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CN-121975849-A - Agrobacterium rhizogenes mediated oleaster non-tissue culture genetic transformation method

CN121975849ACN 121975849 ACN121975849 ACN 121975849ACN-121975849-A

Abstract

The invention discloses a method for agrobacterium rhizogenes-mediated non-tissue culture genetic transformation of elaeagnus angustifolia, and belongs to the field of plant genetic engineering. The method comprises the steps of constructing a recombinant vector containing EGFP and KanR genes, transforming agrobacterium rhizogenes K599, preparing an invasion solution, taking 1-2 month old elaeagnus angustifolia seedlings, beveling first pair of true leaf stems to obtain explants, immersing the base parts of the explants in the invasion solution 30min, dipping a fungus layer at a wound, inserting the explants into a mixed matrix of nutrient soil and vermiculite, dropwise adding the invasion solution, performing dark culture for 2-3 d, transferring to normal illumination condition for culture, regenerating hairy roots at the wound after 20-30 d, and obtaining positive transformed roots through fluorescence microscopy and PCR detection. The method does not need tissue culture, is simple and convenient to operate, short in period and good in repeatability, and provides effective technical support for gene function research and genetic breeding of the elaeagnus angustifolia.

Inventors

  • FU XINXING
  • ZHU XIAOJUAN
  • FENG HANQING
  • SHI YIFAN
  • ZHANG JIAJUN
  • DU TING

Assignees

  • 西北师范大学

Dates

Publication Date
20260505
Application Date
20260308

Claims (7)

  1. 1. A method for agrobacterium rhizogenes mediated non-tissue culture genetic transformation of elaeagnus angustifolia, comprising the steps of: S1, constructing a vector, namely connecting a green fluorescent protein gene EGFP, a kanamycin resistance gene KanR and an expression vector pART by adopting an enzyme digestion and ligase technology to obtain a recombinant vector containing a target gene; S2, agrobacterium transformation and screening, namely transferring the obtained recombinant vector into agrobacterium rhizogenes competent cells, adding the transformed agrobacterium into TY liquid medium which does not contain kanamycin, shaking 2h at 28 ℃ for proliferation culture, and then coating bacterial liquid onto TY solid medium which contains 50 mg/L kanamycin for screening culture, and culturing 40-60 h in an inverted way at 28 ℃ to grow positive agrobacterium single colonies; the formula of the TY liquid culture medium comprises 5 g/L of tryptone, 3 g/L of yeast extract and 10 mM CaCl 2 , and 15 g/L of agar is added into the solid TY culture medium; S3, preparing agrobacterium tumefaciens invasion solution carrying target genes, namely, selecting the monoclonal positive agrobacterium tumefaciens, adding the monoclonal positive agrobacterium tumefaciens into a 3 mL TY liquid culture medium (containing 50 mg/L kanamycin), performing shake culture at 28 ℃ and 220 rpm until the agrobacterium tumefaciens becomes turbid, transferring the turbid agrobacterium tumefaciens into a 35 mL TY liquid culture medium (containing 50 mg/L kanamycin) for culturing for 6-8 hours, centrifuging at 5000 rpm for 10 min, pouring out supernatant fluid, collecting thalli, and adding a heavy suspension to obtain the agrobacterium tumefaciens invasion solution; the formula of the heavy suspension is TY liquid culture medium +10 mM 2- (N-morpholino) ethanesulfonic acid (MES) + mM MgCl 2 +100 mu M acetosyringone, and the pH=6.0; s4, the infection of the explant, namely, taking 1-2 month old elaeagnus angustifolia seedlings, chamfering the stem parts of the first pair of true leaves, leaving 2-3 true leaves as the explant, immersing the basal part of the explant in agrobacterium tumefaciens infection liquid for 30 min, and dipping a layer of TY solid culture medium on the wound of the explant for culturing; S5, performing dark culture on the explant, namely inserting the explant into a moist mixed matrix (nutrient soil: vermiculite=1:2), immediately dripping 3-5 mL of agrobacteria infection liquid resuspended in the soil of a cut part along a stem, covering Miao Pen with a plastic cover for moisturizing, and placing the culture medium into an incubator for dark culture of 2-3 d, wherein the temperature is set to 25 ℃; s6, rooting the explant, namely, after dark culture is finished, culturing the explant in normal conditions, wherein the illumination period is 16 h illumination/8 h darkness, the illumination intensity is 2000 lx, and the temperature is 25 ℃; s7, detecting positive hairy roots, namely culturing 20-30 d to grow hairy roots at the wound, taking the regenerated hairy roots to carry out fluorescence detection by using a body type fluorescence microscope, extracting DNA with the fluorescent hairy roots detected, and carrying out PCR detection.
  2. 2. The method according to claim 1, characterized in that the concentration of the agrobacteria infesting solution is OD 600 0.8-1.0.
  3. 3. The method according to claim 1, wherein 10 mg/L of rooting powder No.1 solution is irrigated during rooting of the explant in step S6.
  4. 4. The method according to claim 1, wherein the primers used in the PCR detection in the step S7 are a forward primer F5'-AGAAGACGTTCCAACCACG-3' and a reverse primer R5'-CGTCGCCGTCCAGCTCGACCAG-3'.
  5. 5. The method according to claim 1, characterized in that the agrobacterium rhizogenes strain is K599.
  6. 6. Use of the method according to any one of claims 1-6 in gene function research of elaeagnus angustifolia.
  7. 7. Use of the method according to any one of claims 1-6 in genetic breeding of elaeagnus angustifolia.

Description

Agrobacterium rhizogenes mediated oleaster non-tissue culture genetic transformation method Technical Field The invention belongs to the field of plant genetic engineering, and particularly relates to an agrobacterium rhizogenes mediated non-tissue culture genetic transformation method of elaeagnus angustifolia. Background The fructus Elaeagni Angustifoliae (Elaeagnus moorcroftii) is deciduous tree of Elaeagnaceae (ELAEAGNACEAE) Elaeagnus (Elaeagnus), and is mainly distributed in northwest arid desert region of China. The oleaster plant has strong vitality, easy propagation, wind sand resistance, drought resistance, high temperature resistance, barren resistance and salt and alkali resistance, and the root and actinomycetes are symbiotic and nodulation, so that the oleaster plant can play roles in biological nitrogen fixation and soil improvement, and is a pioneer tree species for wind prevention, sand fixation, greening and forestation in northwest areas. The fruit is rich in nutritional ingredients such as sugar, vitamin C, amino acid, trace elements and the like, and flavonoid, polyphenol, tannin, alkaloid and other compounds, so that the fruit is a medicinal and edible homologous fruit which people like to eat in northwest areas. The oleaster plant is an important arbor tree species integrating ecological and economic benefits in northwest areas, has important positions in a desert ecosystem, is a special rural economic plant in the areas, and has potential application value. Genetic transformation technology is the basis of biological breeding, and can be used for rapid and accurate improvement of crop varieties. The existing plant genetic transformation method comprises an agrobacterium-mediated method, a flower organ infection method, a viral vector transfer method, a particle bombardment method, a pollen tube channel method, a nano transfer method and the like. Among them, agrobacterium-mediated genetic transformation is most widely used due to its high efficiency, low copy number, high reliability. The agrobacterium for genetic transformation of plants mainly comprises two types of agrobacterium tumefaciens (Agrobacterium tumefaciens) and agrobacterium rhizogenes (Agrobacterium rhizogenes), wherein the two types of agrobacterium tumefaciens and agrobacterium rhizogenes respectively comprise Ti plasmids and Ri plasmids, a section of T-DNA is arranged on the Ti plasmids, and the agrobacterium is inserted into a plant genome after entering cells through infected plant wounds, so that transfer and integration of exogenous genes into host plant cells are realized. The two types of Agrobacterium-induced organs are different, with Agrobacterium rhizogenes inducing mainly regeneration positive roots and Agrobacterium tumefaciens inducing regeneration positive shoots. The receptor materials of traditional agrobacterium-mediated plant genetic transformation are mostly in vitro plant tissues, including sterile seeds, young embryos, leaves, epicotyls, hypocotyls, calli and the like. However, the method is limited by various factors, firstly, an efficient and stable in-vitro regeneration system needs to be established, secondly, the method has certain preference on plant genotypes, and the transformed cell culture process is expensive, complex, long in duration and low in transformation efficiency. At present, only the tissue culture of elaeagnus angustifolia is reported in the literature, but a tissue culture system of elaeagnus angustifolia is not reported yet, and an agrobacterium-mediated genetic transformation system based on the tissue culture is not established yet. The plant grown under the non-tissue culture condition has strong regeneration capacity of axillary bud meristem and stem meristem, and can be used as a receptor material for genetic transformation. In recent years, non-tissue culture genetic transformation methods have been established and optimized on a wide variety of plants. The agrobacterium-mediated non-tissue culture genetic transformation commonly used at present mainly comprises a flower organ infection method, a cut-dip-bud delivery method, a living body injection delivery method depending on regeneration activity, a seed infection method and the like. The root and stem of fructus Elaeagni Angustifoliae have strong regeneration capability. Therefore, under the condition that the tissue culture regeneration system of the elaeagnus angustifolia is still immature, the establishment of the agrobacterium-mediated non-tissue culture genetic transformation system is an important means for breaking through the genetic breeding bottleneck of the elaeagnus angustifolia. Disclosure of Invention Aiming at the defects of the prior art, the invention provides an agrobacterium rhizogenes mediated non-tissue culture genetic transformation method of elaeagnus angustifolia. The invention aims to provide a method for agrobacterium rhizogenes-mediated non-tissue culture genetic transformation of elaeag