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Lilium regale Wilson Dirigent similar protein gene LrDIR1 and application thereof

A protein and gene technology, applied in Minjiang lily Dirigent similar protein gene LrDIR1 and its application field, can solve the problems affecting the yield and quality of lily cut flowers, the decline of bulb quality, the scale rot and fall off, etc., achieve broad market application prospects and shorten the breeding cycle , cost-saving effect

Active Publication Date: 2021-11-05
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fusarium infection of lily bulbs causes basal necrosis and scale rot and falls off, resulting in a decline in bulb quality; leaves turn yellow, wilt and droop after plants are infected with Fusarium, and plants wither and die early, seriously affecting the yield and quality of lily cut flowers

Method used

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  • Lilium regale Wilson Dirigent similar protein gene LrDIR1 and application thereof
  • Lilium regale Wilson Dirigent similar protein gene LrDIR1 and application thereof
  • Lilium regale Wilson Dirigent similar protein gene LrDIR1 and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1: LrDIR1 Gene cloning and sequence analysis

[0021] Lilium Minjiang was inoculated with Fusarium oxysporum, and total RNA was extracted from the roots 24 hours after inoculation. The inoculated roots of Lily Minjiang were ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and the total RNA was extracted by guanidine isothiocyanate method. For RNA, reverse transcriptase M-MLV (promega) was used to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process were as follows: take 5 μg TotalRNA, add 50ng oligo (dT), 2 μL dNTP (2.5mM each), and Add DEPC water to a reaction volume of 14.5 μL; after mixing, heat denaturation at 70°C for 5 minutes, then quickly cool on ice for 5 minutes, then add 4 μL 5×First-standbuffer, 0.5 μL RNasin (200 U), 1 μL M-MLV (200 U ), mix well and centrifuge for a short time, incubate at 42°C for 1.5h, take it out and heat at 70°C for 10min to terminate the re...

Embodiment 2

[0024] Embodiment 2: plant overexpression vector construction

[0025] Use the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert LrDIR1 coli plasmid pGEM-T- LrDIR1 As well as the plasmid of the plant expression vector pCAMBIA2300s, take 1 μL for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid; Xba Ⅰ (TaKaRa) and Eco RI (TaKaRa) for plasmid pGEM-T- LrDIR1 and pCAMBIA2300s for double enzyme digestion (100μL system), the reaction system and operation process are as follows: take 20μL pGEM-T- LrDIR1 and pCAMBIA2300s plasmid, add 10μL 10×K buffer, 4μL Xba I, 6μL Eco RI, 60 μL ddH 2 O, mix well and then centrifuge for a short time, and place it at 37°C for overnight reaction; spot all the digested products on agarose gel for electrophoresis, and then LrDIR1 The fragments and the large fragments of the pCAMBIA2300s vector were gel-recovered separately, and the SanPrep column DNA gel recove...

Embodiment 3

[0028] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0029] The transgenic recipient in this experiment is tobacco. Soak the tobacco seeds in 75% alcohol for 30s, wash them with sterile water and wash them with 0.1% HgCl 2 Soak for 8 minutes, then wash several times with sterile water, sow on 1 / 2 MS medium, culture in dark at 28°C for 6 days, transfer to light incubator (25°C, 16 h / d light) after germination, and then monthly Subculture once with 1 / 2 MS medium.

[0030] Take out the pCAMBIA2300s- containing pCAMBIA2300s- LrDIR1 The Agrobacterium LBA4404 strain of the plasmid was inoculated in 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampicin, and cultivated at 28°C until the medium was cloudy. Pipette 1mL of turbid bacterial solution onto LB solid medium containing 50mg / L Km, and incubate at 28°C for 48h; then scrape off an appropriate amount of Agrobacterium on LB solid medium and inoculate it into...

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Abstract

The invention discloses a Lilium regale Wilson Dirigent similar protein gene LrDIR1. The nucleotide sequence of the Lilium regale Wilson Dirigent similar protein gene LrDIR1 is as shown in SEQ ID NO: 1, and a protein with an amino acid sequence as shown in SEQ ID NO: 2 is encoded. Through the related technology research of the functional genomics, the LrDef1 gene is proved to have a function of improving the antifungal ability of plants. The antifungal LrDIR1 gene disclosed by the invention is constructed on a plant expression vector and transferred into tobacco for overexpression, a transgenic tobacco plant has a very strong antifungal infection ability, and experimental results show that the transgenic tobacco subjected to LrDIR1 overexpression has resistance to infection of fusarium oxysporum and Phoma herbarum.

Description

technical field [0001] The invention relates to molecular biology and genetic engineering related technical fields, in particular to a Minjiang lily Dirigent-like protein gene with the ability to resist fungal infection LrDIR1 and applications. Background technique [0002] During the growth process, plants are more or less under the stress of biotic or abiotic factors, which limit their growth and development to varying degrees, thus affecting the formation of final plant economic and yield traits. Among a variety of adverse stress factors, diseases caused by bacteria, fungi, and viruses are the main factors that endanger plant growth and development. Breeding resistant plant varieties and using pesticides are currently the main methods to solve the problem of plant diseases. The traditional breeding method has the disadvantages of long cycle, difficult to obtain the source of resistance, and the resistance of resistant varieties is easy to lose, so it cannot fundamentall...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/84A01H5/00A01H6/82
CPCC07K14/415C12N15/8282Y02A40/146
Inventor 刘迪秋王自娥梁婷婷邓婕苏琳琳陈虹均葛锋
Owner KUNMING UNIV OF SCI & TECH
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