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CN-121975716-A - Efficient preparation method of peanut leaf protoplast

CN121975716ACN 121975716 ACN121975716 ACN 121975716ACN-121975716-A

Abstract

The invention relates to a high-efficiency preparation method of peanut protoplast, which comprises the steps of culturing a yellowing seedling, carrying out enzymolysis on the protoplast, purifying and re-suspending, converting the protoplast and the like, wherein blade cutting is adopted in the separation process of the protoplast, enzymolysis liquid and treatment modes are optimized, vacuum auxiliary enzymolysis and gradient purification technology are combined, enzymolysis time is shortened to 2h, a large number of protoplasts are obtained through enzymolysis, the yield of the protoplast is improved, centrifugation, re-suspending and standing are carried out for many times, the purity and activity of the protoplast are ensured, the reagent dosage and reaction conditions are accurately controlled in the conversion process, the conversion efficiency is improved, and the prepared protoplast is round, full and complete.

Inventors

  • ZHANG XINYOU
  • ZHANG ZHONGXIN
  • DONG WENZHAO
  • HAN SUOYI
  • ZHANG LILI
  • ZHANG YU
  • CUI MENGJIE
  • ZHANG MENG
  • XU XIANGRU
  • DU PEI
  • SHI LEI

Assignees

  • 神农种业实验室

Dates

Publication Date
20260505
Application Date
20260210
Priority Date
20251202

Claims (6)

  1. 1. The efficient preparation method of the peanut leaf protoplast is characterized by comprising the following specific steps: (1) Cultivation of yellowing seedlings Preparing a plurality of peanut seeds, placing the peanut seeds in a culture container, culturing for 1 week in a dark environment at 25 ℃, and selecting young leaves which are just unfolded and are tender and yellow at the top ends of the peanuts in the 2-leaf period as experimental materials; (2) Protoplast enzymolysis Selecting a second leaf of tender yellow young leaves, cutting the second leaf as close as possible to the leaf sheath, stacking the leaves in order, slightly pressing, cutting the leaves into strips with the width of 0.5-1 mm, transferring the strips of the leaves to a 250 mL culture dish, adding 10-20 mL enzymolysis solution, and uniformly mixing; Placing the culture dish into an opaque container, then placing into a pot, starting a vacuum pump to enable the vacuum degree in the pot to reach 0.06 Mpa-0.07 Mpa, keeping 10min under the pressure, taking out the culture dish, placing into a dark environment for shake cultivation at a rotating speed of 40-50 rpm, continuously carrying out enzymolysis for 1-2 hours, observing through a microscope, and stopping enzymolysis if most cells are separated from the leaves; (3) Protoplast purification and resuspension Adding precooled W5 solution into the protoplast after enzymolysis for dilution, wherein the volume ratio of the W5 solution to the enzymolysis solution is 1:1, and uniformly mixing the two; Taking a 100-mesh sieve, fully wetting the sieve by using a precooled W5 solution, filtering the diluted enzymolysis liquid by the sieve, and removing larger impurities and tissue blocks which are not completely subjected to enzymolysis; Transferring the enzymolysis liquid into a 50 mL centrifuge tube after filtering, centrifuging 2 min by using a centrifugal force of 100 Xg, removing supernatant after centrifuging, adding a 15 mL precooled W5 solution into the centrifuge tube, lightly blowing by using a pipette, and lightly resuspending protoplasts; repeating the steps of centrifugation and re-suspension once to further remove impurities in the protoplast; Placing the resuspended protoplast on ice, standing under dark condition for 20-40 min, placing the centrifuge tube in a centrifuge pre-cooled to 4deg.C, centrifuging for 2min with 100×g centrifugal force to precipitate the protoplast at the bottom of the tube, discarding supernatant, adding pre-cooled MMG solution 500-1000 μl into the centrifuge tube, and gently resuspending cells; Sucking a small amount of cell suspension, placing on a glass slide, observing whether the background has impurities under a microscope, centrifuging again to suck supernatant, and re-suspending protoplast with MMG solution if the background has impurities; (4) Protoplast transformation Preparing a clean 2.0 mL centrifuge tube, adding 110 mu L of freshly prepared PEG/Ca 2+ buffer solution, then adding 10 mu g of plasmid, finally slowly adding 100 mu L of protoplast suspension with the concentration of 10 5 per mL, and lightly blowing by a liquid transfer device to fully mix the three; Placing the centrifuge tube in a light-proof environment at room temperature for standing for 15-20 min; Adding 440 mu L of W5 solution into the centrifuge tube, reversing the centrifuge tube upside down to uniformly mix the solution, thereby diluting the conversion mixed solution and stopping the conversion reaction; Placing the centrifuge tube into a normal temperature centrifuge, centrifuging 3 min by using a centrifugal force of 200 Xg, carefully sucking the supernatant by using a pipettor after the centrifugation is finished, repeating the centrifugation once, sucking the supernatant after the centrifugation, adding 1 mL of W5 solution, and gently mixing to re-suspend the protoplast; and (3) placing the resuspended protoplast in a light-shielding environment for standing induction culture for 12-18 h to obtain the peanut protoplast.
  2. 2. The preparation method according to claim 1, wherein the enzymolysis liquid comprises 0.30 g/10mL of cellulase, 0.15 g/10mL of eductase, 0.03 g/10mL of pectase, 0.6M of mannitol, 10 mM of MES, 10 mM of CaCl 2 , 0.25% of BSA and 5mM of beta-mercaptoethanol.
  3. 3. The process of claim 1 wherein the W5 solution comprises 0.9% NaCl, 125 mM CaCl 2 , 5 mM KCl, 2mM MES and anhydrous glucose 4 mM, wherein the pH is adjusted to 5.8 with KOH solution after adding sterile water to a volume of 50 mL, and the solution is filtered and stored at room temperature.
  4. 4. The preparation method of claim 1, wherein the MMG solution comprises 0.6M mannitol, 15 mM MgCl 2 and 4 mM MES, wherein the pH is adjusted to 5.7 by KOH solution after volume fixing, and the MMG solution is stored at room temperature after filtration.
  5. 5. The preparation method according to claim 1, wherein the PEG/Ca 2+ buffer is mannitol of 0.2M and CaCl 2 of 0.1M, adding sterile water for dissolving, adding PEG-4000 4g, and adding sterile water to constant volume to 10 mL.
  6. 6. The method according to claim 1, wherein the method for observing the background under the microscope in the step (3) is characterized in that, if the background is clean and no obvious impurities exist, the subsequent operation is continued, if the background contains impurities, 4mL of MMG solution is gently added into a centrifuge tube, the mixture is gently mixed by a pipette, the mixture is centrifuged for 2min for 30 s by a centrifugal force of 250 Xg, after the centrifugation is finished, the supernatant is gently sucked, and the protoplast is resuspended by the MMG solution until the concentration of the protoplast reaches 10 5 per mL.

Description

Efficient preparation method of peanut leaf protoplast Technical Field The invention relates to the technical field of plant cell engineering, in particular to an improved preparation method of peanut leaf protoplast. Background Protoplast refers to naked spherical cells obtained by removing plant cell walls by an enzymolysis method, and has irreplaceable roles in research of plant gene functions, genetic transformation, cell fusion, signal transduction and the like. Compared with the whole plant cells, protoplasts can absorb exogenous DNA, protein or small molecular compounds more efficiently due to the removal of the physical barrier of the cell wall, and therefore become an important experimental system for plant molecular biology and biotechnology research. However, peanut (Arachis hypogaea l.) as an important oil and commercial crop, its protoplast preparation still faces significant technical bottlenecks. The plant leaf cell wall has complex structure and mainly consists of polysaccharides with different structures and various varieties, and plays an important role in the plant structure, physiology, growth and development process. These polysaccharides are generally classified into three types, cellulose, hemicellulose (mainly xylan, xyloglucan, mannan) and pectin. The existence of the polysaccharide with a complex structure makes the enzymolysis solution difficult to permeate, and the dissociation of cells is incomplete, so that the protoplast extraction difficulty is increased. The peanut cuticle and the wax layer are thicker, so that the protoplast extraction work is more difficult. The existing method has lower extraction concentration, and the cell activity is reduced by too long enzymolysis time (> 3 h) or intense centrifugation (> 200 Xg), so that the subsequent transformation is difficult. In recent years, although attempts have been made to optimize the enzymatic hydrolysate formulation (e.g., adjust the ratio of cellulase to eductase), the efficiency improvement has been limited and the above problems have not been systematically solved. Therefore, the development of a protoplast preparation method which is efficient, stable and suitable for the characteristics of peanut leaves is very important to promote the researches of peanut gene editing, transient expression analysis, somatic hybridization and the like. Disclosure of Invention The invention provides a preparation method of an improved peanut leaf protoplast, which is used for improving the concentration and activity of the peanut leaf protoplast and meeting the requirement of peanut related research on high-quality protoplast. The technical scheme of the invention is as follows: the efficient preparation method of the peanut leaf protoplast comprises the following specific steps: (1) Cultivation of yellowing seedlings Preparing a plurality of peanut seeds, placing the peanut seeds in a culture container, culturing for 1 week in a dark environment at 25 ℃, and selecting young leaves which are just unfolded and are tender and yellow at the top ends of the peanuts in the 2-leaf period as experimental materials; (2) Protoplast enzymolysis Selecting a second leaf of tender yellow young leaves, cutting the second leaf as close as possible to the leaf sheath, stacking the leaves in order, slightly pressing, cutting the leaves into strips with the width of 0.5-1 mm, transferring the strips of the leaves to a 250 mL culture dish, adding 10-20 mL enzymolysis solution, and uniformly mixing; Placing the culture dish into an opaque container, then placing into a pot, starting a vacuum pump to enable the vacuum degree in the pot to reach 0.06 Mpa-0.07 Mpa, keeping 10min under the pressure, taking out the culture dish, placing into a dark environment for shake cultivation at a rotating speed of 40-50 rpm, continuously carrying out enzymolysis for 1-2 hours, observing through a microscope, and stopping enzymolysis if most cells are separated from the leaves; (3) Protoplast purification and resuspension Adding precooled W5 solution into the protoplast after enzymolysis for dilution, wherein the volume ratio of the W5 solution to the enzymolysis solution is 1:1, and uniformly mixing the two; Taking a 100-mesh sieve, fully wetting the sieve by using a precooled W5 solution, filtering the diluted enzymolysis liquid by the sieve, and removing larger impurities and tissue blocks which are not completely subjected to enzymolysis; Transferring the enzymolysis liquid into a 50 mL centrifuge tube after filtering, centrifuging 2 min by using a centrifugal force of 100 Xg, removing supernatant after centrifuging, adding a 15 mL precooled W5 solution into the centrifuge tube, lightly blowing by using a pipette, and lightly resuspending protoplasts; repeating the steps of centrifugation and re-suspension once to further remove impurities in the protoplast; Placing the resuspended protoplast on ice, standing under dark condition for 20-40 min, placing the centrifu