesticides not only leads to the development of drug resistance of pathogenic bacteria, but also causes damage to the ecological environment. Therefore, the selection and breeding of disease-resistant varieties has attracted more and more attention, and has become one of the effective control measures for plant diseases. However, due to the long cycle of conventional breeding, limited breeding resources, and rapid mutation of pathogenic bacteria, it is difficult to obtain resistant plants and meet the needs of...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/20
Inventor 符勇耀杨利平杨韦朱艺勇郭雅萌
Owner YANGTZE NORMAL UNIVERSITY
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