Liriodendron sino-americanum adventitious root promoting factor LhWRKY1 gene and application thereof

A mandarin jacket and gene technology, applied in the field of genetic engineering, can solve problems such as the unclear mechanism of high rooting rate, and achieve the effects of improving the survival rate of cuttings, increasing the number of lateral roots, and increasing the length of lateral roots

Active Publication Date: 2022-07-22
INST OF BIOLOGICAL RESOURCES JIANGXI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This high rooting rate clone has been popularized and applied in more than 20 counties and cities in many provinces in China, and has achieved remarkable economic, social and ecological benefits, but the mechanism of its high rooting rate is still unclear

Method used

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  • Liriodendron sino-americanum adventitious root promoting factor LhWRKY1 gene and application thereof
  • Liriodendron sino-americanum adventitious root promoting factor LhWRKY1 gene and application thereof
  • Liriodendron sino-americanum adventitious root promoting factor LhWRKY1 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Mining and cloning of LhWRKY1 gene

[0029] 1) Sequence mining and alignment

[0030] Four different growth periods were selected for high rooting rate of the cuttings of A. chinensis, and RNA was extracted and the transcriptome sequencing data of the adventitious root cDNA library was constructed. Through bioinformatics methods, a differentially expressed EST sequence (LH -SXY-1962), Blast this EST sequence in the NCBI database, and found that it has the highest similarity with the WRKY gene of other species, and it is speculated that it may be the WRKY transcription factor gene of A. chinensis.

[0031] 2) Extraction of total RNA from leaves of A. japonica

[0032] Take 1 g of fresh young leaves of P. japonica, quickly grind into powder in liquid nitrogen, extract total RNA of P. japonicus with TRizol kit according to the instructions, dissolve in sterilized ultrapure water, and add 1% agarose. Gel electrophoresis detection ( figure 1 ), stored in a -80°...

Embodiment 2

[0046] Example 2: Construction of a plant expression vector for the LhWRKY1 gene of A. chinensis

[0047] 1) Extraction of total RNA from leaves of A. chinensis

[0048] With embodiment 1 step 2);

[0049] 2) Synthesis of first-strand cDNA

[0050] Synthesis of the first-strand cDNA of A. japonica with TaKaRa PrimeScript TM II 1st Strand cDNASynthesis Kit was performed. The reaction system is as follows: total RNA 2μL (including RNA 2μg), Oligo dT Primer 1μL, dNTP Mixture 1μL, RNase free dH 2 O 6μL, the total system is 10μL. The reaction conditions were incubated at 65°C for 5 minutes and then quickly placed on ice. Then according to the kit instructions, add the following reaction solution: 5×Buffer 4μL, 200U / μL RTase 1μL, 40U / μL RNase Inhibitor 0.5μL and RNase free dH 2 O 4.5 μL, mix slowly. The reaction conditions were: 42°C for 45 minutes, 95°C for 5 minutes, and after cooling on ice, stored in a -80°C ultra-low temperature refrigerator.

[0051] 3) Amplification ...

Embodiment 3

[0062] Example 3: Agrobacterium-mediated genetic transformation of Arabidopsis and functional analysis

[0063] 1) Sow wild-type Arabidopsis seeds (Colombia Col-0) in previously watered nutrient soil (organic matter: vermiculite: perlite = 1:1:1), cover with plastic wrap for 3 days, and wait until the seeds germinate. , Peel off the film, the culture environment of the artificial climate room is 25 ℃, the light time is 14h, and cultivate to the flowering stage.

[0064] 2) Add 1 μL of the positive plasmid of pBWA(V)BS:LhWRKY1 expression vector containing the target fragment detected by PCR to 50 μL of EHA105 Agrobacterium competent cells stored at -80°C, mix well and electroporate, and add 1 mL LB after electroporation. The liquid medium was shaken and cultured at 30° C. and 180 rpm for 30 minutes for activation, and the activated Agrobacterium was cultured on LB solid medium. Pick a single colony from the plate medium, then inoculate it into the liquid medium containing Bast...

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Abstract

The invention discloses an adventitious root promoting factor LhWRKY1 gene of liriodendron sino-americanum. The full-length nucleotide sequence of the LhWRKY1 gene is shown as SEQ ID NO.1, and the CDS sequence of the LhWRKY1 gene is shown as SEQ ID NO.2; a WRKY gene is separated and cloned through an RACE method, an expression vector is constructed and is transformed into arabidopsis thaliana to verify the function of the WRKY gene, and we find that the growth advantage of a main root of the transgenic arabidopsis thaliana is weakened, the number of lateral roots is increased, and the root length is increased. Therefore, the liriodendron sino-americanum LhWRKY1 gene has a good application prospect in promoting the growth and development of the root system and improving the cutting rooting rate and the survival rate. The invention provides a basis for the application of the liriodendron sino-americanum LhWRKY1 gene in molecular breeding.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and more particularly to an adventitious root-promoting factor LhWRKY1 gene of A. chinensis and its application. Background technique [0002] Liriodendron L. is a fast-growing deciduous tree with straight trunks, excellent material, beautiful flowers and leaves, few pests and diseases, strong anti-pollution ability, and high ornamental value. The species of this genus showed a typical East Asia-North America discontinuous distribution pattern, with only Liriodendron in East Asia and Liriodendron in North America. Through artificial hybridization, Chinese scientists successfully carried out the interspecific hybridization of these two species for the first time, and obtained Liriodendron sino-ame ricanum, the interspecific hybrid offspring of Liriodendron genus, formerly known as hybrid horseshoe. Or hybrid Liriodendron. Amygdalina has obvious heterosis, and has stronger growth perf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/84C12N15/66C07K14/415A01H5/06A01H6/20
CPCC07K14/415C12N15/8261C12N15/66
Inventor 杨爱红刘立盘钟永达周华刘淑娟胡萍刘腾云余发新
Owner INST OF BIOLOGICAL RESOURCES JIANGXI ACAD OF SCI
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