Cellulase transient expression in alfalfa and its application in extraction of active substances from xanthoceras leaves

Abstract

The present application relates to the field of biotechnology, and more particularly to a kind of alfalfa transient expression cellulase and its application in Xinguan fruit leaf active substance extraction, comprising the following steps: (1) cellulase coding gene construction vector, transformation agrobacterium, culture, centrifugation, collection of bacteria, suspended in buffer, dilution;(2) the bacteria solution obtained in step (1) is used to carry out vacuum infiltration on alfalfa leaf, culture, and 1-500 μM melatonin is sprayed on alfalfa leaf, and spraying is carried out once a day;(3) the alfalfa leaf obtained in step (2) is ground, homogenized, centrifuged, and supernatant is extracted to obtain cellulase enzyme solution;Melatonin is applied in the process of expression, and the activity of cellulase obtained is significantly improved, the problems of low activity of plant expressed cellulase and extraction of leaf active substance biological enzyme are solved;And the steps are simple, the cellulase prepared does not contain endotoxin, can be used for Xinguan fruit leaf active substance extraction, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a kind of alfalfa transient expression cellulase and its application in Xiongguan fruit leaf active substance extraction. BACKGROUND

[0002] Xiongguan fruit tree belongs to Sapindaceae, and is an important economic crop in China. Xiongguan fruit leaf is rich in protein, amino acids and various trace elements; in addition, Xiongguan fruit leaf also contains flavonoids, polysaccharides and saponins and other bioactive components, has high nutritional value and biological efficacy, is a potential extraction material of many active substances, and is also a potential high-quality non-antibiotic breeding feed additive. However, the active substances in Xiongguan fruit leaf mainly exist in the cytoplasm and are protected by the cell wall. Therefore, the utilization of Xiongguan fruit leaf needs to break the cell wall of Xiongguan fruit leaf to release the active substances. At present, there is still a lack of economic and effective technology to solve the above problems.

[0003] Plant cell wall is mainly composed of cellulose, hemicellulose, lignin, pectin and glycoprotein. Among them, cellulose, hemicellulose and lignin are the main components of plant cell wall. The application of cellulase can effectively hydrolyze the fiber structure of plant cell wall and destroy the integrity of cell wall, thereby promoting the release of effective components in plant cells. However, cellulase is mainly produced by bacteria and fungi at present, which needs complex preparation and preparation process and equipment, and also has the problems of bacterial and fungal endotoxin pollution, which limits the application of cellulase in the field of phytochemistry to some extent. Agrobacterium-mediated plant gene transient expression technology is to insert the target gene into the expression vector, transform the vector into Agrobacterium, and use the penetration technology to make Agrobacterium infect plant host cells. The exogenous gene can be expressed without being integrated into the genome of the plant, so it does not need to form a stable transgenic plant to quickly express the exogenous gene in a short period.

[0004] Currently, cellulase expression mainly uses bacteria or fungi. For example, invention patent CN104404067A discloses the expression of cellulase using Lactobacillus, invention patent CN108624613A discloses the expression of cellulase using industrial strains of Saccharomyces cerevisiae, and invention patent CN104845954A discloses the expression of lignocellulose degradation and cellulase, and hemicellulase using Neurospora, Aspergillus, Trichoderma, Penicillium, Fusarium, or Lateralis. However, cellulase expressed by bacteria and fungi contains potential bacterial and fungal endotoxin contamination. If used in industrial production, it has no or very little impact. However, if it is used for the extraction of active substances and then used in animals or humans, the endotoxins may enter the animal's body and cause disease (see Zhang Jikun. Biological activity of endotoxins and their harm to pigs [J]. Pig Science, 2020, 37(11):134-137.). Therefore, how to obtain cellulase without endotoxins is a key technical problem that those skilled in the art need to solve.

[0005] Because plants do not contain endotoxins, transient expression of cellulase in plants can solve the technical problem of endotoxins in cellulase. Currently, only tobacco has been reported as a plant for expressing cellulase. However, tobacco contains harmful substances such as nicotine, requiring purification of tobacco cellulase. Otherwise, it is not suitable for extracting active substances, which increases the production cost of plant-derived cellulase. At the same time, the activity of transiently expressed cellulase in plants is low, and there is still a lack of simple and effective methods to improve the activity of transiently expressed cellulase in plants. Summary of the Invention

[0006] To address the aforementioned technical problems, the primary objective of this invention is to provide a method for transient transexpression of cellulase in alfalfa, comprising the following steps:

[0007] (1) Construct a vector for the cellulase encoding gene, transform Agrobacterium, culture, centrifuge, collect the bacteria, suspend in buffer, and dilute the bacterial solution to an OD value of 0.6-1.0;

[0008] (2) Vacuum permeation and culture of alfalfa leaves using the bacterial solution obtained in step (1), and spray alfalfa leaves with 1-500 μM melatonin once a day.

[0009] (3) Grind the alfalfa leaves obtained in step (2), homogenize them, centrifuge them, and extract the supernatant to obtain cellulase solution.

[0010] Preferably, the melatonin concentration in step (2) is 20-500 μM.

[0011] Preferably, the Agrobacterium in step (1) is LBA4404 strain, which is first cultured into single colony on YEB agar plate containing antibiotics, and then inoculated into YEB liquid medium for culture.

[0012] Preferably, the OD value in step (1) is at 600 nm.

[0013] Preferably, the buffer in step (1) is 10 mM magnesium sulfate; 10 mM MES-KOH, pH 5.5; 100 μΜ acetosyringone.

[0014] Preferably, the culture condition in step (2) is 25℃ for 4-9 days.

[0015] Preferably, the centrifugal speed in step (3) is 14000 rpm for 20 minutes.

[0016] The second object of the present application is to provide the use of the cellulase obtained by the method in the degradation of lignocellulose.

[0017] The third object of the present application is to provide a cellulase for degrading lignocellulose of plants to extract active ingredients, which is prepared by the method.

[0018] The fourth object of the present application is to provide the use of the cellulase in the extraction of active substances of Xanthoceras sorbifolia Bunge leaves, which include flavonoids, saponins and polysaccharides.

[0019] The present application has the following advantages: (1) the present application provides a method for expressing cellulase by transiently transforming alfalfa leaves, which applies exogenous melatonin in the expression process to improve the activity of the obtained cellulase, and solves the technical problem of low activity of plant-expressed cellulase; (2) the method is simple in steps, and the obtained cellulase does not contain toxic substance endotoxin, and can be used for the extraction of plant active substances; (3) the cellulase prepared by the method effectively increases the extraction efficiency of active substances of Xanthoceras sorbifolia Bunge leaves. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Activity of cellulase in crude enzyme solution of transiently expressed alfalfa leaves.

[0021] non-transform: crude enzyme solution of non-transiently transformed alfalfa leaves (the activity of cellulase therein is set as 100); transform: crude enzyme solution of alfalfa leaves which is transiently transformed with cellulase (without applying melatonin).

[0022] Figure 2Effects of different concentrations of melatonin on cellulase activity in crude enzyme solution of transiently expressed alfalfa leaves.

[0023] Control: Cellulase transiently converts alfalfa leaves (no exogenous substance applied, and cellulase activity set to 100). 1, 20, 100, 200, 500 μM: Cellulase transiently converts alfalfa leaves with melatonin at concentrations of 1, 20, 100, 200, and 500 μM, respectively.

[0024] Figure 3 Effects of different treatment methods on flavonoid extraction from *Xanthoceras sorbifolium* leaves

[0025] 1: Flavonoid extraction yield of *Xanthoceras sorbifolium* leaves without crude enzyme treatment; 2: Flavonoid extraction yield of *Xanthoceras sorbifolium* leaves after crude enzyme treatment with alfalfa leaves without transient conversion; 3: Flavonoid extraction yield of *Xanthoceras sorbifolium* leaves after crude enzyme treatment with alfalfa leaves with transient conversion; 4: Flavonoid extraction yield of *Xanthoceras sorbifolium* leaves after crude enzyme treatment with alfalfa leaves with transient conversion and 200 μM melatonin treatment.

[0026] Figure 4 Saponin extraction yield from Sapindus mukorossi leaves

[0027] 1: Saponin extraction yield of *Xanthoceras sorbifolium* leaves without crude enzyme treatment; 2: Saponin extraction yield of *Xanthoceras sorbifolium* leaves after treatment with crude enzyme solution of alfalfa leaves without transient conversion; 3: Saponin extraction yield of *Xanthoceras sorbifolium* leaves after treatment with crude enzyme solution of alfalfa leaves with transient conversion; 4: Saponin extraction yield of *Xanthoceras sorbifolium* leaves after treatment with crude enzyme solution of alfalfa leaves with transient conversion and 200 μM melatonin.

[0028] Figure 5 Polysaccharide extraction rate of Sapindus mukorossi leaves

[0029] 1: Polysaccharide extraction yield of *Sapindus mukorossi* leaves without crude enzyme treatment; 2: Polysaccharide extraction yield of *Sapindus mukorossi* leaves after crude enzyme treatment with alfalfa leaves without transient conversion; 3: Polysaccharide extraction yield of *Sapindus mukorossi* leaves after crude enzyme treatment with alfalfa leaves with transient conversion; 4: Polysaccharide extraction yield of *Sapindus mukorossi* leaves after crude enzyme treatment with alfalfa leaves with transient conversion and 200 μM melatonin treatment. Detailed Implementation

[0030] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0031] Unless otherwise specified in the following examples, the conditions shall be performed according to the standard conditions or the conditions recommended by the manufacturer.

[0032] Unless otherwise specified, all reagents or instruments used in the following examples are commercially available conventional products.

[0033] Example 1: Preparation of Cellulase

[0034] (1) The cellulase encoding gene was constructed in a binary vector and transformed into LBA4404 Agrobacterium. The transformed LBA4404 strain was inoculated on YEB agar plates containing antibiotics and cultured at 26 degrees Celsius for 2 days. The cultured single colonies were inoculated into YEB liquid medium and cultured at 28 degrees Celsius and 180 rpm for 1 day. The bacteria were collected by centrifugation at 5000 rpm for 10 minutes and resuspended in buffer. The bacterial concentration was determined by measuring the OD value at 600 nm and then diluted to an OD value between 0.6 and 1.0.

[0035] The buffer solution is: 10 mM magnesium sulfate; 10 mM MES-KOH, pH 5.5; 100 μM acetylsuccinone;

[0036] The cellulase described is the gene encoding endoglucanase II from Trichoderma reesei, the gene sequence of which is shown in SEQ ID No. 1 and the amino acid sequence of which is shown in SEQ ID No. 2.

[0037] (2) Vacuum permeation of alfalfa leaves with the bacterial solution obtained in step (1), and culture the permeated alfalfa leaves at 25°C for 4-9 days. During this period, spray the alfalfa leaves with 1-500 μM melatonin once a day.

[0038] (3) After the treatment period, the alfalfa leaves obtained in step (2) were thoroughly ground with a tissue grinder, phosphate buffer was added, the leaves were homogenized in phosphate buffer, centrifuged at 14000 rpm for 20 minutes, the supernatant was extracted, crude cellulase solution was obtained, and enzyme activity was measured.

[0039] Example 2: Enzyme activity assay

[0040] (1) Transient expression of cellulase activity in crude enzyme solution of alfalfa leaves

[0041] Cellulase activity was determined using carboxymethyl cellulose as a substrate via the 3,5-dinitrosalicylic acid method. 1.2 mL of enzyme solution was added to 0.8 mL of a 1.0% carboxymethyl cellulose aqueous solution, mixed, and incubated. Then, 2.0 mL of 3,5-dinitrosalicylic acid reagent was added, mixed, and heated in boiling water for 10 min. After appropriate dilution with distilled water, the absorbance was measured at 540 nm to reflect cellulase activity.

[0042] Enzyme solution obtained from alfalfa leaves that did not undergo transient conversion was used as a control, and its cellulase activity was set to 100.

[0043] Test results as follows Figure 1 As shown, without the application of melatonin, the cellulase activity of alfalfa leaves subjected to transient cellulase gene conversion was about four times that of the control group, indicating that a certain level of cellulase was expressed in alfalfa leaves subjected to transient cellulase gene conversion.

[0044] (2) Effects of different concentrations of melatonin on cellulase activity in crude enzyme solution of transiently expressed alfalfa leaves

[0045] The effects of different concentrations of melatonin on the activity of cellulase in the crude enzyme solution of transiently transexpressed alfalfa leaves were further investigated. The detection method was the same as above.

[0046] Using alfalfa leaves that underwent transient cellulase gene conversion but were not treated with exogenous substances as a control (with cellulase activity set to 100), the effect of different concentrations of melatonin on the cellulase activity in the crude enzyme solution of transiently converted alfalfa leaves was investigated.

[0047] Experimental results are as follows Figure 2 As shown, compared to the control group, the application of 1 μM melatonin had no significant effect on the activity of cellulase transiently expressed in alfalfa leaves. The application of 20 μM, 100 μM, 200 μM, and 500 μM melatonin all significantly increased cellulase activity. Within the range of 200 μM melatonin, cellulase activity increased with increasing dosage; above 200 μM, cellulase activity decreased. The increase in cellulase activity was most significant with the application of 200 μM melatonin.

[0048] Example 3: Cellulase degrades *Sapindus mukorossi* leaves and extracts active substances.

[0049] The leaves of *Xanthoceras sorbifolium* were air-dried in a cool place and ground into powder. The powdered leaves were mixed with the crude cellulase solution prepared in Example 1. 100 ml of crude enzyme solution and 20 ml of water were added to every 10 g of *Xanthoceras sorbifolium* leaf powder, and the mixture was incubated for 8 hours.

[0050] (1) Flavonoid Extraction

[0051] The incubation material was added to 60% ethanol (final concentration) and extracted at 70°C for 1 hour. After extraction, the mixture was filtered, and the filtrate was collected. The residue was extracted again using the same method, and the filtrates were combined. The solution was concentrated and then diluted to volume with 60% ethanol (final concentration) to obtain the test solution.

[0052] Experimental results are as follows Figure 3As shown, there was no significant difference in the flavonoid extraction yield between *Xanthoceras sorbifolium* leaves without crude enzyme treatment and those treated with crude enzyme solution from alfalfa leaves without transient conversion. This indicates that the enzyme activity or cellulase content in the crude enzyme solution from alfalfa leaves without transient conversion was low, resulting in insufficient cell wall breakdown of *Xanthoceras sorbifolium* leaves and failure to release the active substance, flavonoids. The flavonoid extraction yield of *Xanthoceras sorbifolium* leaves treated with crude enzyme solution from alfalfa leaves after transient conversion was significantly increased, indicating that the crude enzyme solution obtained through transient conversion had high cellulase activity, enabling the release of active substances from the leaves. The flavonoid extraction yield of *Xanthoceras sorbifolium* leaves treated with crude enzyme solution from alfalfa leaves after transient conversion and 200 μM melatonin treatment was further improved, indicating that the cellulase in the crude enzyme solution from alfalfa leaves after transient conversion can improve the extraction efficiency of flavonoids from *Xanthoceras sorbifolium* leaves. Furthermore, this effect can be further enhanced by exogenous application of 200 μM melatonin, indicating that exogenous application of melatonin can increase cellulase activity and promote the release of active substances from *Xanthoceras sorbifolium* leaves.

[0053] (2) Saponin Extraction

[0054] The incubation material was added to 70% ethanol (final concentration), extracted at 60°C for 60 minutes, and then filtered to collect the filtrate. The residue was extracted again using the same method, and the filtrates were combined. The extract was concentrated to obtain a paste. After dissolving and diluting the paste, it was centrifuged at 3500 r / min for 15 min, and the supernatant was collected. The supernatant was extracted three times with equal volumes of ethyl acetate and water-saturated n-butanol. The n-butanol phases were combined, concentrated to obtain the paste, and then freeze-dried to obtain total saponins.

[0055] Experimental results are as follows Figure 4 As shown, there was no significant difference in the flavonoid extraction rate of *Xanthoceras sorbifolium* leaves without crude enzyme treatment and the saponin extraction rate of leaves treated with crude enzyme solution from alfalfa leaves without transient conversion. This indicates that the enzyme activity or cellulase content in the crude enzyme solution from alfalfa leaves without transient conversion was low, resulting in insufficient cell wall decomposition of *Xanthoceras sorbifolium* leaves and failure to release active substances. However, the saponin extraction rate of *Xanthoceras sorbifolium* leaves treated with crude enzyme solution from alfalfa leaves with transient conversion was significantly increased. The saponin extraction rate of *Xanthoceras sorbifolium* leaves treated with crude enzyme solution from alfalfa leaves with transient conversion and 200 μM melatonin was further increased, indicating that the cellulase in the crude enzyme solution from alfalfa leaves with transient conversion can improve the extraction efficiency of saponins from *Xanthoceras sorbifolium* leaves. This effect can be further enhanced by the exogenous application of 200 μM melatonin, indicating that the exogenous application of melatonin can increase the enzyme activity of cellulase and promote the release of active substances from *Xanthoceras sorbifolium* leaves.

[0056] (3) Polysaccharide extraction

[0057] The incubation material was defatted with petroleum ether, depigmented with 95% ethanol, and extracted with water under 40W ultrasound for 30 minutes. After filtration, the filtrate was concentrated and 4 times its volume of ethanol was added. The mixture was allowed to stand at 4°C, filtered, and the residue was washed with alcohol, freeze-dried, and weighed.

[0058] Experimental results are as follows Figure 5 As shown, there was no significant difference in polysaccharide extraction yield between *Xanthoceras sorbifolium* leaves without crude enzyme treatment and those treated with crude enzyme solution from alfalfa leaves without transient conversion. This indicates that the enzyme activity or cellulase content in the crude enzyme solution from alfalfa leaves without transient conversion was low, resulting in insufficient cell wall breakdown of *Xanthoceras sorbifolium* leaves and failure to release active substances. However, the polysaccharide extraction yield of *Xanthoceras sorbifolium* leaves treated with crude enzyme solution from alfalfa leaves with transient conversion was significantly increased. The polysaccharide extraction yield of *Xanthoceras sorbifolium* leaves treated with crude enzyme solution from alfalfa leaves with transient conversion and 200 μM melatonin was further improved. This indicates that the cellulase in the crude enzyme solution from alfalfa leaves with transient conversion can improve the extraction efficiency of polysaccharides from *Xanthoceras sorbifolium* leaves, and this effect can be further enhanced by exogenous application of 200 μM melatonin. This suggests that exogenous application of melatonin can increase cellulase activity and promote the release of active substances from *Xanthoceras sorbifolium* leaves.

[0059] In summary, this invention provides a method for expressing cellulase from alfalfa leaves using a transient cellulase gene. The method involves applying exogenous melatonin during the expression process, which enhances the activity of the resulting cellulase and solves the technical problem of low cellulase activity in plant expression. Furthermore, the method is simple, and the prepared cellulase does not contain toxic endotoxins, making it suitable for extracting active substances from plants. The cellulase prepared using this method effectively increases the extraction efficiency of active substances from *Sapindus mukorossi* leaves.

Claims

1. A method for transient transexpression of cellulase in alfalfa, characterized in that, Includes the following steps: (1) Construct a vector using the cellulase encoding gene shown in SEQ ID No.1, transform Agrobacterium, culture, centrifuge, collect bacteria, suspend in buffer, and dilute the bacterial solution to an OD value of 0.6-1.0; (2) Vacuum permeation of alfalfa leaves with the bacterial solution obtained in step (1), culture, and spray alfalfa leaves with 20-500 μM melatonin once a day; (3) Grind the alfalfa leaves obtained in step (2), homogenize them, centrifuge them, and extract the supernatant to obtain cellulase solution.

2. The method as described in claim 1, characterized in that, The Agrobacterium mentioned in step (1) is strain LBA4404. First, a single colony is cultured on a YEB agar plate containing antibiotics, and then the cultured single colony is inoculated into YEB liquid medium for culture.

3. The method as described in claim 1, characterized in that, The OD value mentioned in step (1) is at 600 nm.

4. The method as described in claim 1, characterized in that, The buffer solution described in step (1) is 10 mM magnesium sulfate; 10 mM MES-KOH, pH 5.5; 100 μM acetylsuccinone.

5. The method as described in claim 1, characterized in that, The culture conditions described in step (2) are to culture at 25°C for 4-9 days.

6. The method as described in claim 1, characterized in that, The centrifugation speed in step (3) is 14,000 rpm and the time is 20 minutes.

Images