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CN-121991880-A - Application of complex enzyme in preparation of China rose protoplast

CN121991880ACN 121991880 ACN121991880 ACN 121991880ACN-121991880-A

Abstract

The invention provides an application of complex enzyme in preparing China rose protoplast, and belongs to the technical field of plant protoplast preparation. The compound enzyme is used by compounding the cellulase R-10 and the educing enzyme R-10, wherein the cellulase R-10 mainly acts on cellulose components in the cell wall of the China rose leaf to start disintegration of the cell wall structure, and the educing enzyme R-10 can act on pectin and other components in the cell wall to act cooperatively with the cellulase R-10 to further destroy the cell wall structure and promote the release of protoplast. The preparation of the China rose protoplast by adopting the complex enzyme not only maintains the high activity of the protoplast, but also obtains a large number of protoplasts. The invention has the outstanding beneficial effects of keeping the high activity of the China rose protoplast and increasing the quantity of the China rose protoplast, and opens up a wider way for relevant research and application of the China rose protoplast.

Inventors

  • ZHAO YIYANG
  • ZHONG YIN
  • ZHANG DEQIANG

Assignees

  • 北京农学院

Dates

Publication Date
20260508
Application Date
20260407

Claims (10)

  1. 1. The application of the compound enzyme in preparing China rose protoplast comprises the steps of calculating the volume of an enzymolysis working system, wherein the working concentration of the compound enzyme is that cellulose R-10-4 g/100mL and eductive enzyme R-10-2 g/100mL.
  2. 2. The use according to claim 1, wherein said rose comprises 'lunar powder' rose and/or 'blue manic' rose.
  3. 3. The use according to claim 1, wherein the cellulase R-10 comprises Yakult cellulase R-10 and the eductase R-10 comprises Yakult eductase R-10.
  4. 4. An enzymatic hydrolysate for preparing China rose protoplasts, which is characterized by comprising the following components in terms of total volume of the enzymatic hydrolysate: The dosage of 2-4 g/100mL of cellulase R-10, 1-2 g/100mL of educing enzyme R-10, 0.3-0.8 mol/L, KCl-20 mmol/L, MES is that the pH value of the enzymolysis liquid is adjusted to 5.5-6.0, caCl 2 -20 mmol/L, 0.01-0.02 g/100mL of PVP and 0.1-0.5 g/100mL of BSA.
  5. 5. The enzymatic hydrolysate of claim 4, wherein the enzymatic hydrolysate is used for preparing China rose protoplast, increasing yield of China rose protoplast and increasing activity of China rose protoplast; the Chinese rose comprises 'month powder' Chinese rose and/or 'blue fan' Chinese rose.
  6. 6. A method for preparing China rose protoplasts, comprising: The enzymatic hydrolysate of claim 4 is used for enzymatic hydrolysis of Chinese rose leaves to obtain Chinese rose protoplast.
  7. 7. The method according to claim 6, wherein the rose leaves comprise leaves of rose cutting seedlings and/or leaves of rose tissue culture seedlings.
  8. 8. The method of claim 6, wherein the enzymolysis temperature is 25-28 ℃, and the enzymolysis time is 14-16 h.
  9. 9. The method according to any one of claims 6 to 8, wherein the rose leaves are subjected to pretreatment before enzymolysis, and the pretreatment method comprises the following steps: Cutting China rose leaves into strips, and then soaking the strips in mannitol solution to obtain a tissue material treated by the mannitol solution; And mixing the tissue material treated by the mannitol solution with the enzymolysis solution for vacuum treatment.
  10. 10. A reagent for preparing China rose protoplast is characterized by comprising the enzymolysis liquid, a reagent B and a reagent C according to claim 4; The reagent B comprises a W5 solution; The reagent C comprises FDA working fluid.

Description

Application of complex enzyme in preparation of China rose protoplast Technical Field The invention belongs to the technical field of plant protoplast preparation, and particularly relates to application of a complex enzyme in preparation of China rose protoplast. Background In the field of plant biotechnology, the preparation of protoplasts is a crucial basic operation, which plays a key role in plant cell fusion, genetic transformation, gene function research and other aspects. China rose is a well-known ornamental flower in the world and occupies a significant position in the flower industry. However, there are a number of urgent problems to be solved in China rose protoplast extraction. Most of the existing China rose protoplast extraction methods use other plant protoplast extraction technologies for reference, and are adaptively adjusted on the basis of the extraction technologies. However, due to the unique physiological characteristics and cell structure of China rose, the methods often have difficulty in achieving the ideal extraction effect. On the one hand, the extraction efficiency is not high, which is a more prominent problem. In the extraction process of many traditional methods, the enzymolysis conditions are not properly controlled, so that cell wall breaking is incomplete, a large number of protoplasts cannot be released from cells, so that the number of finally obtained protoplasts is far lower than expected, and the development of subsequent related experiments is greatly limited. For example, some conventional methods employ a single cellulase enzyme for enzymolysis, which, although being capable of partially destroying the cell wall, for complex structural components in the cell wall of China rose, it is difficult for the single enzyme to be completely decomposed, thereby affecting the release efficiency of protoplasts. On the other hand, the low activity of protoplasts is also a big problem for researchers. In the extraction process, the activities of protoplasts are rapidly reduced after separation due to inaccurate control of temperature, osmotic pressure and other conditions. The reduced activity of protoplasts can lead to reduced survival in subsequent cell fusion, culture, etc., severely affecting the reliability and reproducibility of experimental results. For example, enzymatic hydrolysis at too high a temperature may increase the cell wall decomposition rate, but may damage the cell membrane and other structures of the protoplast, thereby reducing the activity. In addition, the existing methods have disadvantages in terms of cost control. The partial extraction method requires the use of expensive special enzymes or reagents or complicated equipment and operation procedures in the extraction process, which clearly increases the experimental cost and limits the wide application of the related technology in actual production and scientific research. In view of the above, the existing China rose protoplast extraction technology has obvious disadvantages in terms of extraction efficiency, protoplast activity, cost control and the like, and there is an urgent need for an extraction method that is more efficient and economical and can ensure high activity of protoplasts. Disclosure of Invention Aiming at the defects in the prior art, the invention aims to provide an application of complex enzyme in preparing China rose protoplast, and the complex enzyme can obviously improve the yield of the China rose protoplast and the activity of the protoplast on the basis of reducing the cost. The invention aims at realizing the following technical scheme: the invention provides an application of a compound enzyme in preparation of China rose protoplast, wherein the working concentration of the compound enzyme is that cellulase R-10-4 g/100mL and eduction enzyme R-10-2 g/100mL based on the volume of an enzymolysis working system. Preferably, the China rose comprises 'moon powder' China rose and/or 'blue fan' China rose. Preferably, the cellulase R-10 comprises Yakult cellulase R-10, and the eductase R-10 comprises Yakult eductase R-10. Preferably, the working concentration of the compound enzyme is that the cellulase R-10 g/100mL and the eductase R-10.5 g/100mL are calculated by the volume of the enzymolysis working system. The invention provides an enzymolysis liquid for preparing China rose protoplast, which comprises the following components in terms of the total volume of the enzymolysis liquid: The dosage of 2-4 g/100mL of cellulase R-10, 1-2 g/100mL of educing enzyme R-10, 0.3-0.8 mol/L, KCl-20 mmol/L, MES is that the pH value of the enzymolysis liquid is adjusted to 5.5-6.0, caCl 2 -20 mmol/L, 0.01-0.02 g/100mL of PVP and 0.1-0.5 g/100mL of BSA. The invention provides application of the enzymatic hydrolysate in preparation of China rose protoplast, improvement of yield of China rose protoplast and improvement of activity of China rose protoplast, wherein the China rose comprises 'month p