AU2020103127A4

AU2020103127A4 - A soybean leaf protoplast separation method for subcellular localization

Info

Publication number: AU2020103127A4
Application number: AU2020103127A
Authority: AU
Inventors: Lei Chen; Qingyuan HE; Shoucheng Huang; Ruining Li; Yingjie Shu; Shuang Wang; Yuli Zhou
Original assignee: Anhui University of Science and Technology; Langfang Normal University
Current assignee: Anhui University of Science and Technology; Langfang Normal University
Priority date: 2020-10-30
Filing date: 2020-10-30
Publication date: 2021-01-07
Anticipated expiration: 2028-10-30

Abstract

The invention relates to a soybean leaf protoplast separation method for subcellular localization. The most suitable conditions for isolation of soybean leaf protoplasts are selected by the following factors: adopting the young leaves of Xiangdou No. 3 as materials. In addition, the regulation of the plasmolysis time, the ratios and concentrations of cellulase Onozuka R 10, Hemicellulase, Macerozyme R-10 and Pectolyase Y-23, as well as the regulation of the concentration of mannitol in the the enzymatic hydrolysate, the pH of the enzymatic hydrolysate, and the enzymatic time. The method described in the present invention obtains a large number of free protoplasts with high activity and it is effective for subcellular localization, which is a simple and fast method to isolate the protoplasts of Xiangdou No. 3 leaves.

Description

A soybean leaf protoplast separation method for subcellular localization

TECHNICAL FIELD

[01] The invention relates to a soybean leaf protoplast separation method for subcellular localization, which belongs to the field of biotechnology.

BACKGROUND

[02] Plant genotype has great influence on protoplast isolation. Xiangdou No. 3 is the main soybean material of the current study, but the method of protoplast isolation of Xiangdou No. 3 has not been reported yet. Therefore, we used the young leaves of Xiangdou No. 3 as materials to establish the protoplast separation system of Xiangdou No. 3 leaves. This invention will provide important technical support to carry out the research of gene transient expression and protein subcellular localization.

SUMMARY

[03] An Purpose of the invention: providing a method for separating soybean leaf protoplasts for subcellular localization.

[04] Technical scheme: selecting the most suitable conditions for isolating the protoplasts of Xiangdou No. 3 leaves through the regulation of the plasmolysis time, the ratios and concentrations of cellulase Onozuka R-10, Hemicellulase, Macerozyme R-10 and Pectolyase Y-23, as well as the regulation of the concentration of mannitol in the the enzymatic hydrolysate, the pH of the enzymatic hydrolysate, and the enzymatic time. The purpose is realized as follows: a soybean leaf protoplast separation method for subcellular localization specifically comprises the following embodiments.

[05] (1) Sample selection: taking the fully expanded young leaves from the Xiangdou No. 3 seedlings with the seedling age of 10-30 days. The leaves are required to be strong and moderate in size. Cleaning the surface of the leaves to removing dust and impurities;

[06] (2) Cutting the cleaned leaves into thin strips of 0.5-1 mm with a blade, and placing them in a petri dish containing plasmolysis solution in the dark for plasmocytosis separation, while the plasmolysis solution is CPW + 13% mannitol;

[07] (3) Transferring the leaf strips after plasmolysis to a petri dish containing enzymatic hydrolysate, and placing them on the shaker in the dark for enzymatic hydrolysis. After a while, it is found that there are no complete leaf strips in the enzymatic hydrolysate. After the solution turns green, the solution is sliced and observed under the microscope, then a large number of round protoplasts will appear in the field of view; The enzymatic hydrolysis solution is a compound of CPW +0.1%MES+0.5% cellulase Onozuka R-10+0.8% Hemicellulase+0.8% Macerozyme R-10+0.4% Pectolyase Y-23.

[08] According to the above method, it is characterized in that the material is preferably the young leaves of Xiangdou No. 3.

[09] Preferably, the plasmolysis time in the step (2) is 2h.

[010] Preferably, the enzymatic hydrolysis solution is a compound of CPW +0.1%MES+0.5% cellulase Onozuka R-10+0.8% Hemicellulase+0.8% Macerozyme R-10+0.4% Pectolyase Y-23.

[011] Preferably, the pH of the enzymatic hydrolysis solution in the step (3) is 6.0.

[012] Preferably, the enzymolysis time in the step (3) is 6 h.

[013] Preferably, the mannitol concentration in the enzymolysis solution in step (3) is 10%.

[014] Beneficial effect: the method is simple and practical, and the separation material is easy to obtain; The enzymatic hydrolysis method is also simple, the protoplasts obtained is plentiful with high activity. Therefore, this technology will be an important technical support for gene transient expression and protein subcellular localization.

BRIEF DESCRIPTION OF THE FIGURES

[015] Figure 1. The protoplast of XiangdouNo. 3 leaves.

[016] Figure 2. The protoplast diagram of Xiangdou No. 3 leaves stained with trypan blue.

[017] Figure 3. The schematic diagram of Xiangdou No. 3 leaf protoplasts used for subcellular localization.

DESCRIPTION OF THE INVENTION

[018] In order to deepen the understanding of the present invention, the present invention will be described in further detail below in conjunction with examples. These examples are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[019] Materials: tender leaves with the age of 10-30 days or young leaves on mature seedlings of XiangdouNo. 3.

[020] In this example, the formula of the CPW solution is as follows:

Compounds Concentration (mgL-1

) KH 2Po 4 27.2 KNO 3 101.0 CaCI2-2H 20 1480.0 MgSO 4 -7H2 0 246.0 KI 0.16 CuSO 4 -5H2 0 0.025

[021] In this example, the formula of W5 solution is as follows:

Compounds Concentration (mgL- 1 )

NaCI 9 CaCI2-2H20 18.375 KCI 0.37 MES 0.4

[022] In this example, the formula of MMG solution is as follows:

Compounds Concentration (mgL- 1 )

MgCI 2 3.05 MES 0.8 Mannitol 72.88

[023] In this example , the formula of the WI solution is as follows:

Compounds Concentration (mgL-1

) Mannitol 91.1 MES 0.78 KCI 1.49

[024] Adjusting the pH of W5 solution, MMG solution and WI solution to 5.7 with KOH, which are sterilized at 121°C for 20 min, and stored at 4°C for later use.

[025] Protoplast isolation

[026] (1) Taking the young leaves of XiangdouNo. 3 soybean, cleaning them with tap water and then with pure water for later use.

[027] (2) Cutting the young leaves of Xiangdou No. 3 soybean mentioned above into thin strips of 0.5-1 mm, and placing them in the plasmolysis solution (CPW+13% mannitol) for plasmocytosis separation under room temperature and dark conditions. The leaves are completely immersed in the solution with tweezers. Performing plasmolysis for 2 h.

[028] (3) Transferring the leaf strips in step (2) to the enzymolysis solution, performing the enzymolysis for 6 hours in the dark while the temperature is 27°C, the rotation speed is 45 rpm. The enzymolysis solution is composed of the following materials: cell protoplast washing solution CPW+10% Mannitol+0.1% MES+0.5% Cellulase Onozuka R-10 + 0.8% Hemicellulase + 0.8% Macerozyme R-10 +0.4% Pectolyase Y -23, and the pH value is 6.0.

[029] (4) Detecting the leaf protoplasts separated in step (3): taking 10 pL of protoplast solution with 2 pL of 0.4% trypan blue dye solution, which are mixed evenly. After making a slice with a blood cell counting plate, it is observed under an optical microscope. In the visual field, the unstained protoplasts are living protoplasts, and the dyed blue protoplasts are inactive protoplasts. The yield of protoplasts (protoplast yield = total number of protoplasts in 25 squares /0.1xOOOx1O/g-FW) is 1.75x107/g-FW,

and the survival rate is 82.86%.

[030] Protoplast transformation.

[031] (5) Filtering the enzymatically hydrolyzed mixture with three layers of 75 pm nylon mesh, and the filter residue is discarded; Slowly adding isotonic W5 solution to the upper layer of the filtrate, which is placed on ice for more than 30 minutes until the solution separates.

[032] (6) Aspirating 10 mL of the middle layer of protoplasts, then washing it with 10 mL of W5 solution (centrifuging at 200 g for 2 min, and discarding the supernatant) for 2-3 times to obtain relatively pure protoplasts.

[033] (7) Protoplast transformation: taking PEG as the medium to transform plasmid pA7 with 35S-driven green fluorescent protein GFP into protoplasts: the transformation system is demonstrated as follows: 15 pL of plasmid, 100 pL of protoplasts are dissolved in MMG solution, 115 pL of 40% PEG-CaC2; Placing them at room temperature for 10-20 min, then adding 400-440 pL of W5 solution to terminate the reaction; Centrifuging at 200 g for 2 min, and removing the supernatant; then adding 1 mL of WI solution to suspend the protoplasts and placing it at 28°C in the dark for incubation for 12-24 hours.

[034] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, in keeping with the broad principles and the spirit of the invention described herein.

[035] The present invention and the described embodiments specifically include the best method known to the applicant of performing the invention. The present invention and the described preferred embodiments specifically include at least one feature that is industrially applicable

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. The method for quickly and efficiently preparing leaf protoplasts of Xiangdou No. 3, is characterized in that the method comprises the following steps:

(1) Sample selection: taking the fully expanded young leaves from the Xiangdou No. 3 seedlings with the seedling age of 10-30 days. The leaves are required to be strong and moderate in size. Cleaning the surface of the leaves to removing dust and impurities;

(2) Cutting the cleaned leaves into thin strips of 0.5-1 mm with a blade, and placing them in a petri dish containing plasmolysis solution in the dark for plasmocytosis separation, while the plasmolysis solution is CPW + 13% mannitol;

(3) Transferring the leaf strips after plasmolysis to a petri dish containing enzymatic hydrolysate, and placing them on the shaker in the dark for enzymatic hydrolysis. After a while, it is found that there are no complete leaf strips in the enzymatic hydrolysate. After the solution turns green, the solution is sliced and observed under the microscope, then a large number of round protoplasts will appear in the field of view; The enzymatic hydrolysis solution is a compound of CPW +0.1%MES+0.5% cellulase Onozuka R-10+0.8% Hemicellulase+0.8% Macerozyme R-10+0.4% Pectolyase Y-23;

The plasmolysis time in the step (2) is 2h;

The pH of the enzymatic hydrolysis solution in the step (3) is 6.0;

The enzymolysis time in the step (3) is 6 h.

2. The method according to claim 1, it is characterized in that: adopting the young leaves of Xiang Soybean No. 3 as materials.

3. The method according to claim 1, it is characterized in that: the enzymatic hydrolysis solution in (3) is a compound of CPW +0.1%MES+0.5% cellulase Onozuka R-10+0.8% Hemicellulase+0.8% Macerozyme R-10+0.4% Pectolyase Y-23.

The method according to claim 1, it is characterized in that: the mannitol concentration in the enzymolysis solution in step (3) is 10%.