Application of AeMYB30 and / or AeUF3GaT1 in improving the fruit setting rate of golden sunflower

Abstract

The present invention provides the application of AeMYB30 and / or AeUF3GaT1 in improving the fruit setting rate of golden sunflower, belonging to the field of plant cultivation and genetic engineering. In the present invention, the hyperoside improves the plant fruit setting rate through the mediation of the complex MYB30-UF3GaT1, and the plant fruit setting rate can be simply, quickly and efficiently improved by spraying the hyperoside preparation on the plant. Spraying the hyperoside preparation of the present invention can significantly improve the fruit setting rate and thousand-grain weight of understory economic plants. Taking golden sunflower as an example, the fruit setting rate can reach up to 87.4% and the thousand-grain weight can reach up to 80.3g.

Description

[0001] This application is a divisional application with an application date of April 13, 2021, application number 202011460615.4, and invention name "Application of hyperoside in improving plant fruit set rate and / or 1,000-grain weight, hyperoside preparations and methods of use thereof". Technical Field

[0002] The invention belongs to the field of plant cultivation and genetic engineering, and particularly relates to the application of AeMYB30 and / or AeUF3GaT1 in improving the fruit setting rate of golden sunflower. Background Art

[0003] In economic planting under the forest, the traditional method of increasing the fruit set rate by promoting wind or insect pollination to increase pollination rate and reducing the development of other nutritional organs is not only ineffective and complicated to operate, but also relatively costly. Therefore, the research on new methods to promote fruit set that are simple to operate, low in cost and effective is urgent.

[0004] Hyperoside is a flavonoid secondary metabolite in forest economic plants. It has good antioxidant capacity and can not only promote the growth and development of plants themselves, but also treat some human diseases, such as blood circulation and cardiovascular and cerebrovascular symptoms. It has very important medicinal value. At present, the role of hyperoside in plant flowering and fruiting is not clear. Summary of the invention

[0005] In view of this, the object of the present invention is to provide an application of hyperoside in improving plant fruit setting rate and / or thousand-grain weight, a hyperoside preparation and a method of using the same. In the present invention, the hyperoside improves the plant fruit setting rate through the mediation of the complex MYB30-UF3GaT1, and the plant fruit setting rate can be simply, quickly and efficiently improved by spraying the hyperoside preparation on the plant.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides application of hyperoside in improving plant fruit setting rate and / or thousand-grain weight.

[0008] Preferably, the plant is an economic plant under the forest.

[0009] Preferably, the economic plants under the forest include golden sunflower, golden sunflower, long-haired sunflower, hibiscus and arrow-leaf okra.

[0010] Preferably, the application concentration of the hyperoside is 5 to 100 mg / L.

[0011] The invention provides a hyperoside preparation for improving plant fruit setting rate and / or thousand-grain weight, comprising hyperoside and a buffer solution.

[0012] Preferably, the buffer solution uses water as solvent and comprises the following components per liter: 0.19g KNO3, 1.65g NH4NO3, 0.17g KH2PO4, 0.00178g FeSO4·7H2O, 0.0373g Na2·EDTA, 0.004g glycine, 0.0002g VB ammonium hydrochloride sulfate, 0.001g vitamin B6, 0.1g inositol, 0.0062g H3BO3, 0.00083g KI and 10g sucrose.

[0013] Preferably, the pH value of the buffer solution is 5.0-5.2.

[0014] Preferably, the concentration of hyperoside in the hyperoside preparation is 20 to 100 mg / L.

[0015] The invention provides a method for using the hyperoside preparation, which comprises spraying the hyperoside preparation on plants.

[0016] Preferably, the spraying is whole-plant spraying, the total number of spraying is 3 to 6 times, and the interval between two adjacent sprayings is 1 to 2 days.

[0017] Beneficial effects of the present invention: The present invention provides the use of hyperoside in improving the fruit setting rate and / or thousand-grain weight of plants, a hyperoside preparation and a method of using the same. In the present invention, the hyperoside improves the fruit setting rate of plants through the mediation of the complex MYB30-UF3GaT1, exogenous application of hyperoside promotes the up-regulated expression of the MYB30 gene, and MYB30 improves the transcription level by binding to the UF3GaT1 promoter, thereby improving the synthesis of hyperoside affected by the UF3GaT1 gene; spraying the hyperoside preparation on the plant can simply, quickly and efficiently improve the fruit setting rate of the plant.

[0018] Spraying the hyperoside preparation of the present invention can significantly increase the fruit setting rate and thousand-grain weight of economic plants under forests. Taking golden sunflower as an example, the fruit setting rate can reach up to 87.4% and the thousand-grain weight can reach up to 80.3g. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effects of three different treatments of blank control, application of buffer solution only, and application of hyperoside preparation on the fruiting of Helianthus annuus in the present invention; A is the comparison of fruit size, B is the comparison of seed size, and C is the comparison results of average fruit weight, length, width, fruiting rate, number of seeds, thousand-seed weight and seed size;

[0020] Figure 2 This is the validation result of the MYB30-UF3GaT1 complex in the present invention, indicating that MYB30 can directly bind to the UF3GaT1 promoter;

[0021] Figure 3 The effects of overexpression and inhibition of expression of MYB30 or UF3GaT1 gene on average fruiting rate in the present invention; wherein A represents overexpression and inhibition of expression of MYB30, and B represents overexpression and inhibition of expression of UF3GaT1;

[0022] Figure 4 This is the mechanism of action of the hyperoside-mediated MYB30-UF3GaT1 complex in the present invention in affecting fruit set. DETAILED DESCRIPTION

[0023] The present invention provides the use of hyperoside in improving the fruit setting rate and / or thousand-grain weight of plants. In the present invention, the plant is preferably an understory economic plant; the understory economic plants include golden sunflower, long-haired sunflower (Abelmoschuscrinitus Wall.), yellow hibiscus (Abelmoschus manihot (Linn.) Medicus), and arrow-leaf okra (Abelmoschus muliensis Feng). In the specific implementation of the present invention, golden sunflower is taken as an example. In the present invention, the application concentration of the hyperoside is preferably 5 to 100 mg / L, more preferably 20 to 80 mg / L, and most preferably 50 mg / L.

[0024] In the present invention, the hyperoside improves the plant fruiting rate through the mediation of the complex MYB30-UF3GaT1. The specific mechanism of action is as follows: Figure 4 Exogenous spraying of hyperoside solution promoted the up-regulation of MYB30 transcription factor in plants. MYB30 directly binds to the promoter of UF3GaT1 to increase the transcriptional expression of UF3GaT1. UF3GaT1 is a key enzyme in the hyperoside biosynthesis pathway, so the up-regulation of UF3GaT1 increases the content of hyperoside in plants. See the results for details. Figure 2 The present invention verifies and supports the role of hyperoside in promoting reproductive development through the understanding of the above-mentioned complete mechanism of action, thereby achieving the purpose of promoting the fruiting of economic plants under the forest. The result data in the examples also prove that the hyperoside preparation and use method provided by the present invention can achieve the purpose of simply, quickly and efficiently promoting the fruiting of economic plants under the forest.

[0025] The present invention provides a hyperoside preparation for improving plant fruit setting rate and / or 1000-grain weight, comprising hyperoside and a buffer solution. In the present invention, the concentration of hyperoside in the preparation is preferably 20 to 100 mg / L, more preferably 40 to 60 mg / L, and most preferably 50 mg / L.

[0026] In the present invention, the buffer solution uses water as solvent, and preferably includes the following components per liter: 0.19g KNO3, 1.65g NH4NO3, 0.17g KH2PO4, 0.00178g FeSO4·7H2O, 0.0373g Na2·EDTA, 0.004g glycine, 0.0002g VB ammonium hydrochloride sulfate, 0.001g vitamin B6, 0.1g inositol, 0.0062g H3BO3, 0.00083g KI and 10g sucrose. In the present invention, the pH value of the buffer solution is preferably 5.0-5.2, more preferably 5.1. The present invention does not specifically limit the source of the reagents in the buffer solution, and conventional commercial products in the art can be used. In the specific implementation of the present invention, the reagents are purchased from Sigma (Sigma Aldrich Trading Co., Ltd.).

[0027] The present invention has no special limitation on the preparation method of the hyperoside preparation, and the conventional liquid preparation preparation method in the art can be adopted to dissolve hyperoside in the buffer solution.

[0028] The present invention also provides a method for using the hyperoside preparation, and the hyperoside preparation is used to spray plants. In the present invention, the spraying is selected to spray leaves, flowers or whole plants, preferably the whole plants. The present invention has no special restrictions on the specific spraying amount of the spraying, and the spraying is performed until the plants are moistened. In the present invention, taking golden sunflower as an example, preferably 10 plants are sprayed with each liter of hyperoside preparation until the leaves of the whole plant are moistened. In the present invention, the total number of spraying is preferably 3 to 6 times, more preferably 4 to 5 times, and the time interval between each spraying is preferably 1 to 2 days, more preferably 1 day. In the present invention, taking golden sunflower as an example, preferably when the plant height reaches 1m and the crown width reaches 50cm, the first spraying is performed. The present invention has no special restrictions on the method of spraying, and the conventional spraying method in the art can be used, and preferably an automatic sprayer is used for spraying.

[0029] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1

[0031] (1) Selection of economic plants under the forest: The experimental object was sunflower, and good quality sunflower seeds of the "Jin Guang" variety were selected (the standard of good quality is that the seeds are full, without damage, and have consistent shape and color). After cultivation in a greenhouse (25°C during the day and 20°C at night, with a temperature difference of 2°C; air humidity 70%) for 20 days, they were transplanted to the field. After growing for 3 months after transplanting, sunflower plants with a plant height of 95 to 105 m, a crown size of 50 cm, and strong and consistent growth were selected for subsequent operations.

[0032] (2) Preparation of the hyperoside preparation of the present invention: The hyperoside preparation is prepared by mixing hyperoside with a buffer solution, wherein 50 mg of hyperoside is mixed in every 1 L of the buffer solution, and the preparation is performed immediately before use. The buffer solution uses water as a solvent, and the following components are added per liter: 0.19 g of KNO3, 1.65 g of NH4NO3, 0.17 g of KH2PO4, 0.00178 g of FeSO4·7H2O, 0.0373 g of Na2·EDTA, 0.004 g of glycine, 0.0002 g of VB ammonium hydrochloride sulfate, 0.001 g of vitamin B6, 0.1 g of inositol, 0.0062 g of H3BO3, 0.00083 g of KI, and 10 g of sucrose, and the pH value of the buffer solution is adjusted to 5.1.

[0033] (3) Carry out spraying operation according to the following method:

[0034] Spraying part: Use hyperoside preparation to spray the whole plant of golden sunflower;

[0035] Spraying time: Start spraying when the plant height reaches 1m and the crown width reaches 50cm, spray four times in total;

[0036] Spraying cycle: spray once every other day;

[0037] Spraying dosage: Spray 10 plants evenly with each liter of hyperoside solution until all the leaves are moistened.

[0038] On the basis of the above scheme, an automatic sprayer is used to evenly spray the prepared hyperoside preparation on the surface of the entire understory economic plants.

[0039] After 3 months, the seeds were harvested to determine the fruit set rate and thousand-grain weight. The fruit set rate was determined by manual judgment. Empty grains were judged by manual subjective methods, and empty grains and full grains were counted at the same time. The ratio of full grains to the total number of seeds was the fruit set rate. The experiment was repeated three times, and the average value of the three results was taken. The thousand-grain weight was determined by randomly selecting three 1,000 seeds, weighing them separately, and calculating the average value. The fruit set rates of the three experiments were: 85.9%, 90.5% and 85.8%, and the average fruit set rate was 87.4%. The results of the three thousand-grain weight measurements were: 70.9g, 89.2g and 80.8g, and the average thousand-grain weight was 80.3g.

[0040] Example 2

[0041] The concentration of hyperoside in the hyperoside preparation was 5 mg / L, and the remaining steps and parameters were consistent with those in Example 1.

[0042] Example 3

[0043] The concentration of hyperoside in the hyperoside preparation was set to 10 mg / L, and the remaining steps and parameters were consistent with those in Example 1.

[0044] Example 4

[0045] The concentration of hyperoside in the hyperoside preparation was set to 20 mg / L, and the remaining steps and parameters were consistent with those in Example 1.

[0046] Example 5

[0047] The concentration of hyperoside in the hyperoside preparation was set to 100 mg / L, and the remaining steps and parameters were consistent with those in Example 1.

[0048] The comparison results of the fruit setting rate and thousand-grain weight of Examples 1 to 5 are shown in Table 1.

[0049] Table 1 Effects of different concentrations of hyperoside solution on hyperoside promoting the fruiting rate of golden sunflower

[0050]

[0051]

[0052] It can be seen from Table 1 that when the spraying concentration is 50 mg / L, the best purpose of promoting the fruit setting rate of golden sunflower can be achieved.

[0053] Example 6

[0054] The spraying interval was 2 days, and the other steps and parameters were consistent with those in Example 1.

[0055] Comparative Example 1

[0056] The spraying interval was 3 days, and the other steps and parameters were consistent with those in Example 1.

[0057] Comparative Example 2

[0058] The spraying interval was 4 days, and the remaining steps and parameters were consistent with those in Example 1.

[0059] The results of Example 1 and Comparative Examples 1-2 are compared in Table 2.

[0060] Table 2 Effects of different hyperoside solution spraying cycles on hyperoside promoting the fruiting rate of golden sunflower

[0061] interval Fruiting rate (%) Thousand-grain weight (g) 1 day interval 87.4 80.3 2 days interval 75.1 66.4 3 days interval 73.2 64.2 4 days interval 69.3 62.4

[0062] It can be seen from Table 2 that when the spraying cycle is once every other day, the best goal of promoting the fruit setting rate of golden sunflower can be achieved.

[0063] Example 7

[0064] The spraying part is the flower of golden sunflower, and the other steps and parameters are consistent with those in Example 1.

[0065] Example 8

[0066] The spraying site was the leaves of Helianthus annuus, and the remaining steps and parameters were consistent with those in Example 1.

[0067] The results of Example 1, Example 7 and Example 8 are compared in Table 3.

[0068] Table 3 Effects of different hyperoside solution spraying positions on hyperoside promoting the fruiting rate of golden sunflower

[0069]

[0070]

[0071] It can be seen from Table 3 that when the spraying site is the whole plant of Helianthus annuus, the purpose of promoting the fruit setting rate of Helianthus annuus can be best achieved.

[0072] Example 9

[0073] The sprayed plants are the whole plants of long-haired sunflower, yellow hibiscus and arrow-leaved okra, and the other steps and parameters are consistent with those in Example 1.

[0074] Table 4 Effects of hyperoside preparations on the fruiting rate of different understory plants

[0075] Fruiting rate (%) Sunflower Hibiscus tiliaceus Arrowleaf Okra Comparison 66.4 70.1 54.3 Buffer solution 64.1 68.9 52.2 Hyperoside preparations 70.5 75.8 65.1

[0076] As shown in Table 4, hyperoside preparations have the effect of promoting the fruiting of many understory plants.

[0077] Example 10

[0078] To prove that MYB30 can bind to the promoter of UF3GaT1, a yeast one-hybrid experiment was used for verification. The gene sequence of 1235bp before the start codon of the UF3GaT1 gene was selected as its promoter region, and the region was divided into 4 parts (P: -1235–0; P1: -411–0; P2: -801–-411; P3: -1235–-801). The P, P1, P2, and P3 fragments were constructed into the pAbAi vector and transformed into YIHGOLD yeast cells. At the same time, the transcription factor MYB30 was constructed into the pGADT7 vector and transformed into the above yeast cells. After transformation, it was spread on the isoleucine-deficient medium, and the presence or absence of plaques in 200μg / ml AbA was used to determine whether the target gene interacted with MYB30. The results showed that P and P2 showed an interaction relationship with MYB30. The specific results are shown in Figure 2 .

[0079] Embodiment 11

[0080] To verify the role of AeMYB30 and AeUF3GaT1 in fruiting in Helianthus annuus, the fruiting rate was determined in stable transgenic Helianthus annuus plants with overexpression and suppression of AeMYB30 and AeUF3GaT1. To overexpress AeMYB30 and AeUF3GaT1 in Helianthus annuus, AeMYB30 and AeUF3GaT1 were ligated into GFP-pROKII vector, respectively, to generate overexpression vectors: pROKII-AeMYB30-GFP-OE and pROKII-AeUF3GaT1-GFP-OE. In addition, a 253 bp AeMYB30 fragment and a 268 bp AeUF3GaT1 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-AeUF3GaT1-RNAi. The recombinant plasmids were transformed into K599 Agrobacterium for subsequent use.

[0081] Select plump, undamaged, uniformly shaped and colored mature seeds, cut them open and culture them with Agrobacterium on callus induction medium for 10-15 days. Then, transfer the mature callus to bud induction medium to induce bud formation. After budding, transfer 3-5 cm buds to root induction medium to make them root. Finally, transfer the rooted buds to the greenhouse and obtain seeds after 3 months.

[0082] Table 5 Effect of AeMYB30 on the fruit setting rate of Helianthus annuus

[0083] Average fruit setting rate (%) Comparison 59.4 Empty vector 51.3 MYB30 represses expression of-1 20.3 MYB30 represses expression of-2 37.6 MYB30 overexpression-1 83.3 MYB30 overexpression-2 87.1

[0084] Table 6 Effect of AeUF3GaT1 on the fruit setting rate of golden sunflower

[0085] Average fruit setting rate (%) Comparison 62.2 Empty vector 57.9 UF3GaT1 inhibits expression of-1 29.7 UF3GaT1 inhibits expression of-2 35.5 UF3GAT1 overexpression-1 83.2 UF3GAT1 overexpression-2 91.0

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of AeMYB30 and / or AeUF3GaT1 in improving the fruit setting rate of Helianthus annuus.

2. The use according to claim 1, characterized in that: Overexpression of AeMYB30 and / or AeUF3GaT1 can improve the fruit setting rate of Helianthus annuus.

3. Application of recombinant vectors overexpressing AeMYB30 or AeUF3GaT1 in improving the fruit setting rate of Helianthus annuus.

4. The use according to claim 3, characterized in that: The original vector of the recombinant vector includes GFP-pROKII.

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