CN115669470A - Application of AemYB30 and/or AeUF3GaT1 in improving seting rate of Abelmoschus manihot - Google Patents
Application of AemYB30 and/or AeUF3GaT1 in improving seting rate of Abelmoschus manihot Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C21/00—Methods of fertilising, sowing or planting
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/25—Root crops, e.g. potatoes, yams, beet or wasabi
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/60—Flowers; Ornamental plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05B—PHOSPHATIC FERTILISERS
- C05B7/00—Fertilisers based essentially on alkali or ammonium orthophosphates
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G5/00—Fertilisers characterised by their form
- C05G5/20—Liquid fertilisers
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
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- Life Sciences & Earth Sciences (AREA)
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Forests & Forestry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Ecology (AREA)
- Pest Control & Pesticides (AREA)
- Soil Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides application of AemYB30 and/or AeUF3GaT in improving the setting percentage of abelmoschus manihot, belongs to the field of plant cultivation and genetic engineering, and in the invention, the hyperoside is used for improving the setting percentage of plants through mediation of a complex MYB30-UF3GaT, and the plant setting percentage can be simply, quickly and efficiently improved by spraying a hyperoside preparation on the plants. By spraying the hyperin preparation disclosed by the invention, the setting rate and the thousand seed weight of understory economic plants can be remarkably improved, taking goldenrod flower as an example, the setting rate can reach 87.4% at most, and the thousand seed weight can reach 80.3g at most.
Description
The application is a divisional application with the application date of 2021, 13/04, the application number of 202011460615.4, the invention name of hyperin application in improving plant maturing rate and/or thousand seed weight, hyperin preparation and use method thereof.
Technical Field
The invention belongs to the field of plant cultivation and genetic engineering, and particularly relates to application of AemYB30 and/or AeUF3GaT in improving the setting percentage of abelmoschus manihot.
Background
In the under-forest economic planting, the traditional method for improving the maturing rate by promoting anemophily or entomophily to improve the pollination rate and reducing the development of other nutritive organs has the defects of unobvious effect, complicated operation and high cost, so that the research on the new method for promoting the maturing rate, which has the advantages of simple operation, low cost and remarkable effect, is not slow.
Hyperin is a flavonoid secondary metabolite in the under-forest economic plants, has good oxidation resistance, can promote the growth and development of the plants, can treat human diseases such as blood circulation and cardiovascular and cerebrovascular symptoms, has very important medicinal value, and has unclear effect on flowering and fructification of the plants at present.
Disclosure of Invention
In view of the above, the present invention aims to provide an application of hyperin in improving plant seed setting rate and/or thousand seed weight, a hyperin preparation and a use method thereof. In the invention, the hyperoside is mediated by the MYB30-UF3GaT complex to improve the plant setting percentage, and the plant setting percentage can be simply, quickly and efficiently improved by spraying a hyperoside preparation on the plant.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides application of hyperin in improving plant setting percentage and/or thousand seed weight.
Preferably, the plant is an under-forest economic plant.
Preferably, the under forest economic plants comprise Abelmoschus manihot, and the under forest economic plants comprise Abelmoschus manihot, around Mao Huangkui, abelmoschus manihot and Abelmoschus esculentus.
Preferably, the application concentration of the hyperin is 5-100 mg/L.
The invention provides a hyperin preparation for improving plant setting percentage and/or thousand seed weight, which comprises hyperin and buffer solution.
Preferably, the buffer solution takes water as a solvent and comprises the following components per liter: 0.19g KNO 3 、1.65g NH 4 NO 3 、0.17g KH 2 PO 4 、0.00178g FeSO 4 ·7H 2 O、0.0373g Na 2 EDTA, 0.004g of glycine, 0.0002g of ammonium VB hydrochloride, 0.001g of vitamin B6, 0.1g of inositol, 0.0062g H 3 BO 3 0.00083g KI and 10g sucrose.
Preferably, the pH value of the buffer solution is 5.0-5.2.
Preferably, the concentration of the hyperin in the hyperin preparation is 20-100 mg/L.
The invention provides a using method of the hyperin preparation, and the hyperin preparation is used for spraying plants.
Preferably, the spraying is whole-plant spraying, the total spraying times are 3-6 times, and the time interval between two adjacent spraying times is 1-2 days.
The invention has the beneficial effects that: the invention provides application of hyperin in improving plant setting percentage and/or thousand seed weight, a hyperin preparation and a using method thereof. In the invention, the hyperoside is mediated by a complex MYB30-UF3GaT1 to improve plant fructification rate, the exogenous application of hyperoside promotes the up-regulation expression of MYB30 genes, and MYB30 improves the transcription level by combining with an UF3GaT promoter, so that the synthesis of hyperoside influenced by UF3GaT genes is improved; the plant setting rate can be simply, quickly and efficiently improved by spraying the hyperin preparation on the plants.
By spraying the hyperin preparation disclosed by the invention, the setting rate and the thousand seed weight of understory economic plants can be remarkably improved, taking goldenrod flower as an example, the setting rate can reach 87.4% at most, and the thousand seed weight can reach 80.3g at most.
Drawings
FIG. 1 is a graph showing the effect of three different treatments of blank control, application of buffer solution only, and application of hyperin preparation on burdening of Abelmoschus manihot in the present invention; a is the comparison of fruit size, B is the comparison of seed size, C is the comparison result of average fruit weight, length, width, seed setting rate, seed number, thousand seed weight, seed size and the like;
FIG. 2 is a validation result of the MYB30-UF3GaT complex of the present invention, indicating that MYB30 can be directly combined with UF3GaT promoter;
FIG. 3 is the effect of overexpression and suppression of expression of MYB30 or UF3GaT genes on average seed set in the present invention; wherein A is MYB30 overexpression and suppression expression, and B is UF3GaT overexpression and suppression expression;
FIG. 4 is the mechanism of action of hyperoside-mediated MYB30-UF3GaT1 complex in influencing fructification in the present invention.
Detailed Description
The invention provides application of hyperin in improving plant setting percentage and/or thousand seed weight. In the present invention, the plant is preferably an under-forest economic plant; the under forest economic plants include Abelmoschus manihot, abelmoschus manihot Mao Huangkui (Abelmoschus manihot wall.), abelmoschus manihot (Linn.) Medicus, abelmoschus esculentus (Abelmoschus muliensis Feng). In the specific implementation process of the invention, abelmoschus manihot is taken as an example. In the present invention, the concentration of hyperin is preferably 5 to 100mg/L, more preferably 20 to 80mg/L, and most preferably 50mg/L.
In the invention, the hyperoside is used for improving the plant setting percentage through the mediation of a compound MYB30-UF3GaT1, and the specific action mechanism is shown in figure 4. The method is characterized in that a hyperoside solution is externally sprayed to promote up-regulated expression of MYB30 transcription factors in plants, transcription expression of UF3GaT1 is improved by directly combining a promoter of UF3GaT1 for MYB30, UF3GaT is a key enzyme in a hyperoside biosynthesis pathway, so that the up-regulated expression of UF3GaT1 increases the content of hyperoside in the plants, and the result is shown in a result figure 2. The invention verifies and supports the function of the hyperin in promoting reproductive development by understanding the complete action mechanism, thereby achieving the purpose of promoting the fructification of the under-forest economic plants. The result data in the embodiment also proves that the hyperin preparation and the use method provided by the invention can realize the aim of simply, quickly and efficiently promoting the fructification of the under-forest economic plants.
The invention provides a hyperin preparation for improving plant setting percentage and/or thousand seed weight, which comprises hyperin and buffer solution. In the present invention, the concentration of hyperoside in the preparation is preferably 20 to 100mg/L, more preferably 40 to 60mg/L, and most preferably 50mg/L.
In the present invention, the buffer solution uses water as a solvent, and preferably comprises the following components per liter: 0.19g KNO 3 、1.65g NH 4 NO 3 、0.17g KH 2 PO 4 、0.00178g FeSO 4 ·7H 2 O、0.0373g Na 2 EDTA, 0.004g of glycine, 0.0002g of ammonium VB hydrochloride, 0.001g of vitamin B6, 0.1g of inositol, 0.0062g H 3 BO 3 0.00083g KI and 10g sucrose. In the present invention, the pH of the buffer solution is preferably 5.0 to 5.2, and more preferably 5.1. The source of the reagents in the buffer solution is not particularly limited in the present invention and may be any commercially available product conventionally used in the art, and in the practice of the present invention, the reagents are available from Sigma (Sigma aldrich trade, ltd).
The preparation method of the hyperin preparation is not particularly limited, and the hyperin can be dissolved in the buffer solution by adopting a conventional liquid preparation method in the field.
The invention also provides a using method of the hyperin preparation, and the hyperin preparation is used for spraying plants. In the invention, the spraying is carried out by selecting spraying leaves, flowers or whole plants, and preferably spraying the whole plants. The spraying amount of the invention is not specially limited, and the invention can be sprayed to plant moistening. In the present invention, taking sunflower as an example, 10 plants are preferably sprayed per liter of hyperin preparation until the whole plant leaves are wet. In the present invention, the total number of spraying is preferably 3 to 6, more preferably 4 to 5, and the time interval between each spraying is preferably 1 to 2 days, more preferably 1 day. In the invention, taking the golden sunflower as an example, the first spraying is preferably carried out when the height of the plant reaches 1m and the crown width reaches 50 cm. The spraying method is not particularly limited, and the spraying can be carried out by adopting a conventional spraying method in the field, preferably by adopting an automatic spraying machine.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
(1) Selection of under-forest economic plant plants: selecting an experimental object Abelmoschus manihot, selecting Abelmoschus manihot seeds with good quality of 'golden light' varieties (the standard of good quality is full seeds, no damage, consistent shape and color), cultivating in a greenhouse (25 ℃ in the daytime, 20 ℃ in the evening, 2 ℃ in temperature difference between the upper temperature and the lower temperature, and 70% in air humidity), transplanting into the field after 20 days, growing for 3 months after transplanting, and selecting Abelmoschus manihot plants with the plant height of 95-105 m, the crown size of 50cm, and strong and consistent growth vigor for subsequent operation.
(2) The preparation method comprises the following steps: the hyperin preparation is prepared by mixing hyperin and buffer solution, wherein 50mg of hyperin is mixed in 1L of buffer solution, and the preparation is prepared as required. The buffer solution takes water as a solvent, and the following components are added into the buffer solution per liter: 0.19g KNO 3 、1.65g NH 4 NO 3 、0.17g KH 2 PO 4 、0.00178g FeSO 4 ·7H 2 O、0.0373g Na 2 EDTA, 0.004g of glycine, 0.0002g of ammonium VB hydrochloride, 0.001g of vitamin B6, 0.1g of inositol, 0.0062g H 3 BO 3 0.00083g KI, 10g sucrose and adjusting the pH of the buffer to 5.1.
(3) Spraying operation is carried out according to the following method:
spraying the parts: spraying the whole golden flower sunflower plant by using a hyperin preparation;
spraying time: the spraying time is that when the plant height reaches 1m and the crown width reaches 50cm, the spraying is carried out for four times;
and (3) spraying period: spraying the mixture once every 1 day;
spraying dosage: spraying 10 plants per liter of hyperin solution until the leaves of the whole plant are wet.
On the basis of the scheme, the prepared hyperin preparation is uniformly sprayed on the surface of the economic plant plants in the whole forest by using an automatic sprayer.
Seeds were harvested after 3 months for seed set percentage and thousand kernel weight measurements. The maturing rate is determined by a manual judgment method. And judging whether the seeds are empty or not by a manual subjective method, and counting empty or full seeds at the same time, wherein the ratio of the full seeds to the total number of the seeds is the setting rate. The experiment was repeated three times, and the results were averaged over the three results. Thousand seed weight determination three 1000 seeds were counted randomly, weighed separately, and the average was calculated. The fructification rate of the three experiments is as follows: 85.9 percent, 90.5 percent and 85.8 percent, and the average seed setting rate is 87.4 percent. The results of three thousand-grain weight measurements are: 70.9g, 89.2g and 80.8g, and the average thousand-grain weight is 80.3g.
Example 2
The concentration of hyperin in the hyperin preparation was 5mg/L, and the remaining steps and parameters were kept the same as in example 1.
Example 3
The concentration of hyperin in the hyperin preparation was 10mg/L, and the remaining steps and parameters were kept the same as in example 1.
Example 4
The concentration of hyperin in the hyperin preparation was set to 20mg/L, and the remaining steps and parameters were kept the same as in example 1.
Example 5
The concentration of hyperin in the hyperin preparation was set at 100mg/L, and the remaining steps and parameters were kept the same as in example 1.
The results of comparison of the setting percentage and the thousand kernel weight of examples 1 to 5 are shown in Table 1.
TABLE 1 Effect of different concentrations of hyperin solutions on hyperin-promoted golden flower seed setting percentage
As can be seen from Table 1, when the spraying concentration is 50mg/L, the purpose of optimally promoting the setting rate of the Abelmoschus manihot can be achieved.
Example 6
The spraying interval was 2 days, and the remaining steps and parameters were in accordance with example 1.
Comparative example 1
The spraying interval was 3 days, and the remaining steps and parameters were in accordance with example 1.
Comparative example 2
The spraying interval was 4 days, and the remaining steps and parameters were in accordance with example 1.
The results of example 1 and comparative examples 1 to 2 are shown in Table 2.
TABLE 2 influence of different spraying periods of hyperoside solution on hyperoside-promoted Hibiscus manihot setting rate
| Spacer | Percentage of fruit set (%) | Thousand Kernel weight (g) |
| At intervals of 1 day | 87.4 | 80.3 |
| At intervals of 2 days | 75.1 | 66.4 |
| At intervals of 3 days | 73.2 | 64.2 |
| At intervals of 4 days | 69.3 | 62.4 |
As can be seen from table 2, when the spraying cycle is once every 1 day, the purpose of optimally promoting the setting rate of the abelmoschus manihot can be achieved.
Example 7
The spraying part is a flower of Abelmoschus manihot, and the other steps and parameters are consistent with those of the embodiment 1.
Example 8
The spraying part is the leaf of the abelmoschus manihot, and the other steps and parameters are consistent with those of the embodiment 1.
The results of example 1, example 7 and example 8 are compared in table 3.
TABLE 3 influence of different spraying parts of hyperin solution on the hyperoside-promoted golden flower burry setting rate
As can be seen from table 3, when the spraying part is the whole abelmoschus manihot plant, the purpose of optimally promoting the setting rate of the abelmoschus manihot can be achieved.
Example 9
The sprayed plants are Mao Huangkui, and the whole plants of abelmoschus manihot and okra, and the rest steps and parameters are consistent with those of example 1.
TABLE 4 Effect of hyperin formulations on seed set percentage of different under-forest plants
| Percentage of fruit set (%) | Length Mao Huangkui | Abelmoschus manihot (L.) Medicus | Okra leaf |
| Control | 66.4 | 70.1 | 54.3 |
| Buffer solution | 64.1 | 68.9 | 52.2 |
| Hyperoside preparation | 70.5 | 75.8 | 65.1 |
As can be seen from Table 4, the hyperin preparation has the effect of promoting the setting of various under-forest plants.
Example 10
To demonstrate that MYB30 was able to bind to the promoter of UF3GaT, it was verified by yeast single-hybrid experiments. Selecting a gene sequence 1235bp before an initiation codon of UF3GaT1 gene as a promoter region, and dividing the region into 4 parts (P: -1235-0, P1: 411-0, P2: 801-411. The P, P, P2, P3 fragments were constructed separately on pabal vector and transformed into YIHGOLD yeast cells. At the same time, the transcription factor MYB30 was constructed on pGADT7 vector and transformed into the yeast cells described above, respectively. After transformation, it was plated in isoleucine deficient medium and whether the gene of interest interacted with MYB30 was judged based on the presence of plaque in 200 μ g/ml AbA. It was found that P and P2 show an interaction with MYB30, and the specific results are shown in fig. 2.
Example 11
In order to verify the effect of AemYB30 and AeUF3GaT1 in Abelmoschus manihot in fructification, the fructification rate of stable transgenic Abelmoschus manihot plants in which the AemYB30 and AeUF3GaT are overexpressed and expression is inhibited is determined. To overexpress aemeyb 30 and AeUF3GaT in abelmoschus manihot, aemeyb 30 and AeUF3GaT were ligated into GFP-pROKII vectors, respectively, to generate overexpression vectors: pROKII-AemYB30-GFP-OE and pROKII-AeUF3 GaT-GFP-OE. In addition, a 253bp AemYB30 fragment and 268bp AeF 3GaT fragment were cloned into the NcoI/SwaI and Xbal/BamHI restriction sites of pROKII, respectively, to generate RNA interference vectors: pROKII-AemYB30-RNAi and pROKII-AeUF3 GaT-RNAi. The recombinant plasmid was transferred into K599 Agrobacterium for subsequent use.
And selecting full mature seeds without damage and with consistent shape and color, cutting the seeds, and culturing the seeds and the agrobacterium on a callus induction culture medium for 10-15 days. Then, the mature callus was transferred to a shoot induction medium to induce the formation of shoots. After sprouting, transferring 3-5 cm sprouts into a root induction culture medium to root the sprouts. Finally, the rooted shoots were transferred to a greenhouse and seeds were obtained after 3 months.
TABLE 5 Effect of AemYB30 on Hibiscus manihot setting percentage
| Average setting percentage (%) | |
| Control | 59.4 |
| Empty vector | 51.3 |
| MYB30 inhibition of expression-1 | 20.3 |
| MYB30 suppression expression-2 | 37.6 |
| MYB30 overexpression-1 | 83.3 |
| MYB30 overexpression-2 | 87.1 |
TABLE 6 influence of AeUF3GaT1 on Hibiscus manihot setting percentage
| Average setting percentage (%) | |
| Control | 62.2 |
| Empty vector | 57.9 |
| UF3GaT inhibits expression-1 | 29.7 |
| UF3GaT inhibits expression-2 | 35.5 |
| UF3GaT overexpression-1 | 83.2 |
| UF3GaT overexpression-2 | 91.0 |
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 (4)
- Application of AemYB30 and/or AeUF3GaT1 in improving seting rate of Abelmoschus manihot.
- 2. The use of claim 1, wherein AemYB30 and/or AeUF3GaT achieve an increase in Hibiscus manihot setting rate by overexpression.
- 3. Application of a recombinant vector for over-expressing AemYB30 or AeUF3GaT1 in improving the setting rate of Abelmoschus manihot.
- 4. The use according to claim 3, wherein the original vector of the recombinant vector comprises GFP-pROKII.
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