Lilium regale chitinase gene LrCHI2 and application thereof

A chitinase gene, lily technology, applied in the fields of molecular biology and genetic engineering, can solve the problems affecting the yield and quality of lily cut flowers, the quality of seed bulbs, the decay and falling off of scales, etc., and achieve broad market application prospects and breeding cycles. The effect of shortening, reducing usage

Active Publication Date: 2021-02-12
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 chitinase gene LrCHI2 and application thereof
  • Lilium regale chitinase gene LrCHI2 and application thereof
  • Lilium regale chitinase gene LrCHI2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: LrCHI2 cDNA cloning and sequence analysis

[0019] Total RNA was extracted from the root of Lily Minjiang, ground into powder with liquid nitrogen, then transferred to a centrifuge tube, total RNA was extracted by guanidine isothiocyanate method, and total RNA was extracted by reverse transcriptase M-MLV (promega) Synthesize the first strand of cDNA as a template. The reaction system and operation process are as follows: Take 5 μg Total RNA, add 50ng oligo(dT), 2μL dNTP (2.5mmol / L each), and DEPC water in sequence until the reaction volume is 14.5μL; , heat denaturation at 70°C for 5 minutes, then quickly cool on ice for 5 minutes, then add 4μL 5×First-stand buffer, 0.5μL RNasin (200U), 1μL M-MLV (200U) in sequence, mix well and centrifuge for a short time, and incubate at 42℃ After 1.5 hours, take it out and heat it at 70°C for 10 minutes to terminate the reaction. After the first strand of cDNA is synthesized, store it at -20°C for later use.

[0020] Use...

Embodiment 2

[0022] Embodiment 2: plant overexpression vector construction

[0023] Use the SanPrep column type plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert LrCHI2 coli plasmid pGEM-T- LrCHI2 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; Bam HI (TaKaRa) and Eco RI (TaKaRa) for plasmid pGEM-T- LrCHI2 Carry out double digestion with pCAMBIA2300s (100μL system), the reaction system and operation process are as follows: take 20μL pGEM-T- LrCHI2 and pCAMBIA2300s plasmid, add 10μL 10×K buffer, 4μL BamHI , 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 LrCHI2 The fragments and the large fragments of the pCAMBIA2300s vector were gel-recovered separately, and the SanPrep column DNA gel...

Embodiment 3

[0026] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0027] 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.

[0028] Take out the pCAMBIA2300s- containing pCAMBIA2300s- LrCHI2 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 nucleotide sequence of the lilium regale chitinase gene LrCHI2 is shown as SEQ ID NO: 1, and the lilium regale chitinase gene LrCHI2 encodes a protein with an amino acid sequence shown as SEQ ID NO: 2. Functional genomics related technical researches prove that the LrCHI2 gene has the function of improving the resistance of plants to pathogenic fungi. The antifungal gene LrCHI2 disclosed by the invention is constructed on a plant expression vector and transferred into tobacco for overexpression, and the transgenic tobacco for overexpression of LrCHI2 has relatively strong capability of resisting infection of alternaria oryzae, aspergillus oryzae and fusarium oxysporum.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and in particular relates to a chitinase gene of Minjiang lily with the ability to resist fungal infection LrCHI2 and its application. Background technique [0002] Plants possess a variety of mechanisms to protect themselves from various pathogens. When plants are attacked by pathogens, plants respond to pathogens and external stress by rapidly changing gene expression, and induce the resynthesis of some special proteins, most of which are pathogenesis-related proteins (PRs). Proteins are an important part of the plant defense system. PRs in plants mainly manifest as chitinase (CHI), dextranase (dextranase) and thaumatin-like proteins (TLPs). According to amino acid sequence, serological relationship and biological activity, PR proteins are divided into 17 classes (Kaur A, Pati PK, Pati AM, Nagpal AK. In-silico analysis of cis-acting regulatory elements of p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/82A01H5/00A01H6/82
CPCC12N9/2442C12N15/8282C12Y302/01014
Inventor 刘迪秋李珊邓婕王自娥苏琳琳梁婷婷
Owner KUNMING UNIV OF SCI & TECH
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