CN112251395A - Separation method of loquat protoplast - Google Patents
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Abstract
The invention provides a method for improving drought or/and cold stress resistance of loquat seedlings, which adopts melatonin solution with specific concentration to irrigate the loquat seedlings before the loquat seedlings are subjected to drought or/and cold stress, and the result shows that: the melatonin treatment obviously improves the content and fluorescence parameters of endogenous melatonin in the loquat seedling leaves; the contents of SOD, POD, CAT and APX in the loquat seedling leaves are obviously enhanced, the content of MDA is reduced, and the integrity of the tissue structure of the leaves is maintained. Researches find that the melatonin solution with specific concentration can improve the drought stress resistance of the loquat seedlings and the cold stress resistance of the loquat seedlings, the input cost in the planting process is low, the survival rate of the seedlings is high, the popularization speed and efficiency of new varieties are improved, the survival rate of offspring in the cross breeding process is improved, the process and efficiency of breeding the new varieties are accelerated, and the method has important strategic significance on the sustainable development of the loquat industry in China.
Description
Technical Field
The invention belongs to the technical field of biology, relates to research on preparation of loquat protoplasts through enzymolysis, and particularly relates to a separation method of loquat protoplasts.
Background
Loquat (Eriobotrya japonica Lindl) is a rose family, loquat genus plant, and is a perennial woody evergreen small tree with the height of 10 meters; strong, yellow-brown, dense rust-colored or grayish-brown fuzz. Loquat originates from China, and has a cultivation history for thousands of years. The fruit of the fruit tree is ripe in the late spring and early summer, and is an important economic fruit tree in China. At present, the loquat is mainly bred by the traditional methods such as natural seed selection, sexual hybridization screening and later generation, and the like, and the method has long time consumption and low success rate. With the increasing maturity of biotechnology, biotechnology breeding combining genomics and molecular biology gradually breaks some limitations of conventional breeding and becomes a new breeding mode. However, loquat still has no efficient and stable genetic system established at present, which directly hinders the development of biotechnology breeding, functional genomics and the like.
Protoplasts are a collective term for various structures within the cell wall, and also a morphological structural unit that constitutes a cell, where various metabolic activities in the cell are carried out. Protoplasts include cell membranes, cytoplasm, nuclei, organelles, and the like. The chemical components of the protoplast are very complex, the components of the protoplast are constantly changed along with the metabolic activity of cells, and the relative component proportion is 85-90 percent of water, 7-10 percent of protein, 1-2 percent of lipid, 1-1.5 percent of other organic matters (including nucleic acid) and 1-1.5 percent of inorganic matters. Among them, a complex mainly composed of a protein and a nucleic acid is the most important component related to life activities. Compared with cells containing cell walls, protoplasts can not only maintain totipotency of cells, but also accept exogenous genetic materials more easily, so that exogenous DNA can be expressed rapidly in plant protoplasts. The successful separation of the loquat protoplast is the first step of research and application of loquat in the aspects of functional gene research by further utilizing genetic transformation, germ plasm resource creation by cell fusion, cell clone breeding and the like.
The existing protoplast separation method mainly comprises a mechanical separation method and an enzymatic hydrolysis method. Mechanical separation is the release of protoplasts from a wound by treating the cells in a hypertonic sugar solution to effect a degree of plasmolysis of the cells and grinding or cutting the corresponding plant tissue. The operation is time-consuming and labor-consuming, the yield of protoplasts is low, and the separation material is limited to highly vacuolated cells, so that the use is greatly limited. The enzymolysis method is to release protoplast by decomposing cellulose of cell wall and pectin in intercellular space with biological enzyme. In the research of loquat protoplasts disclosed in the prior art, callus or suspension culture is often used as starting material for culturing protoplasts (see the article "callus formation by loquat protoplast culture" of Linshun et al, proceedings of Fujian agricultural college, 20(2):179- "184, 1991; article" isolation and culture of loquat protoplasts of folium Toosendan ", proceedings of Fujian agricultural university, 23(l):26-29,1994). The methods are used for specially processing materials, firstly, callus or suspension culture is obtained, and then, the loquat protoplast is obtained through enzymolysis separation.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a separation method of loquat protoplasts, which takes loquat leaves as materials, is convenient for obtaining samples and does not need to carry out special treatment on the materials; meanwhile, only two enzyme preparations of cellulase and eductase are needed in the enzymolysis process, and the enzyme solution is simple to prepare; a considerable amount of protoplasts can be obtained in one step without complicated test steps.
Except for special description, the percentages are mass percentages.
In order to achieve the purpose, the technical scheme of the invention is as follows:
since the prior art mostly adopts callus or suspension culture as starting material to culture protoplast, it is unknown to those skilled in the art whether the protoplast can be successfully prepared by using loquat leaf as material. Meanwhile, the loquat protoplast is prepared by separating the loquat leaves serving as the material through an enzymatic hydrolysis method, and whether enzymes used by the conventional enzymatic hydrolysis method, such as cellulase, macerozyme, pectinase, collapsing enzyme, helicase, hemicellulase and the like, can successfully prepare the protoplast is not known. Therefore, the inventors conducted a series of investigations involving the kind and concentration of the enzyme. It was found that when the selection of the type and concentration of the enzyme was inappropriate, a series of cases such as obtaining cell debris, obtaining irregular shapes of cells, observing that the cell wall was not removed, obtaining no cell, obtaining no protoplast of loquat, and the like were liable to occur. Surprisingly, the protoplast can be successfully separated from the loquat (No. 6 early-season, Imperial concubine and Hua Bai No. 1) leaves by using the mixed enzyme liquid consisting of the cellulase and the eductase.
According to one embodiment of the invention, the loquat protoplast separation method, which uses loquat leaves as materials and prepares the loquat protoplast by an enzymatic separation method, is characterized in that: the enzyme is a mixed enzyme consisting of cellulase and eductase.
In order to increase the yield of loquat protoplasts, according to one embodiment of the present invention, the cellulase is cellulase RS and the macerase is macerase R-10.
Further, the concentration of the cellulase RS is 1-5.4%, and the concentration of the macerozyme R-10 is 1.8-4.7%, in mass%. Furthermore, the concentration of the cellulase RS is 5.0 percent, and the concentration of the macerozyme R-10 is 3.6 to 4.0 percent in percentage by mass.
According to an embodiment of the present invention, the loquat is selected from loquat No. 6, Imperial concubine, and Huabai No. 1.
According to one embodiment of the present invention, the above method comprises pretreating folium Eriobotryae before enzymolysis, wherein the pretreatment comprises washing with running water to remove villi on the inner and outer surface of folium Eriobotryae, cutting the folium Eriobotryae into strips, and soaking in 0.6-1.0M mannitol hypertonic solution for 2-4 h.
According to an embodiment of the invention, in the above method, the loquat leaf is subjected to a result observation after enzymolysis, and the result observation is that the enzymolysis solution is added with the same volume of the protoplast cleaning solution to stop enzymolysis, the protoplast cleaning solution is screened through a 300-mesh screen, the liquid is centrifuged, the supernatant is discarded, the protoplast cleaning solution is added into the precipitate, and the protoplast is observed through heavy suspension.
According to an embodiment of the present invention, the above loquat protoplast separation method comprises the following steps:
(1) pretreatment: cleaning villi on the inner and outer epidermis surfaces of young loquat leaves with running water, cutting the leaves into fine strips with the size of about 0.5mm by a knife, and soaking in 0.6-1.0M mannitol hypertonic solution for about 2-4 h;
(2) enzymolysis: removing the mannitol solution, adding the enzymolysis solution, placing on a shaker at 50-100rpm/min, and performing enzymolysis and oscillation in the dark for 10-12 h; the enzyme in the enzymolysis liquid is mixed enzyme consisting of cellulase RS and eductase R-10, the concentration of the cellulase RS is 5.0 percent, the concentration of the eductase R-10 is 3.6 to 4.0 percent, and the weight percentage is calculated;
(3) and (4) observing results: adding a protoplast cleaning solution with the same volume as the enzymolysis solution, stopping enzymolysis, sieving the protoplast-enzyme mixed solution with a 300-mesh sieve, centrifuging, removing the supernatant, adding the protoplast cleaning solution into the precipitate, and resuspending and observing the protoplast.
According to an embodiment of the present invention, the above method for separating loquat protoplasts comprises the following steps:
(1) pretreatment: placing young loquat leaves in a beaker, washing the inner and outer surface fluff of the leaves with running water, cutting the leaves into thin strips with the size of about 0.5mm by using a sharp blade, and soaking in 0.6-1.0M mannitol hypertonic solution for 3 hours;
(2) enzymolysis: removing the mannitol solution, adding the enzymolysis solution, placing on a shaker at 50-100rpm/min, and performing enzymolysis and oscillation in the dark for 10-12 h; the preparation method of the enzymolysis liquid comprises the following steps: adding 10mM MES, 0.6M mannitol, 5.0% cellulase RS, and 3.6-4.0% segregation enzyme R-10 into 10ml enzymolysis solution, stirring, heating at 50-60 deg.C for 10min, naturally cooling, sequentially adding 0.01-1% BSA and 1mM CaCl2The first-level water is used for fixing the volume to 10 ml;
(3) and (4) observation: adding a protoplast cleaning solution with the same volume as the enzymolysis solution to stop enzymolysis, sieving with a 300-mesh sieve, transferring into a centrifuge tube, centrifuging at 700r/min for 12 minutes, discarding the supernatant, adding the protoplast cleaning solution into the precipitate, and resuspending and observing the protoplast.
Has the advantages that:
without intending to be bound by any theory, it is believed that although some loquat protoplasts can be cultured from callus or suspension cultures as starting materials, these methods are not readily available and are complicated in terms of process (see Linshun et al, "callus formation from loquat protoplast culture", proceedings of the Fujian college of agriculture, 20(2): 179. 184, 1991; Linqing et al, "isolation and culture of loquat protoplasts, proceedings of the Fujian university of agriculture, 23(l):26-29,1994).
The inventor conducts an experiment for preparing the protoplast by taking loquat (No. 6, Imperial concubine and Huabai No. 1) leaves as starting materials through enzymolysis and separation, and unexpectedly discovers that when the enzyme in the enzymolysis liquid is a mixture of cellulase RS and macerozyme R-10 and the concentration of the cellulase RS of 5.0 percent and the macerozyme R-10 of 3.6 to 4.0 percent (calculated by mass percentage), the protoplast can be better separated from the loquat leaves. According to the invention, the loquat leaf material is taken outdoors, the sample is convenient to take, and special treatment is not required to be carried out on the material; only two enzyme preparations of cellulase and eductase are needed in the enzymolysis process, and the preparation of enzyme solution is simple; a considerable amount of protoplasts can be obtained in one step without complicated test steps. The invention saves cost and labor force, and does not need special instruments: the enzymolysis liquid has simple components and lower cost; the preparation process of the protoplast only needs conventional instruments such as a shaking table, a centrifugal machine and the like, and does not need special instruments.
Drawings
FIG. 1 shows the leaves of Eriobotrya japonica taken as materials, and the variety is "Zao-Zhong No. 6" Eriobotrya japonica;
FIG. 2 is a loquat leaf obtained from raw materials, and the variety is "Imperial concubine" loquat;
FIG. 3 shows the leaves of Eriobotrya japonica taken as materials, the variety is "Huabai No. 1" Eriobotrya japonica;
FIG. 4 is a microscopic observation result of the protoplast of loquat No. 6, wherein a large number of protoplasts can be observed, with the magnification: 4 x 10;
FIG. 5 is a result of microscopic observation of protoplasts of the loquat highest ranked imperial concubine, wherein a large number of protoplasts can be observed, with magnification: 4 x 10;
FIG. 6 is a microscopic observation result of protoplast of Eriobotrya japonica Hua Bai No. 1, in which a large number of protoplasts can be observed with a magnification of: 4 x 10;
fig. 7 is a graph of the result of FDA staining of the protoplast of loquat early No. 6, where high-activity protoplasts can be observed at magnification: 2 × 10;
FIG. 8 is the result of FDA staining of protoplast of loquat Imperial concubine, wherein high-activity protoplast can be observed, and the magnification is as follows: 2 × 10;
fig. 9 is a graph of the result of FDA staining of protoplast of loquat huabai No. 1, where high-viability protoplasts can be observed, magnification: 2X 10.
Detailed Description
The present invention is described in detail below with reference to specific examples, which are given for the purpose of further illustrating the invention and are not to be construed as limiting the scope of the invention, and the invention may be modified and adapted by those skilled in the art in light of the above disclosure. MES (2-N-morpholinohexanesulfonic acid) of the present invention was purchased from sigma aldrich trade ltd, brand: SIGMA, goods number: M3671-50G; mannitol was purchased from Tianjin Schiens Aupportkok, Inc., brand: jones discount, good number: a100122-0500; cellulose RS (cellulase RS), available from Happy Biotechnology Inc., of Shanghai, under the brand name: YAULT, Cat number: AOV 0108; macerozyme R-10 (isolation enzyme R-10), available from Shanghai Haoyang bright-color Biotech Co., Ltd., brand: YAULT, Cat number: l0021-10 g; pectolyase (pectinase Y-23), available from Shanghai, a bright and bright Biotech Co., Ltd., brand: YAULT, Cat number: AOV 0095; BSA (bovine serum albumin), purchased from vein biotechnology, llc of chongqing, brand: elabscience, cat #: E-IR-R108;crashed enzymes, purchased from Chongqing Baiyan Biotech, Inc., brand: ding Guo; the goods number is: DH 115-2; CaCl2(calcium chloride), available from Shanghai-derived leaf Biotech, Inc., cat #: s24109-500 g.
Example 1
1. Seed germination
(1) Seeds of the early-bell No. 6 loquat variety are sown in soil containing vermiculite;
(2) about 40 days, the seeds can be selected when they sprout out of the young leaves (as shown in figure 1).
2. Test procedure
(1) Pretreatment: placing young plant leaves in a beaker, washing the inner and outer epidermis surface fluff of the leaves with running water, cutting the leaves into fine strips with the size of about 0.5mm by a sharp blade, and immediately soaking in 0.6-1.0M mannitol hypertonic solution for 2-4 h.
(2) Enzymolysis: removing mannitol solution, adding mixed enzyme solution, placing on a shaker at 50-100rpm/min, and performing enzymolysis and shaking overnight (about 12h) in dark. The mixed enzyme preparation solution (10ml) of the invention is as follows: firstly, adding 10mM MES, 0.6M mannitol, 5.0% cellulase RS and 3.6% segregation enzyme R-10, stirring uniformly, heating at 55 ℃ for 10min, and after natural cooling, sequentially adding: 0.01-1% BSA, 1mM CaCl2And the volume is adjusted to 10ml by first-grade water.
(3) And (4) observation: adding equal volume of W5 solution (ready for use), stopping enzymolysis, sieving with 300 mesh sieve, transferring into 15ml centrifuge tube, 700r/min, centrifuging for 12 min, removing supernatant (discarding), adding 1ml W5 solution into the precipitate, and resuspending and observing protoplast.
The W5 solution, the cleaning solution for the protoplast, can be used for stopping the enzymolysis reaction or cleaning the protoplast.
W5 solution of the invention (50 ml): 5mL of 1.54M NaCl, 6.25mL of 1M CaCl2，1.25mL 0.2M KCl，1mLMES-KOH(pH＝5.7)。
Referring to example 1, the inventors conducted an enzyme search.
(1) First, the enzyme preparation was prepared as follows: 1%, 2% and 3% of cellulase and 0.1%, 1% and 2% of pectinase are used as concentrations, a three-factor three-level difference table is set, and as shown in the following table 1, plant cells without cell walls can be observed by a microscope, a large number of cell fragments are obtained, and no loquat protoplast is obtained.
TABLE 1 Low concentration cellulase + pectinase separation of protoplasts from loquat leaves
(2) The inventor increases the concentrations of cellulase and pectinase, changes the concentrations of cellulase and pectinase to 3.6%, 4.2%, 4.8% and pectinase to 1.8%, 2.1% and 2.4%, sets a three-factor three-level difference table, and as shown in the following table 2, no cells or almost all cell fragments can be observed by a microscope, and no loquat protoplast can be obtained.
TABLE 2 high concentration cellulase + pectinase separation of protoplasts from loquat leaves
(3) The inventors changed pectinase to the eductase in the group (2) without changing the cellulose concentration, and set a three-factor three-level difference table, as shown in table 3 below, in which, by microscopic observation, no cells or cell fragments were observed in the individual groups, and a smaller number of protoplasts were observed in the remaining groups.
TABLE 3 cellulase + Segregation enzyme separation of protoplasts from loquat leaves
(4) The enzyme preparation was changed to three enzymes: as shown in Table 4 below, pectinase was added to the group (3) at the same concentration as that of the eductase, and thus, almost all of the plant cells without cell walls removed were observed by microscopic observation, and many of the cell debris were observed, so that loquat protoplasts were not obtained.
TABLE 4 cellulase + Segregation enzyme + pectinase for protoplast separation in loquat leaves
(5) On the basis of the group (3), a collapsing enzyme was added, as shown in Table 5 below, and by microscopic observation, no cells were observed under the microscope, and no loquat protoplast was obtained.
TABLE 5 cellulase + Segregation enzyme + collapse enzyme separation of protoplasts from loquat leaves
(6) Based on group (3), the concentration of the isolated enzyme was set to be in the range of 1.8% to 4.7% without changing the concentration of the cellulase, and as shown in Table 6 below, the loquat protoplasts were observed under a microscope.
TABLE 6 cellulase + Segregation enzyme separation of protoplasts from loquat leaves
(7) Based on group (6), the concentration of the isolated enzyme was found to be 3.6%, and the cellulase concentration was found to be 4% -5.4% without the isolated enzyme, and a three-factor three-level difference table was set, as shown in table 7 below, in which the loquat protoplasts were observed under a microscope.
TABLE 7 cellulase + Segregation enzyme separation of protoplasts from loquat leaves
Experiments show that in the process of preparing protoplast by using leaves as materials and using an enzymolysis method, mixed enzyme formed by cellulase and pectinase is obtained, and neither cell fragments nor plant cells without cell walls are obtained, so that loquat protoplast is not obtained; a smaller number of protoplasts can be seen with a mixture of cellulase + macerase; the mixed enzyme formed by cellulase, pectinase and eductase is used to obtain the plant cell without cell wall removed; with cellulase + eductase + crashed enzyme, almost no cells were observed under the microscope. A large number of experiments show that in the process of preparing the protoplast by using the leaf as the material and using the enzymolysis method, the enzyme preparation only needs two enzymes of cellulase RS and macerozyme R-10, and compared with the combination of cellulose, macerozyme and pectinase or the combination of cellulose, macerozyme and breakdown enzyme, the combination of the two enzymes of cellulase and macerozyme is obviously better than the combination of the three enzymes. Further research shows that the optimal enzyme concentration range for separating the loquat leaf protoplast is as follows: 5.0% of cellulase RS and 3.6-4.0% of macerozyme R-10. The results of microscopic observation of the 5.0% cellulase RS and 3.6% macerase R-10 set are shown in FIG. 4 (microscopic magnification 4X 10), and a large number of protoplasts can be observed clearly.
FDA dyeing: a proper amount of protoplasts are taken, 5mg/ml FDA (fluorescent diacetate) staining solution is added, the ratio of the number of cells which emit green fluorescence in one visual field to the total number of the cells is counted under a fluorescence microscope and is shown in figure 7 (multiple of the microscope is 2 multiplied by 10), and the staining result shows that the protoplast FDA staining result graph of loquat early clock No. 6 can observe high-activity protoplasts.
Referring to example 1 above, a mixed enzyme consisting of 5.0% cellulase RS and 3.6% macerase R-10 was used for the leaf protoplast separation of Imperial concubine loquat and Eriobotrya japonica Hua Bai No. 1. The young and tender leaves of loquat Imperial concubine are shown in figure 2; the result of protoplast microscopic observation of the loquat highest ranking imperial concubine is shown in figure 5 (the microscope multiple is 4 multiplied by 10), and a large number of protoplasts can be obviously observed; the result of FDA staining of protoplast of loquat highest ranked imperial concubine is shown in FIG. 8 (microscope magnification: 2X 10), and high-activity protoplast can be observed. The loquat Huabai No. 1 young leaf is shown in figure 3; the microscopic observation result of the protoplast of loquat Huabai No. 1 is shown in FIG. 6 (the microscope multiple is 4X 10), and a large number of protoplasts can be obviously observed; the result of FDA staining of loquat Hua Bai No. 1 protoplast is shown in FIG. 9 (microscopic magnification: 2X 10), and high-activity protoplasts were observed. In conclusion, the method is suitable for separating the protoplasts of the three loquats of Zaoshu No. 6, Imperial concubine and Huabai No. 1, and the activity of the protoplasts of the loquats after separation is high.
Claims (9)
1. A separation method of loquat protoplast uses loquat leaves as material, and prepares the loquat protoplast by separating with an enzymolysis method, which is characterized in that: the enzyme is a mixed enzyme consisting of cellulase and eductase.
2. The method of claim 1, wherein: the cellulase is cellulase RS, and the isolation enzyme is isolation enzyme R-10.
3. The method of claim 2, wherein: the concentration of the cellulase RS is 1-5.4%, and the concentration of the macerozyme R-10 is 1.8-4.6%, calculated by mass percentage.
4. The method of claim 3, wherein: the concentration of the cellulase RS is 5.0 percent, and the concentration of the macerozyme R-10 is 3.6-4.0 percent in percentage by mass.
5. The method of claim 1, wherein: the loquat is selected from one of loquat No. 6, Imperial concubine and Huabai No. 1.
6. The method of any one of claims 1-5, wherein: the loquat leaves are pretreated before enzymolysis, wherein the pretreatment is to wash down fluff on the inner and outer surface of the loquat leaves by running water, cut the loquat leaves into strips by a knife and put the strips into 0.6 to 1.0M mannitol hypertonic solution for soaking treatment for 2 to 4 hours.
7. The method of any one of claims 1-5, wherein: and carrying out result observation after enzymolysis on the loquat leaves, wherein the result observation is to add protoplast cleaning fluid with the same volume as that of the enzymolysis liquid to stop enzymolysis, sieving the protoplast cleaning fluid with a 300-mesh sieve, carrying out liquid centrifugation, discarding supernatant, adding the protoplast cleaning fluid into the precipitate, and carrying out resuspension observation on the protoplast.
8. The method of claim 1, wherein the following steps are employed:
(1) pretreatment: cleaning villi on the inner and outer epidermis surfaces of young loquat leaves with running water, cutting the leaves into fine strips with the size of about 0.5mm by a knife, and soaking in 0.6-1.0M mannitol hypertonic solution for about 2-4 h;
(2) enzymolysis: removing the mannitol solution, adding the enzymolysis solution, placing on a shaker at 50-100rpm/min, and performing enzymolysis and oscillation in the dark for 10-12 h; the enzyme in the enzymolysis liquid is mixed enzyme consisting of cellulase RS and eductase R-10, the concentration of the cellulase RS is 5.0 percent, and the concentration of the eductase R-10 is 3.6 percent in percentage by mass;
(3) and (4) observing results: adding a protoplast cleaning solution with the same volume as the enzymolysis solution, stopping enzymolysis, sieving the protoplast-enzyme mixed solution with a 300-mesh sieve, centrifuging, removing the supernatant, adding the protoplast cleaning solution into the precipitate, and resuspending and observing the protoplast.
9. A separation method of loquat protoplasts comprises the following steps:
(1) pretreatment: placing young loquat leaves in a beaker, washing the inner and outer surface fluff of the leaves with running water, cutting the leaves into thin strips with the size of about 0.5mm by using a sharp blade, and soaking in 0.6-1.0M mannitol hypertonic solution for 3 hours;
(2) enzymolysis: removing the mannitol solution, adding the enzymolysis solution, placing on a shaker at 50-100rpm/min, and performing enzymolysis and oscillation in the dark for 10-12 h; the preparation method of the enzymolysis liquid comprises the following steps: adding 10mM MES, 0.6M mannitol, 5.0% cellulase RS, and 4.0% segregation enzyme R-10 into 10ml enzymolysis solution, stirring, heating at 55-60 deg.C for 10min, naturally cooling, sequentially adding 0.01-1% BSA and 1mM CaCl2Using primary water to fix the volume to 10ml, and measuring the volume by mass percent;
(3) and (4) observation: adding a protoplast cleaning solution with the same volume as the enzymolysis solution to stop enzymolysis, sieving with a 300-mesh sieve, transferring into a centrifuge tube, centrifuging at 700r/min for 12 minutes, removing the supernatant, adding the protoplast cleaning solution into the precipitate, and resuspending and observing the protoplast.
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