CN109089604B

CN109089604B - Method for improving natural wax in citrus and application thereof

Info

Publication number: CN109089604B
Application number: CN201810842582.6A
Authority: CN
Inventors: 程运江; 何义仲; 毕光林; 朱晨桥; 许让伟; 邓秀新
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2018-07-27
Filing date: 2018-07-27
Publication date: 2021-01-08
Anticipated expiration: 2038-07-27
Also published as: CN109089604A

Abstract

The invention relates to the field of citrus biology, and provides a method for improving natural wax in citrus, wherein 0.1-200 mu mol/L metazachlor solution is sprayed to citrus leaves in a short time when young shoots of citrus plants are taken for 12-15 days. The research of the invention shows that after the metazachlor solution is stopped being sprayed, the plant and the fruit show that the surface crystal compensativity is increased after the growth is recovered, the gene expression level of a wax synthesis path is obviously improved, the surface glossiness and smoothness of the citrus leaves and the fruit are increased, and the resistance of the citrus plant and the postharvest storage and fresh-keeping capability of the citrus fruit are effectively improved through the increase of the natural wax.

Description

Method for improving natural wax in citrus and application thereof

Technical Field

The invention relates to the field of citrus biology, in particular to a method for improving natural wax in citrus and application thereof.

Background

Citrus is an important fresh fruit, and wide-skinned Citrus typified by ponkan (Citrus reticulata Blanco cv. ponkan), Citrus typified by shatian pomelo (c.grandis Osbeck cv. shatianyu), and Citrus typified by lemon (c.limon cv. eureka) are very important in the Citrus industry in china and even the world.

The cuticle of the plant epidermis is composed of cutin and wax, and is a hydrophobic barrier covering all the contact parts of the terrestrial plants and the air. The wax on the surface of the plant has the functions of preventing non-porous water loss in plant tissues, preventing ultraviolet injury, maintaining surface cleanness and surface water drainage, resisting insect pest invasion and the like, and plays a very important role in adapting to the external environment of the plant. The waxy layer of the citrus is the first barrier for plant self-protection, and has the functions of preventing the water loss of tissues, maintaining the cleanness and smoothness of the surface, resisting the invasion of germs and insects and the like.

In addition, the citrus fruits are important fresh-eating fruits, and after being harvested, the fresh-eating citrus fruits usually need to be subjected to commercialized treatment such as fruit washing and single fruit packaging (or waxing) to enter the sales market. The waxing process is complex, the cost is high, and the behaviors of using additives such as dyeing and the like, industrial wax, inferior wax and the like exist. Improper waxing causes the generation of peculiar smell in fruits, and the difference is mainly caused by the composition and structure difference between natural wax and synthetic wax.

At present, relevant reports for effectively improving the synthesis of natural citrus wax to improve the resistance of citrus plants and the preservation performance of picked fruits are lacked.

Disclosure of Invention

The invention aims to solve the problem that the synthesis of natural wax of citrus cannot be simply and effectively improved in the prior art, and provides a method for improving the natural wax in citrus, which effectively promotes the synthesis amount of the natural wax of citrus leaves and fruits, improves plant resistance and improves the storage and preservation performance of the harvested citrus.

In order to solve the above problems, the present invention provides the following technical solutions:

the method for improving the natural wax in the citrus is characterized in that 0.1-200 mu mol/L metazachlor solution is sprayed to citrus leaves when young tips of the citrus shoot are taken for 12-15 days.

Preferably, the concentration of the metazachlor solution is 50-180 mu mol/L.

Preferably, the solvent of the metazachlor solution comprises ethanol and water, and the volume ratio of the ethanol to the water is 1: 299-599.

Preferably, the metazachlor solution is sprayed for 1-7 times.

Preferably, the single spraying amount of the metazachlor solution is 25-40 mL/plant.

The invention also provides application of the method in the technical scheme in improving the resistance of the citrus plants.

The invention also provides application of the technical scheme in improving the postharvest storage and fresh-keeping capacity of the citrus fruits.

Compared with the prior art, the technical scheme provided by the invention has the following advantages:

the invention provides a method for improving natural wax in citrus, which is to spray 0.1-200 mu mol/L metazachlor solution to citrus leaves when young citrus shoots are taken for 12-15 days. The research of the invention shows that after the plant and the fruit are recovered to grow after the metazachlor solution is stopped spraying for 2 weeks, the compensatory increase of the surface crystals (namely the increase of the synthesis of natural wax) is shown, the gene expression level of a wax synthesis path is obviously improved, the glossiness and smoothness of the surfaces of citrus leaves and the fruit are effectively improved, and the plant resistance and the fresh-keeping performance of the harvested citrus fruit are obviously improved.

The research of the invention shows that the gene expression quantity of 3-beta-Ketoacyl CoA Synthetase (KCS) in a natural wax synthesis path of the citrus is remarkably increased compared with that of a normal plant after the citrus is treated by the method. The KCS participates in the generation reaction of super-long chain saturated fatty acids (VLCFAs) in the synthesis process of natural waxes in citrus, namely malonyl-CoA is taken as a substrate to catalyze C16 or C18 fatty acids to extend and condense to generate VLCFAs with sufficient carbon chain length, and KCS is the key for determining the reaction speed of the catalytic reaction and the acyl chain degree of the finally obtained acyl coenzyme A product. In the reaction for producing VLCFAs, the gene expression levels of beta-ketoacyl-CoA reductase (KCR), beta-hydroxyacyl-CoA Hydrolase (HCD) and enoyl-CoA reductase (ECR) participating in FAE cycle together with KCS are not significantly different from those of normal plants.

The research of the invention also shows that after the metazachlor solution with the concentration is applied, the expression quantity of related genes such as CER4 and CER3 genes in a natural wax synthesis pathway of citrus is remarkably improved, and the method can simultaneously promote the expression of related enzyme genes for synthesizing wax in a primary alcohol pathway and an alkane pathway.

The metazachlor is low in toxicity and easy to degrade, the half-life period is 10.92-12.68 d, metazachlor residues are basically not existed when citrus fruits are on the market, and the metazachlor is safe to human bodies. The method provided by the invention only needs to spray the metazachlor solution, and has the advantages of low cost, simplicity, feasibility and convenience for popularization.

Drawings

FIG. 1 shows the growth of citrus seedlings under different treatments according to example 1;

FIG. 2 is a microscopic image of citrus fruit leaves under different treatments as in example 1;

FIG. 3 is a graph showing a comparison of the expression levels of genes related to the citrus wax synthesis pathway under different treatments in example 1;

FIG. 4 shows the decay of the harvested fruits under different treatments in example 2.

Detailed Description

The invention provides a method for improving natural wax in citrus, which is characterized in that when young tips of the citrus shoot are taken for 12-15 days, 0.1-200 mu mol/L metazachlor solution is sprayed to citrus leaves.

The results of the examples of the invention show that the natural wax content in the leaves from 2d is significantly increased and the number of wax crystals is significantly increased when the spraying of the metazachlor solution is finished.

Compared with normal leaves, the young leaves sprayed by the method have the advantages of small area, increased thickness, greener and brighter color, and the mature leaves have no obvious difference from the normal leaves in shape. The citrus plant fruits sprayed with the metazachlor solution and clear water have no obvious difference in surface color and appearance and no malformed fruits, namely the method does not damage the appearance and commodity value of the citrus fruits.

The method for improving the natural wax in the citrus can be used for citrus seedlings and mature plants.

According to the invention, the metazachlor solution is preferably sprayed to the citrus leaves when the young citrus shoots are taken for 13-14 days. The invention selects about 2 weeks of young sprout treetop spraying to intervene the natural wax synthesis way in the early growth stage of the citrus and compenstately promote the mass expression of the related genes in the natural wax synthesis way of the citrus, thereby continuously synthesizing a large amount of natural wax in the later stage and improving the content of the total natural wax.

Metazachlor belongs to chloroethaneAnilide herbicide, rat acute oral LD₅₀2150mg/kg, acute percutaneous LD₅₀More than 6810mg/kg, and low toxicity. The half-life period is 10.92-12.68 d, and the degradation is easy. The source of the metazachlor is not specially limited, and the metazachlor can be obtained from commercial products.

In the invention, the concentration of the metazachlor solution is preferably 50-180 mu mol/L, and more preferably 100-150 mu mol/L. In the invention, metazachlor solution at low concentration (below 200 mu mol/L) has obvious promotion effect on expression of citrus natural waxy synthesis pathway related genes such as KCS, CER4, CER3 and the like, and inhibition effect is possible when the metazachlor solution exceeds an effective concentration range.

In the present invention, the solvent of the metazachlor solution comprises ethanol and water, and the ethanol and the water are used as the solvent and have no effect of promoting the synthesis of citrus natural wax. According to the invention, the volume ratio of ethanol to water in the metazachlor solution is preferably 1: 299-599.

In the present invention, the preparation method of the metazachlor solution is preferably as follows: dissolving metazachlor by using ethanol to obtain metazachlor mother liquor; and then diluting the metazachlor mother liquor with water until the concentration of the metazachlor solution is 0.1-200 mu mol/L.

In the invention, the spraying frequency of the metazachlor solution is preferably 1-7 times, and more preferably 2-5 times. According to the invention, preferably, when the spraying times are multiple, the time interval between two adjacent spraying times of the metazachlor solution is 1-5 days, and more preferably 3-4 days. The invention aims to recover the tree vigor of plants and promote compensatory synthesis of fatty acid metabolic pathways of the plants by spraying the metazachlor solution for many times.

In the invention, the single spraying amount of the metazachlor solution is preferably 25-40 mL/plant, and more preferably 30-35 mL/plant.

When the method is used for spraying, the front and the back surfaces of the citrus plant leaves are all sprayed until water drops.

The invention also provides application of the method in improving the resistance of citrus plants, and the natural wax in the citrus is improved by spraying 0.1-200 mu mol/L metazachlor solution to citrus leaves when young tips of the citrus shoot for 12-15 days so as to improve the resistance of the citrus plants to pest and disease attack, the resistance of adverse environments and the like.

The invention also provides application of the formula in improving the postharvest storage and fresh-keeping capacity of citrus fruits. According to the method, 0.1-200 mu mol/L metazachlor solution is sprayed to citrus leaves from young tips of the citrus fruits to 12-15 days, so that the synthetic amount of natural wax of citrus plants is promoted, the content of the natural wax of the fruits of the citrus plants is increased, and the postharvest storage and fresh-keeping capacity of the citrus fruits is enhanced. The research of the invention shows that the rotting rate of the fruits of the citrus plants treated by the method disclosed by the invention stored for 2 months at 4 ℃ is reduced by 13.33% compared with that of the fruits of the conventional citrus, the fresh-keeping time of the picked citrus can be effectively prolonged, and the requirements on fresh-keeping operations such as manual waxing and the like are reduced.

In order to further illustrate the present invention, the following embodiments are described in detail, but they should not be construed as limiting the scope of the present invention.

Example 1

1. Selecting materials: selecting a new-hol navel orange seedling growing for 2 weeks;

2. preparing a medicament: mixing metazachlor and absolute ethyl alcohol, dissolving and preparing into 60mmol/L stock solution. The stock solution was diluted with clear water to 100. mu. mol/L metazachlor solution.

3. Experimental treatment: two seedlings of the New Heier navel orange with the same size are selected, and at the beginning of young sprout treetop 14d, the leaf is sprayed with metazachlor solution and clear water respectively, 30 mL/plant is sprayed each time, and the leaf is sprayed every other day for 7 times (2 weeks in total).

And (3) observing the wax condition on the surface of the plant after stopping spraying for 2 weeks in the spraying process, determining the wax content and the expression condition of the related genes of the wax synthetic pathway, and repeating the steps three times biologically.

4. Results

(1) As shown in FIG. 1, the upper part of the figure shows the growth of the citrus seedlings sprayed with clear water and the lower part shows the growth of the citrus seedlings sprayed with 100. mu. mol/L metazachlor solution. When the metazachlor solution is sprayed on citrus seedlings, the leaves of the seedlings are relatively yellow, thickened and reduced. When the spraying is stopped for 2 weeks, the growth potential of the leaves of the metazachlor-sprayed Newhall navel orange seedlings is recovered and turned green, the leaf surfaces are bright, but the areas are smaller than the normal leaves.

(2) As shown in fig. 2, AB is a microscope image of the clear water treated citrus leaf at different magnifications; CD is a microscopic observation of metazachlor treated citrus leaves at different magnifications; the magnification of AC is 2000 times and that of BD is 5000 times.

After stopping spraying the metazachlor solution for 2 weeks, the growth of the leaves is recovered, the leaves become green and thick, and the wax is increased rapidly. As can be seen from FIG. 2, the amount of crystals in the citrus leaves treated with the metazachlor solution was significantly greater than that of the citrus leaves treated with clear water, i.e. the amount of waxy crystal structure in the leaves treated with the metazachlor solution of the concentration according to the present invention was greater. By statistical analysis, the number of crystals increased by as much as 2-fold.

(3) The gene detection kit is used for detecting the related gene expression conditions in the citrus wax synthesis pathway, namely, the expression quantities of KCS (3-beta-ketoacyl CoA synthetase), ACC (acetyl coenzyme A carboxylase), KCR (3-beta-ketoacyl CoA reductase), HCD (beta-hydroxyacyl coenzyme A hydrolase), ECR (enoyl CoA reductase), CER (fatty acyl reductase) and MAH (medium-chain alkane hydroxylase) in citrus leaves after the clear water treatment and the metazachlor solution treatment are stopped for 2 weeks are respectively detected, and the specific result is shown in figure 3.

In each histogram shown in fig. 3, non-black color indicates the expression level of the relevant gene in the citrus leaf sprayed with metazachlor, black color indicates the expression level of the relevant gene in the citrus leaf sprayed with clear water, p is <0.05, and p is < 0.01.

As shown in figure 3, acetyl CoA generates malonyl-CoA through the action of acetyl CoA carboxylase (ACCase), ACC1 gene expression is related to the acetyl CoA carboxylase, ACC1 gene expression level in citrus leaves sprayed with metazachlor is obviously higher than that of clear water treatment, and raw materials for synthesizing waxy direct precursors VLCFAs (extra-long chain inclusion fatty acids) are improved.

3-beta-Ketoacyl CoA Synthetase (KCS) participates in the condensation reaction of the extension of the ultra-long chain fatty acid, has the phenomena of substrate specificity and functional redundancy and ensures the diversity of VLCFAs; compared with clear water treated leaves, the metazachlor treated leaves KCS4, KCS6 and KCS10 are all up-regulated by more than 2 times. 3-beta-ketoacyl-CoA reductase (KCR), hydroxyacyl-CoA dehydratase (HCD) and enoyl-CoA reductase (ECR) respectively participate in reactions such as wax reduction, dehydration and alkenyl reduction, and compared with leaves treated by clear water, the gene expression of the leaves treated by metazachlor has no significant difference.

KCS, KCR, HCD and ECR together catalyze the production of VLCFAs, where KCS is the rate-limiting enzyme for synthesizing VLCFAs and determines the length of the synthesized carbon chain. The invention can obviously improve the content of VLCFAs by promoting the increase of KCS expression, and further improve the finally synthesized wax content by increasing the precursor substance of wax synthesis.

With VLCFAs as precursor material, there are two waxy synthesis pathways, one of which is the primary alcohol pathway in which VLCFAs well fatty acid reductases FARs (CER4 gene) are reduced to primary alcohols, which are condensed with saturated fatty acids under the catalysis of Waxy Synthetase (WS) to form alkyl esters (wax esters); the second is an alkane pathway, in which VLCFAs are reduced into aldehyde under the action of ester acyl coenzyme reductase FAR (CER3 gene), and are subjected to catalytic decarboxylation by aldehyde decarboxylase to generate alkane, the alkane is catalyzed by medium-chain alkane hydroxylase (MAH) to generate secondary alcohol, and the secondary alcohol is catalyzed by MAH to generate ketone.

As shown in fig. 3, fatty acid reductases FARs (CER4) participate in VLC-acyl CoA reduction reactions to generate primary alcohols with even carbon chains; the gene expression of CER4 was up-regulated by more than 5-fold in metazachlor-treated leaves as analyzed by comparison with clear water-treated leaves. Wax Synthetase (WS) catalyzes primary alcohol and carbon chain length to be more than or equal to C16 VCL-acyl CoA to form wax esters, and through comparative analysis with leaves treated by clean water, the gene expression of WSD1 of the leaves treated by metazachlor is up-regulated by more than 2 times.

As shown in fig. 3, acyl CoA reductase (CER3) up-regulated long-chain fatty acyl CoA to intermediate state ultra-long-chain acyl aldehydes by 7.41-fold in the expression of pyr 3 in treatment with metazachlor; CER1 decarboxylates aldehydes to form ultralong paraffins, and the metazachlor treatment up-regulates the expression by 2.33 times.

Therefore, the wax synthesis related gene expression level of the citrus can be obviously improved after the metazachlor is treated, the natural wax synthesis is promoted, and the resistance of citrus plants is further improved.

Example 2

1. Selecting materials: selecting a three-year-old kumquat plant (bearing fruit);

2. preparing a medicament: mixing metazachlor and absolute ethyl alcohol, dissolving and preparing into 60mmol/L stock solution. The stock solution was diluted with clear water to 200. mu. mol/L metazachlor solution.

3. Experimental treatment: 3 wild oranges with the same size are selected, the fruit bearing amount is about 10, and the fruits are in the green maturity period. The leaves are sprayed with metazachlor and clear water solution respectively, 30 mL/plant is sprayed every 3 days, and the spraying is continuously carried out for 5 times.

After the treatment is finished, observing the growth condition of the fruits, and counting the deformity rate;

after the fruits are ripe, the picked citrus fruits are stored at low temperature of 4 ℃ for 2 months, the change of the fruits during storage is observed, and the rotting rate of the stored fruits is counted.

4. Results

(1) No significant difference in the fruit deformity rate during fruiting and after picking was observed for 2 months in the second year.

(2) The color and the appearance of the picked fruit surface are not obviously different from those of the fruit treated by clear water.

(3) As shown in fig. 4, a is the post harvest fruit storage rot rate of the clear water treatment, B is the post harvest fruit storage rot rate of the metazachlor treatment, p is < 0.05.

As can be seen from FIG. 4, the decay rate of metazachlor treatment was 13.33% lower than that of clear water treatment.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for improving natural wax in citrus is characterized in that 0.1-200 mu mol/L metazachlor solution is sprayed to citrus leaves when young citrus shoots are taken for 12-15 days.

2. The method for improving natural waxiness in citrus according to claim 1, wherein the concentration of the metazachlor solution is 50-180 μmol/L.

3. The method for improving natural waxiness in citrus according to claim 1, wherein the solvent of the metazachlor solution comprises ethanol and water, and the volume ratio of the ethanol to the water is 1: 299-599.

4. The method for improving natural waxiness in citrus according to claim 1, wherein the metazachlor solution is sprayed for 1-7 times.

5. The method for improving natural waxiness in citrus according to claim 4, wherein the single spraying amount of the metazachlor solution is 25-40 mL/plant.

6. The method for improving the natural wax content of citrus fruits according to claim 4 or 5, wherein when the spraying times are more than 1, the time interval between two adjacent spraying times of the metazachlor solution is 1-5 days.

7. Use of a method according to any one of claims 1 to 6 for increasing resistance in a citrus plant.

8. Use of the method according to any one of claims 1 to 6 for improving the postharvest storage and freshness-retaining ability of citrus fruit.