CN115500168B

CN115500168B - Method for regulating camellia oleifera genes by adopting methyl jasmonate

Info

Publication number: CN115500168B
Application number: CN202211287280.XA
Authority: CN
Inventors: 龚文芳; 宋启玲; 袁德义; 于欣冉; 纪珂; 姜毅
Original assignee: Central South University of Forestry and Technology
Current assignee: Central South University of Forestry and Technology
Priority date: 2022-10-20
Filing date: 2022-10-20
Publication date: 2023-10-13
Anticipated expiration: 2042-10-20
Also published as: CN115500168A

Abstract

The application relates to a method for regulating a camellia gene by adopting methyl jasmonate. After methyl jasmonate is sprayed on the whole tea-oil tree, the cell number in the tea-oil camellia seed is increased, and meanwhile, the oil content of the tea-oil camellia seed can be improved, and the fatty acid content in the tea-oil camellia seed is improved; in particular, the expression of key genes in the fatty acid synthesis and metabolic pathways of camellia oleifera, including acetate-CoA carboxylase, long-chain fatty acyl-CoA synthase, and stearoyl-carrier protein desaturase, is also increased. The application provides support for the application of some high-yield cultivation measures of the camellia oleifera, and the oil content of camellia oleifera seeds is improved, and the unit area yield and the total yield of the camellia oleifera are increased.

Description

Method for regulating camellia oleifera genes by adopting methyl jasmonate

Technical Field

The application relates to a method for regulating a camellia gene by adopting methyl jasmonate, and belongs to the technical field of biology.

Background

The camellia oleifera (Camellia oleifera) is a camellia plant of the camellia family, is a special edible oil tree seed in China, has a cultivation and utilization history of 2300 years, is an important woody edible oil tree seed in China, has rich resources and large cultivation area in China, has strong adaptability and is dry and barren resistant, years of income is planted in one year, and plays an important role in ecological construction industry in China and peasants in mountain areas becoming barren and rich. The camellia seed is a camellia seed. The tea oil mainly comprises palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid and arachidonic acid, the content of unsaturated fatty acid in the tea oil seed oil is up to more than 80%, the content of oleic acid is up to more than 70%, the content of oleic acid is close to or even exceeds that of olive oil, and the tea oil is rich in various microelements and active ingredients necessary for human bodies. Increasing oil content of camellia seeds is a great goal and a technical difficulty of camellia breeding for a long time in the future, and is difficult to realize through conventional breeding technology. In order to achieve the aim of improving the variety of the camellia oleifera, one feasible method is to discover key enzymes which can influence the synthesis of fatty acids and grease by various plant regulators, or to research and control the quantity and the area of camellia oleifera seed cells and the like, so as to finally achieve the aim of improving the oil content of camellia oleifera seeds.

It has been demonstrated in bacteria that oil tea lipid metabolism involves a number of enzymes, of which the fatty acid synthesis initiation step is the production of malonate coa by acetate coa carboxylase (ACC, EC 6.4.1.2) with malonyl coa content in the plastid of only 10% of acetyl coa, and thus malonyl coa content may be an important regulator of the fatty acid synthesis pathway, by increasing acetate coa carboxylase activity to promote fatty acid synthesis. Researchers clone cDNA sequences and genome sequences of 4 subunits of ACCase from camellia oleifera, quantitatively study the expression rules of the ACCase in different development stages and different tissues and organs of camellia oleifera seeds through multiplex and real-time fluorescence, and study the influence of over-expression and interference on oil synthesis in Arabidopsis thaliana (see Wang Baoming, cloning and functional study of ACCase genes of camellia oleifera, university of advanced forestry science and technology, doctor's academy of science and technology, publication day: 2012-10-15).

Long chain fatty acyl-CoA synthetases (LACS, long chain fatty acid acyl-CoAsynthetase member, EC 6.2.1) play a key role in the biochemical synthesis pathway of almost all fatty acid derivatives in the organism. At present, the LACS gene and biochemical research thereof are reported in detail in bacteria, yeast and mammals, and LACS is also gradually and deeply studied in higher plants, 9 LACS family members have been identified in Arabidopsis thaliana which is a model of literature report, and it is confirmed that each AtLACS has different expression patterns in different organs and plays an important role in different nodes of fatty acid related lipid metabolism. LACS in camellia oleifera are known in the art to be involved in fatty acid synthesis (Ping Lin et al seed Transcriptomics Analysis in Camellia oleifera Uncovers Genes Associated with Oil Content and Fatty Acid composition. Int. J. Mol. Sci.2018,19,118,1-17). It has also been reported that LACS4 protein and bZIP60 are co-expressed in Camellia oleifera seed germination (see Wenfang Gong et al full-Length Transcriptome from Camellia oleifera Seed Provides Insight into the Transcript Variants Involved in Oil biosystemsis J. Agric. Food chem.2020,68,49,14670-14683).

Vegetable oleic acid (18:1) is catalyzed by a soluble stearoyl carrier protein desaturase (stearoyl acyl-carrier-protein desaturase, SAD, EC 1.14.99.6) using stearic acid as a substrate. SAD family members have specificity for specific substrate chain lengths, double bonds are introduced between carbon atoms of different fatty acid hydrocarbon chains, SAD is the only enzyme for regulating and controlling the unsaturated fatty acid level of plant cells, therefore, SAD plays a key role in regulating and controlling the unsaturated fatty acid level of the plant cells, SAD plays a role in catalyzing the first desaturation of stearic acid to form oleic acid, the first desaturation reaction in the unsaturated fatty acid synthesis pathway is carried out, SAD related genes of plants such as arabidopsis thaliana, camellia oleifera and the like are cloned, the content of stearic acid and palmitic acid is reduced after SAD conversion of arabidopsis thaliana plants, and the content of oleic acid and palmitic acid is increased, and the SAD genes have the function of controlling the conversion of saturated fatty acids to unsaturated fatty acids.

FAD, fatty acid desaturase (fatty acid desaturase), belongs to the class of Acyl-lipid desaturases (Acyl-lipid desaturases) and is an important class of plant fatty acid desaturases. Researchers clone FAD genes from model plant Arabidopsis thaliana at the earliest time, and obtain 5 gene family members which are FAD2, FAD3, FAD6, FAD7 and FAD8 respectively through total separation; subsequently, researchers have cloned different family members of the FAD gene from oil crops such as soybean, sesame, olive tree, etc.

Methyl jasmonate (MeJA) is a phytohormone. Methyl jasmonate exists in plants as a common signal substance, influences plant metabolism, and participates in plant physiological processes such as seed germination, flowering and fruiting, antioxidation in fruit and vegetable storage period, aroma substance formation, disease and pest stress resistance formation and the like.

Melatonin (MT) also known as pinoresinol, chemically known as N-acetyl-5 methoxy tryptamine, is an important class of indole compounds that are widely found in organic bodies such as animals and plants. The exogenous melatonin has important regulation and control effects on the aspects of crop seed germination, root system development, fruit maturation, stress resistance and the like.

Methyl jasmonate and melatonin have the effect of promoting seed germination and plant growth, but the number of seed cells is increased, and the volume of the cells is increased to expand the seeds, so that further research on the influence and the reason of methyl jasmonate and melatonin on the development of oil tea seeds is necessary.

The effects of plant hormones on fatty acid synthesis genes are different and even opposite in different species, and the trend (increase or decrease) of the expression levels of these genes in the same species to the response of the same plant hormone is not always consistent. Therefore, it is hoped to find a plant hormone capable of simultaneously improving the genes related to fatty acid synthesis of camellia oleifera so as to improve the oil content of camellia oleifera and promote the development of camellia oleifera breeding technology.

Disclosure of Invention

In order to solve the problems, the application provides a method for simultaneously improving the expression of a plurality of genes in a fatty acid synthesis pathway of camellia oleifera, wherein methyl jasmonate is sprayed on the whole camellia oleifera, and the plurality of genes are a long-chain fatty acyl CoA synthetase gene LACS, an acetic acid coenzyme A carboxylase gene ACC, a stearoyl carrier protein desaturase gene SAD and a fatty acid desaturase gene FAD.

In some embodiments, the LACS is LACS4, the ACC is ACC1, the SAD is SAD1, SAD6, and the FAD is FAD2, FAD3.

In some embodiments, the period of spraying is the initial oil synthesis period, or the peak oil synthesis period, and/or the late oil synthesis period of the camellia seeds.

In some embodiments, the concentration of the spray is 0.1-5mmol/L, preferably 0.5mmol/L.

The application also provides a method for improving the cell number of the camellia seeds, and methyl jasmonate or melatonin is sprayed on the whole camellia sinensis.

In some embodiments, the concentration of methyl jasmonate is 0.5mmol/L, or the concentration of melatonin is 100 μmol/L.

The application also provides a method for improving the oil content of the camellia seeds, which is characterized in that methyl jasmonate is sprayed on the whole camellia seeds in the oil synthesis peak period and/or the oil synthesis later period of the camellia seeds. In some embodiments, the concentration of the spray is 0.1-5mmol/L, preferably 0.5mmol/L.

Drawings

Fig. 1 shows the fruits and seeds of camellia oleifera at different stages of development according to example 1 of the present application.

FIG. 2 shows the effect of exogenous MeJA (J) and MT (M) treatments of example 1 of the present application on seed volume and weight of camellia oleifera seeds at different concentrations.

FIG. 3 shows the effect of exogenous MeJA (J0.5) and MT (M100) treatments of example 1 of the present application on the seed volume and weight of camellia oleifera seeds.

Fig. 4 shows the observation of oil tea seed development by paraffin section according to example 1 of the present application.

FIG. 5 shows the effect of exogenous MeJA (J0.5) and MT (M100) treatments on the number and cell area of inner and outer seed coat cells of camellia seeds observed in paraffin sections according to example 1 of the present application.

FIG. 6 shows the change of water content and oil content of camellia seeds treated without concentration exogenous MeJA and SHAM according to example 2 of the present application.

FIG. 7 is a graph showing the change in kernel oil body after MeJA treatment of the control and 0.5mmol/L in example 2 of the present application.

FIG. 8 is a color chart showing the variation of differential gene expression levels in the lipid synthesis and metabolism pathway according to example 3 of the present application.

FIG. 9 shows the variation of differential gene expression levels in the lipid synthesis and metabolism pathway according to example 3 of the present application.

FIG. 10 is a schematic diagram showing the relationship between the number of camellia seed cells, the expression of fatty acid synthesis related genes and the like and the content of grease.

Detailed Description

In order to specifically illustrate the general design concept of the present application, specific experimental parameters are shown below as examples, but should not be construed as a reason for limiting the scope of the present application.

The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

One object of the present application is to: the method for improving the expression of related genes in the oil tea fatty acid synthesis pathway is characterized by spraying methyl jasmonate on the whole oil tea tree, wherein the related genes in the oil tea fatty acid synthesis pathway are long-chain fatty acyl CoA synthetase genes (LACS).

According to the above method, the genes further comprise an acetate coenzyme a carboxylase gene (ACC), and/or a stearoyl-carrier protein desaturase gene (SAD), and/or a Fatty Acid Desaturase (FAD).

The application also aims at providing a method for improving the expression of related genes in the fatty acid synthesis path of camellia oleifera, which is characterized in that methyl jasmonate is sprayed on the whole camellia oleifera, wherein the related genes in the fatty acid synthesis path of camellia oleifera are stearoyl carrier protein desaturase genes (SADs).

According to the above method, the genes further include a long chain fatty acyl-CoA synthase gene (LACS), and an acetate CoA carboxylase gene (ACC) and/or a Fatty Acid Desaturase (FAD).

According to the above method, the ACC is ACC1 and/or the LACS is LACS4 and/or the SAD is SAD1, SAD6 and/or the FAD is FAD2, FAD3.

According to the method, the spraying concentration of the methyl jasmonate is 0.1-5mmol/L, preferably 0.5mmol/L. According to the method, the methyl jasmonate is sprayed on the whole plant by a sprayer until the water drops from the leaves. According to the method, the methyl jasmonate is sprayed every month until the fruits are ripe.

The application further aims at providing a method for improving the number of camellia seed cells, which is characterized in that methyl jasmonate and/or melatonin are sprayed on the whole camellia sinensis.

According to the above method, the concentration of methyl jasmonate is 0.5mmol/L, or the concentration of melatonin is 100. Mu. Mol/L. According to the method, the methyl jasmonate or melatonin is sprayed on the whole plant by a sprayer until the plant drips from the leaves. According to the method, the methyl jasmonate or melatonin is sprayed once every month until the fruits are ripe, and the time for improving the expression of the related genes in the fatty acid synthesis pathway of the camellia oleifera seeds occurs in the ripe stage of the camellia oleifera seeds.

The application also aims to provide application of methyl jasmonate in increasing unsaturated fatty acid content of camellia oleifera and/or increasing oil content of camellia oleifera seeds.

The application also aims to provide application of methyl jasmonate in simultaneously improving expression of an acetate coenzyme A carboxylase gene (ACC), a long-chain fatty acyl CoA synthetase gene (LACS), a stearoyl carrier protein desaturase gene (SAD) and a Fatty Acid Desaturase (FAD) in a fatty acid synthesis pathway of oil tea.

According to the above application, wherein the ACC is ACC1 and/or the LACS is LACS4 and/or the SAD is SAD1, SAD6 and/or the FAD is FAD2, FAD3.

According to the application, the spraying concentration of the methyl jasmonate is 0.1mmol/L-5mmol/L, preferably 0.5mmol/L.

The genes of the above fatty acid synthesis pathway are all known in the art, for example, the ACC1 gene may be an acetate-CoA carboxylase gene with Genbank accession number xp_028107774.1, the FAD2 gene may be a fatty acid desaturase gene with Genbank accession number AGH32914.1 and AIN52150.1, the FAD3 gene may be a fatty acid desaturase gene with Genbank accession number xp_028121231.1, the LACS4 gene may be a long-chain fatty acyl CoA synthase gene with Genbank accession numbers xp_028120550.1, xp_028065343.1 and ALF39596.1, the SAD1 may be a fatty acid desaturase gene with Genbank accession number xp_028062579.1, and the SAD6 may be a fatty acid desaturase gene with Genbank accession number xp_ 028092290.1.

The application firstly defines the influence of methyl jasmonate and melatonin on the number of camellia seed cells, the influence of methyl jasmonate on the accumulation of unsaturated fatty acids in camellia seeds, and the influence of methyl jasmonate on the expression level of key genes in the process of synthesizing the fatty acids in the camellia seeds through differential expression, thereby providing support for improving the oil content of the camellia seeds, increasing the yield and the total yield of the camellia in unit area and the application of some high-yield cultivation measures of the camellia, and having important theoretical value and important application value (see figure 10).

Example 1 influence of methyl jasmonate and melatonin treatment on seed development of Camellia oleifera

1.1 hormone spraying

The experiment selects two plant growth regulators of methyl jasmonate (MeJA: 0.1mM, 0.5mM, 2.5mM and 5 mM) and melatonin (MT: 50 mu M, 100 mu M, 150 mu M and 200 mu M), 4 hormone concentrations are respectively set, a single factor experiment design is adopted, 9 treatments are totally set up by the experiment, and the exogenous MeJA, MT and Control (CK) with different concentrations are treated by using equal amount of clean water as a control. For each treatment, 3 trees were sprayed with 0.01% Tween-20, and the whole plant was sprayed with a sprayer until the water was dropped from the leaves. Spraying every other month from the beginning of 3 months until the fruits ripen. The test is a field test, and a periodic sampling method is adopted. Sample collection was performed every 14 days, starting at 28 days 3 months in 2020, until the fruit ripens (10 months 24 days). The sampling part is the upper part of each tree, and is randomly selected. Fresh samples were placed in an ice bin and returned to the laboratory for subsequent experimental determination.

1.2 seed weight and volume determination

Fresh seeds collected at different times were weighed, and each 100 seeds were grouped and repeated 3 times in total. The seed volume was determined using a submerged method after weighing the seed.

1.3 results

The rapid expansion time of the seeds was found to be 7-9 months by investigation. Seeds were predominantly in the seed coat development stage at 3-7 months and in the embryo development stage at 8-10 months (figure 1).

The use of different concentrations of MeJA and MT found that 0.5mmol/L MeJA (J0.5) and 100 μmol/L MT (M100) significantly increased the volume and weight of seeds, by 30% and 15% respectively, during rapid expansion (fig. 2, 3).

The inner and outer beads of the ovule are both formed into the seed coats, which form the inner and outer seed coats, respectively. The outer seed coat cells are radially elongated, the cell walls are continuously lignified and thickened, the cell cavities are reduced, the cells are closely arranged, and the cells are finally developed into black lignified shell-shaped seed coats. Microscopic observations showed that the inner seed coat gradually consumed from day 5 to day 23 and gradually became membranous from day 7 to day 18, between the shell-like seed coat and the hypertrophic cotyledons (fig. 4).

To further facilitate statistics of cell numbers and areas, the seeds are divided into three regions (the point end is above the region, the middle is in the region, and the other end is below the region). The cell number in the inner and outer seed coat regions increased mainly from 3 months 2 days 8 to 5 months 23 days, after which it began to decline. Cell area and number are inversely varying and there is no significant difference between treatments. Exogenous hormones were shown to affect seed size primarily by increasing cell numbers in the seed coat in the early stages of seed development (fig. 5).

Example 2 influence of methyl jasmonate on the moisture and oil content of camellia seeds

2.1 hormone spraying

Methyl jasmonate (MeJA) and methyl jasmonate inhibitor were selected for the test: two plant growth regulators of Salicin Hydroxamic Acid (SHAM) were each set at 4 hormone concentrations, and exogenous MeJA, SHAM and Control (CK) at different concentrations were tested using a single-factor test design for a total of 9 treatments (Table 1) with equal amounts of fresh water as controls. For each treatment, 3 trees were treated, and 0.01% Tween-20 was added during spraying, and the whole plant was sprayed to leaf drops by a sprayer. Spraying every other month from the beginning of 4 months until the fruits ripen. The test is a field test, and a periodic sampling method is adopted. Sample collection was performed every 14 days, starting at 9/4/2020, until the fruit ripens (10/24). The sampling part is the upper part of each tree, and is randomly selected. Fresh samples were placed in an ice bin and returned to the laboratory for subsequent experimental determination.

TABLE 1 design of exogenous growth regulator one-factor experiments

Table 1 Test factor level of growth regulator

2.2 determination of seed moisture and oil content

Fresh seeds collected during different periods were weighed (W1 mass) and dried in an oven at 60 ℃ to constant weight (W2 mass). Experiments were repeated 3 times. Water content (%) = [ (W1-W2)/W1 ]. Times.100%

And extracting oil tea seed oil by adopting a Soxhlet extraction method. Fresh seeds collected at different times were dried to constant weight in an oven at 60 ℃. Grinding the dried seeds into powder by a grinder. About 5g of the camellia seed powder was placed in a folded filter paper tube, and the bag Cheng Youbao was tightly tied up with a fine cotton thread after being sealed with absorbent cotton, and then inserted into a Soxhlet extractor. About 50ml of petroleum ether was added to an aluminum cup connected to a Soxhlet extractor for extraction. The extraction temperature of the machine was set at 75 ℃. The flow of Soxhlet extractor extraction is as follows: the sample was boiled with the cannula in solvent for 30min. And rinsing for 150min, evaporating for 60min to recover the solvent, and finally taking the extracted oil out of the oil cup and storing the oil in a centrifuge tube for standby. Oil content (%) = [ (weight of oil cup before extraction-weight of oil cup after extraction)/weight of powder ] ×100%.

2.3 seed oil body observations

Selecting fresh camellia fruits, cutting out seeds by a single-sided blade, selecting normal and full seeds, removing inner and outer seed coats, cutting the middle part of the kernels, cutting the seeds into slices by a double-sided blade, placing the slices under a common microscope, observing proper cell vision, placing a nile red dye solution (prepared into a mother solution of 1mg/mL by acetone and preserved in a 4-DEG C refrigerator, diluting into 10 mug/mL by sterile water during use) for 10min in a dark place, and properly blowing and beating by a gun head for several times during the use to enable the dyeing to be more sufficient. The dye liquor is then rinsed clean with sterile water several times. And (3) using a sterile water sealing sheet, sucking off excessive water, and then placing under a laser confocal microscope for observation. Observations were made at a wavelength of 512nm and photographed.

2.4 determination of seed fatty acid Components

The determination of the fatty acid component is referred to the national standard GB5009.168-2016. 60.0mg (accurate to 0.1 mg) of the oil sample to be measured is accurately weighed into a test tube with a stopper, and 2.0ml of an internal standard solution (5 mg/ml of undecanotriglyceride carbonate solution) is accurately added. After adding 4ml of isooctane to dissolve the sample, 200. Mu.l of potassium hydroxide methanol solution (2 mol/L) was added to dissolve the sample by slightly heating if necessary, and the mixture was covered with a glass stopper and shaken vigorously for 30 seconds, and then allowed to stand until clear. Approximately 1g sodium bisulfate was added, and the mixture was vigorously shaken to neutralize potassium hydroxide. After salt precipitation, the upper layer solution is moved to an upper machine bottle, and chromatographic analysis is carried out on the upper machine.

The main fatty acid component of the oil was analyzed by gas chromatograph. Chromatographic conditions: a hydrogen flame ionization detector was used, column chromatography (60 m. Times.0.25 mm. Times.0.2 μm). The carrier gas was nitrogen and the split ratio was 1:50.1 μl autosample was introduced. Heating procedure: first, the temperature was maintained at 50℃for 2 minutes, and then the temperature was raised to 170℃and maintained at 10℃for 10 minutes. Thereafter, the temperature was raised to 180 ℃ (2 ℃/min) and maintained for 10 minutes. Finally, the temperature was raised to 220 ℃ (4 ℃/min) and maintained for 22 minutes.

2.5 results

1> influence of exogenous MeJA and SHAM treatment on water content of camellia seeds

As shown in fig. 6-a, the water content of camellia seeds was in a decreasing trend throughout the oil synthesis. Comparing the water contents of the seeds after different treatments, the water content reduction is more obvious after MeJA treatment of 0.1mmol/L and 0.5mmol/L. And the water content after MeJA treatment of 5mmol/L is generally similar to or slightly higher than CK. The water content at each concentration after SHAM treatment is slightly lower than CK as a whole.

2> Effect of exogenous MeJA and SHAM treatments on oil seed oil content

As shown in fig. 6-B. The oil content of the seeds is extremely low and approaches zero before 8 months and 1 day. Seed oil content differences were significant after MeJA treatment at different concentrations. In the whole process of grease synthesis, after MeJA treatment of 0.5mmol/L, the oil content is improved most remarkably, and 43.07% is improved compared with CK in the mature process (24 days of 10 months). Next, the MeJA treatment of 0.1mmol/L increased 28.69% in comparison with CK during maturation. And the inhibition effect on the oil content after the MeJA treatment of 5mmol/L is remarkable.

Comparing the change in oil content of the seed after SHAM treatment at four concentrations, the oil content after SHAM treatment at 4mmol/L and 8mmol/L as a whole was increased compared to CK. And 2mmol/L and 10mmol/L SHAM treated to approximate CK.

3> Effect of exogenous MeJA and SHAM treatments on oil tea seed oil body

As can be seen from FIG. 6-B, the increase in oil content after MeJA treatment at 0.5mmol/L was most remarkable. Therefore, we selected samples treated with CK and MeJA at 0.5mmol/L, and observed changes in oil bodies in their kernel cells. As a result, as shown in FIG. 7, at the beginning of 8 months, the number of oily bodies in the kernel cells after the treatment with CK and MeJA at 0.5mmol/L was extremely small and hardly observed; small amounts of very small volumes of oil have appeared in the kernel cells in mid 8 months and were mainly distributed around the cell walls. On days 8-29 to 9-12, the number of oil bodies increases and accumulates near the cell wall. After 9 late months, the volume of the oil body increased and distributed toward the center of the cells. At maturity (24 days of 10 months), the volume of oil body is maximum and the kernel cells are filled. Compared with the change of CK oil bodies, the oil body morphology in kernel cells after MeJA treatment of 0.5mmol/L is not different from CK, but the number of oil bodies is obviously increased.

4> Effect of exogenous MeJA and SHAM treatment on fatty acid content of Camellia oleifera seeds

a. Influence of exogenous MeJA and SHAM treatment on oleic acid relative content of oil tea seeds

As shown in Table 2, the relative content of oleic acid showed an increasing trend with the synthesis of oil inside the seeds. The oleic acid relative content after MeJA treatment at different concentrations was significantly different. Wherein, the relative content of oleic acid after MeJA treatment of 0.1mmol/L, 0.5mmol/L and 2.5mmol/L is improved compared with CK, and the influence in the early stage of grease synthesis is more obvious. And when the MeJA-treated oleic acid is mature, the oleic acid relative content after the MeJA treatment is increased by 4.04 percent compared with CK. The relative content of oleic acid after the treatment of 5mmol/L MeJA shows an inhibition effect.

Comparing the change of oleic acid relative content after SHAM treatment with different concentrations, the oleic acid relative content of seeds after SHAM treatment with the concentration of 2mmol/L is generally similar to CK. The SHAM treatment effect of 8mmol/L is relatively better, and the oleic acid relative content is improved by 1.82 percent compared with CK during maturation.

TABLE 2 changes in oleic acid relative content of camellia seeds after MeJA and SHAM treatments at different concentrations

b. Influence of exogenous MeJA and SHAM treatment on relative linoleic acid content of camellia seeds

The relative amounts of linoleic acid in seeds after various concentrations of MeJA and SHAM treatments are shown in table 3. The relative content of linoleic acid in the camellia seeds generally has a tendency of descending first, then ascending and then descending in the growth and development process of camellia fruits. In the last ten 8 months, the relative amounts of linoleic acid after treatment with 4 concentrations of exogenous MeJA were higher than the control group, but in smaller increments. The relative content of linoleic acid after treatment with MeJA at 4 concentrations was lower than that of the control group between the last 8 months and the last 9 months. After 10 months, 5mmol/L MeJA treatment, the relative content of linoleic acid was higher than that of the control group, while the remaining concentrations were lower than that of the control group. In combination, the inhibition effect on the relative content of linoleic acid after the treatment of MeJA with different concentrations is more obvious, and the numerical difference between the treatment concentrations is smaller when the treatment is mature.

After SHAM spraying treatment with different concentrations, the relative content of linoleic acid is slightly higher than that of a control group in the middle and upper ten days of 8 months, but the increase amount is smaller. And the relative content of linoleic acid is slightly lower than that of the control group from the last 8 months until the fruits are ripe. Combining the effects of different concentrations of MeJA and SHAM treatments, both exogenous MeJA and SHAM treatments generally exhibited a reduction in the relative amounts of linoleic acid.

TABLE 3 changes in the relative content of linoleic acid in camellia seeds after treatment with MeJA and SHAM at different concentrations

c. Influence of exogenous MeJA and SHAM treatment on relative linolenic acid content of camellia seeds

The relative amounts of linolenic acid in seeds after treatment with different concentrations of MeJA and SHAM are shown in table 4. The relative content of linolenic acid in the camellia seeds gradually decreases along with the growth and development of camellia fruits. The relative content of linolenic acid after MeJA treatment of 0.5mmol/L is higher than that of the control group in the middle and upper ten days of 8 months; the relative content of linolenic acid after MeJA treatment of 4 concentrations is reduced in the middle and late 8 months to the middle and late 9 months compared with that of a control group; the relative content of linolenic acid after being treated by the exogenous MeJA of 5mmol/L is higher than that of the control group in 10 months, and the other concentration treatments are lower than that of the control group; at maturity, there is less numerical difference between treatments.

The relative content of linolenic acid was also reduced overall after treatment with different concentrations of SHAM compared to the control.

TABLE 4 changes in the relative content of linolenic acid in camellia seeds after treatment with MeJA and SHAM at different concentrations

d. Influence of exogenous MeJA and SHAM treatments on the relative content of unsaturated fatty acids in camellia seeds

The change of the relative content of unsaturated fatty acid in seeds treated by MeJA and SHAM with different concentrations is shown in Table 5, and the relative content of unsaturated fatty acid in camellia seed is continuously increased along with the growth and development of camellia fruit. The promoting effect on the relative content of unsaturated fatty acid after MeJA treatment of 0.5mmol/L is more remarkable in 8 months. Wherein the relative content of unsaturated fatty acid is 28.18% higher than CK in 8 months and 15 days. At maturity (24 days of 10 months), the relative content of unsaturated fatty acid is increased by 3.42% compared with CK. The inhibition effect on the relative content of unsaturated fatty acid after the MeJA treatment of 5mmol/L is remarkable.

Comparing the relative content of unsaturated fatty acid in seed after SHAM treatment with different concentrations, the relative content of unsaturated fatty acid after SHAM treatment of 2mmol/L, 4mmol/L, 8mmol/L and 10mmol/L is similar to or slightly lower than CK except 8 months and 15 days.

TABLE 5 variation of unsaturated fatty acid relative content of camellia seeds after MeJA and SHAM treatments at different concentrations

Example 3 Effect of plant hormone on expression of Gene involved in fatty acid Synthesis of Camellia oleifera seed

3.1 sample collection

The results of the oil content, the relative unsaturated fatty acid content and the relative saturated fatty acid content in the camellia seeds after the MeJA treatment are combined show that the 0.5mM MeJA treatment has more remarkable promotion effect on the oil content, the relative unsaturated fatty acid content and the relative saturated fatty acid content of the camellia seeds. In addition, the oil content of the 'Hua Shuo' seed is extremely low in 7 months (initial stage of oil synthesis), 9 months are peak stages of oil synthesis, and 10 months are mature stages of oil tea seeds (late stage of oil synthesis). Therefore, the study selected 0.5mM MeJA treated seeds at 7 months 18, 9 months 12, and 10 months 24 as study material, and clear water as control material. Samples on these 3 days were collected and labeled as CK1 (7 month 18 day water control), J1 (7 month 18 day 0.5mM MeJA treated seed), CK2 (9 month 12 day water control), J2 (9 month 12 day 0.5mM MeJA treated seed), CK3 (10 month 24 day water control), J3 (10 month 24 day 0.5mM MeJA treated seed) were investigated for differential expression of genes in their fatty acid synthesis pathways by means of high throughput transcriptome sequencing.

3.2 transcriptome sequencing and analysis

And (3) performing quality control on the raw reads obtained by sequencing by using fastp, and filtering reads with low quality, wherein the reads with adapter, the reads with N ratio more than 10% and all of A bases and low quality (the number of bases with the quality value Q less than or equal to 20 is more than 50% of the whole reads) are removed, so that clean reads are obtained. And (3) comparing the gene with a reference genome, and then reconstructing the transcript to obtain the complete transcript. The differentially expressed genes were then calculated using RPKM (Reads Per Kilobase per Million mapped reads) values. Gene differential expression analysis was performed according to the general filtration criteria of edge (log 2|fold| >1, FDR < 0.05), fold Change indicating the ratio of RPKM between the two samples.

3.3 Effect of exogenous MeJA treatment on tea seed transcriptome

3.3.1 differential expressed Gene KEGG enrichment analysis

Differentially expressed gene enrichment involved 11 pathways of lipid (table 6). CK1 vs J1 is mainly enriched in the alpha-linolenic acid metabolic pathway. CK2 vs J2 is mainly enriched in unsaturated fatty acid biosynthesis, pantothenic acid and coa biosynthesis, fatty acid metabolism, alpha-linolenic acid metabolism, fatty acid biosynthesis, fatty acid degradation, and glyceride metabolism. Whereas CK3 vs J3 was enriched on all 11 pathways.

TABLE 6 lipid metabolism pathways screened

3.3.2 differential Gene analysis involving fatty acid anabolism

From 11 pathways involved in lipid metabolism, 60 genes related to fatty acid synthesis and metabolism were selected. As shown in fig. 8 and 9, methyl jasmonate can be sprayed in the oil-tea camellia seed oil-fat synthesis peak period and/or the oil-fat synthesis later period to simultaneously increase the expression level of a plurality of genes (such as LACS4, ACC1, SAD6, FAD2, FAD 3), but has little influence or inhibition effect on KAS and FAD 6.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. A method for increasing the cell number of camellia seeds, which is characterized in that methyl jasmonate and/or melatonin are sprayed on the whole camellia sinensis; the concentration of methyl jasmonate is 0.5mmol/L, or the concentration of melatonin is 100 mu mol/L.

2. A method for improving oil content of camellia seeds is characterized in that methyl jasmonate is sprayed on the whole camellia seeds in the oil synthesis peak period and/or the oil synthesis later period of the camellia seeds; the spraying concentration is 0.1-5 mmol/L.

3. The method of claim 2, wherein the concentration of the spray is 0.5mmol/L.

4. A method for simultaneously improving expression of a plurality of genes in a fatty acid synthesis pathway of camellia oleifera is characterized in that methyl jasmonate is sprayed on the whole camellia oleifera, wherein the genes are long-chain fatty acyl CoA synthetase gene LACS, acetic acid CoA carboxylase gene ACC, stearoyl carrier protein desaturase gene SAD and fatty acid desaturase gene FAD; the spraying concentration is 0.1-5 mmol/L.

5. The method of claim 4, wherein the LACS is LACS4, the ACC is ACC1, the SAD is SAD1, SAD6, and the FAD is FAD2, FAD3.

6. The method according to claim 4 or 5, wherein the spraying period is the oil synthesis peak period and/or the oil synthesis later period of the oil tea seeds.

7. The method of claim 4 or 5, wherein the concentration of the spray is 0.5mmol/L.

8. The method of claim 6, wherein the concentration of the spray is 0.5mmol/L.