Application of Lagerstroemia indica LiTCP14 gene in reduction of Lagerstroemia indica leaf size

Abstract

The invention provides application of a Lagerstroemia indica LiTCP14 gene in reducing the size of Lagerstroemia indica leaves. The coding sequence of the LiTCP14 gene is shown as SEQ ID NO: 1. A VIGS system is adopted to silence the expression of the Lagerstroemia indica LiTCP14 gene, a LiTCP14 gene silent strain is obtained, the LiTCP14 gene expression level and the leaf size of the Lagerstroemia indica LiTCP14 gene silent strain are remarkably reduced compared with those of a wild strain, it is indicated that leaflet Lagerstroemia indica can be obtained through silence of the expression of the Lagerstroemia indica LiTCP14 gene, and the Lagerstroemia indica LiTCP14 gene silent strain has a certain practical value in the aspect of

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to application of a crape myrtle LiTCP14 gene in reducing the size of crape myrtle leaves. Background Art

[0002] Lagerstroemia indica L. is a deciduous small tree or shrub belonging to the genus Lagerstroemia L., Lythraceae family. There are about 60 species worldwide, most of which are deciduous green shrubs or trees. Current research on crape myrtle varieties mainly focuses on the collection and evaluation of its genetic resources, the development of new varieties, and in-depth discussions on flower fragrance, flower color, flower shape and plant characterization.

[0003] Cultivating small-leaf crape myrtle is a hot topic that breeders continue to pay attention to, mainly for the following reasons: 1. The leaves of small-leaf crape myrtle are smaller and more delicate, making the whole plant look more compact and beautiful, creating a delicate and exquisite atmosphere in the garden landscape, and having a high ornamental value; 2. The leaves of small-leaf crape myrtle are smaller, and their surface area is relatively small, so the amount of water evaporation is relatively small, which helps to maintain the water balance in the body, so as to better adapt to the arid environment, reduce irrigation needs, and reduce maintenance costs. 3. Compared with crape myrtle varieties with larger leaves, small-leaf crape myrtle has a small leaf area, and relatively speaking, there are fewer hiding places for pests and diseases, and the ventilation and light transmittance are better, which is not conducive to the breeding and spread of pests and diseases, thereby reducing the occurrence rate of pests and diseases to a certain extent, and reducing the cost and difficulty of prevention and control. Therefore, the research and development of genes that regulate the size of crape myrtle leaves and the cultivation of small-leaf crape myrtle by means of genetic engineering are one of the hot topics that technicians in this field pay attention to. Summary of the invention

[0004] The technical problem to be solved by the present invention is to develop a gene for regulating the size of crape myrtle leaves and to cultivate small-leaf crape myrtle by means of genetic engineering.

[0005] In a first aspect, the present invention provides use of a substance that inhibits the expression of a Lagerstroemia indica LiTCP14 gene in reducing the size of Lagerstroemia indica leaves. The coding sequence of the LiTCP14 gene is shown in SEQ ID NO:1.

[0006] Furthermore, inhibition of LiTCP14 gene expression in Lagerstroemia indica was achieved through virus-mediated gene silencing technology.

[0007] Further, inhibiting the expression of the Lagerstroemia indica LiTCP14 gene includes:

[0008] The pTRV2 and the target fragment of the LiTCP14 gene were connected to obtain the recombinant viral plasmid pTRV2-LiTCP14;

[0009] Transforming the recombinant viral plasmid pTRV2-LiTCP14 into Agrobacterium to obtain a recombinant bacterial solution;

[0010] The recombinant bacterial solution and the bacterial solution containing pTRV1 are used to infect the recipient crape myrtle to obtain the target crape myrtle with LiTCP14 gene silenced.

[0011] Further, reducing the size of the crape myrtle leaves includes reducing the length and width of the crape myrtle leaves.

[0012] Furthermore, the crape myrtle is white-flowered crape myrtle.

[0013] A second aspect of the present invention provides a method for reducing the leaf size of Lagerstroemia indica by inhibiting the expression of the Lagerstroemia indica LiTCP14 gene, wherein the coding sequence of the LiTCP14 gene is shown in SEQ ID NO:1.

[0014] The third aspect of the present invention provides a method for cultivating crape myrtle, which inhibits the expression of the receptor crape myrtle LiTCP14 gene to obtain a target crape myrtle having a leaf size smaller than that of the receptor crape myrtle;

[0015] The coding sequence of the LiTCP14 gene is shown in SEQ ID NO: 1.

[0016] Furthermore, inhibition of LiTCP14 gene expression in Lagerstroemia indica was achieved through virus-mediated gene silencing technology.

[0017] Further, inhibiting the expression of the Lagerstroemia indica LiTCP14 gene includes:

[0018] The pTRV2 and the target fragment of the LiTCP14 gene were connected to obtain the recombinant viral plasmid pTRV2-LiTCP14;

[0019] Transforming the recombinant viral plasmid pTRV2-LiTCP14 into Agrobacterium to obtain a recombinant bacterial solution;

[0020] The recombinant bacterial solution and the bacterial solution containing pTRV1 are used to infect the recipient crape myrtle to obtain the target crape myrtle with LiTCP14 gene silenced.

[0021] The fourth aspect of the present invention provides the above-mentioned substance for inhibiting the expression of the Lagerstroemia indica LiTCP14 gene.

[0022] The present invention provides an application of a substance for inhibiting the expression of a crape myrtle LiTCP14 gene in reducing the leaf size of crape myrtle, wherein the coding sequence of the LiTCP14 gene is shown in SEQ ID NO: 1. The present invention uses a VIGS system to silence the expression of the crape myrtle LiTCP14 gene, and obtains a LiTCP14 gene silenced strain, which has a significantly reduced leaf size compared to a wild-type strain, indicating that silencing the expression of the crape myrtle LiTCP14 gene can obtain a small-leaf crape myrtle, and has certain practical value in crape myrtle breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 These are photos of leaves of the 'Japanese White Flower' variety of Crape Myrtles at different stages;

[0024] Figure 2 For Figure 1 The relative expression analysis results of LiTCP14 gene in leaves at different stages; S1-S5 represent different stages of leaf development (ae represents the significant difference between groups);

[0025] Figure 3 This is the identification result of the pTRV2-LiTCP14 recombinant vector;

[0026] Figure 4 The phenotypes of crape myrtle branches infected with pTRV2-GFP and pTRV2-LiTCP14 for 7, 14, and 21 days, respectively; A is the phenotype of the branches infected with wild-type 'Japanese White Flower', with the left side showing the infection result of pTRV2-GFP and the right side showing the infection result of pTRV2-LiTCP14; B is the phenotype of the branches infected with irradiated 'Yuwei', with the left side showing the infection result of pTRV2-GFP and the right side showing the infection result of pTRV2-LiTCP14; the scale bar is 5 cm;

[0027] Figure 5 The statistical results of the width and length of the leaves of Crape Myrtle after the branches of Crape Myrtle were infected with pTRV2-GFP and pTRV2-LiTCP14 for 7, 14 and 21 days respectively; A is the statistical result of the width of the leaves after the branches of wild-type 'Japanese White Flower' were infected; B is the statistical result of the length of the leaves after the branches of wild-type 'Japanese White Flower' were infected; C is the statistical result of the width of the leaves after the branches of irradiated 'Yuwei' were infected; D is the statistical result of the length of the leaves after the branches of irradiated 'Yuwei' were infected;

[0028] Figure 6 This is the result of measuring the expression level of LiTCP14 gene in different varieties of Crape Myrtle by qRT-PCR after the branches of different varieties of Crape Myrtle were infected with pTRV2-GFP and pTRV2-LiTCP14 for 21 days; **** indicates statistical difference among the groups (p<0.0001). DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the reagents and equipment used in the present invention are conventional reagents and equipment in the art. Of course, the use of instruments and materials in the embodiments is not limited to the enumeration of this example, but is based on being able to solve the technical problems of the present invention and achieve corresponding technical effects.

[0031] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The reagents used in the following examples, unless otherwise specified, are all commercially available or publicly available reagents. Various vectors known in the art, such as commercially available vectors, including plasmids, etc., can be selected and used in the following examples.

[0032] Example 1: Verification of the expression profile of the LiTCP14 gene in leaves of Lagerstroemia indica at different stages

[0033] Extract as Figure 1 The crape myrtle leaves at different stages are named S1-S5; the total RNA of the leaves was extracted using the RNAplant kit (Aikerui Biotechnology Co., Ltd.), and the total RNA was reverse transcribed into cDNA using a reverse transcription kit (Aikerui Biotechnology Co., Ltd.). According to the nucleotide sequence of the crape myrtle LiTCP14 gene, nucleotide primer sequences such as SEQ ID NO: 2 and SEQ ID NO: 3 were designed.

[0034] Using cDNA obtained by reverse transcription of crape myrtle leaves at different stages as templates, and using nucleotide primer sequences such as primers of SEQ ID NO: 2 and SEQ ID NO: 3, the expression profile of LiTCP14 in leaves at different stages was verified. The results are as follows: Figure 2 shown.

[0035] according to Figure 2 It can be seen that the expression level of LiTCP14 in leaves at different stages is different. The expression level is higher in the S1 stage, and as the leaves of Lagerstroemia grow, the expression level shows a downward trend.

[0036] Example 2: Silencing and identification of the Lagerstroemia indica LiTCP14 gene using the VIGS system

[0037] 1. Construction of pTRV2-LiTCP14 vector and silencing strain

[0038] Both TRV2 and TRV1 vectors were purchased from the Plasmid Vector Bacteria Cell Gene Collection Center.

[0039] The pTRV2 plasmid was extracted using a plasmid extraction kit, and then the vector pTRV2 was double-digested with EcoRⅠ and XbaⅠ restriction endonucleases, and the gel was recovered after agarose gel electrophoresis to obtain a linearized vector; the LiTCP14 gene coding sequence with a nucleotide sequence of SEQ ID NO:1 was amplified using primers with nucleotide primer sequences such as SEQ ID NO:4 and SEQ ID NO:5, and the amplified product was used as the LiTCP14 interference fragment. The linearized vector and the LiTCP14 interference fragment were connected using a seamless cloning enzyme to obtain a connection product. The connection product was introduced into Escherichia coli DH5α competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.), and after the Escherichia coli was cultured at 37°C overnight, the positive single clone was picked and expanded for culture. According to the sequence of the Lagerstroemia indica LiTCP14 gene, primers with nucleotide sequences shown in SEQ ID NO:6-7 were designed, and PCR identification and extraction of positive plasmids were performed for sequencing verification, such as Figure 3 As shown, a successfully connected recombinant virus vector was obtained, named pTRV2-LiTCP14.

[0040] The pTRV2-LiTCP14 vector was transferred into Agrobacterium GV3101 pSoup p19 (purchased from Shanghai Weidi Biotechnology Co., Ltd.) and cultured to obtain pTRV2-LiTCP14 Agrobacterium culture liquid; at the same time, pTRV2-GFP with a GFP tag was set as a control group, and pTRV2-GFP was transferred into Agrobacterium GV3101pSoup p19 to obtain pTRV2-GFP Agrobacterium culture liquid; in addition, the pTRV1 vector was transferred into Agrobacterium GV3101 pSoup p19 to obtain pTRV1 Agrobacterium culture liquid.

[0041] Take the positively identified Agrobacterium pTRV2-LiTCP14 and the control empty pTRV2-GFP, streak on RFP (rifampicin) and KAN (Kana) dual-resistance LB solid medium, inverted in a 28°C incubator for dark culture for 2 days; use a sterile pipette tip to scrape off the long strip plaques, and evenly spread them on new RFP and KAN dual-resistance LB solid medium, inverted in a 28°C incubator for dark culture for 1 day; use a sterile pipette tip to scrape off all plaques, and resuspend them with a 1M MgCl2 solution to an OD600 of 1.0-1.2. pTRV1 Agrobacterium was cultured and activated in the same way and resuspended to the same concentration. The pTRV2-LiTCP14 / GFP Agrobacterium suspension was mixed with an equal volume of pTRV1 Agrobacterium suspension, and a 1 M 2-morphoethanesulfonic acid (MES) solution (pH = 5.6) was added at a volume ratio of 1:100, and a 100 μM acetosyringone (AS) solution was added at a volume ratio of 1:1000. The mixture was evenly mixed and placed in a 28°C incubator for dark culture for 2-3 h to complete the preparation of the infection solution.

[0042] The wild type white flower crape myrtle variety 'Japanese White Flower' was taken, and the irradiated type obtained after irradiating it with 60Co-γ rays was named 'Yuwei'. 'Japanese White Flower' and 'Yuwei' were pruned into cuttings and the buds were retained for later use.

[0043] Dispense 20-30mL of the infection solution into a 50mL centrifuge tube sterilized by high temperature, place the cuttings upside down in the infection solution, cover all the wounds, and use the vacuum negative pressure method to infect the cuttings, repeat 3 times; rinse with clean water and culture in the dark at room temperature for 2 days. After the culture is completed, place them in a plant incubator and change the water every 1-2 days. The culture conditions are a light intensity of 5000LUX, a humidity of 60%, a temperature of 26℃, and 16h of light and 8h of darkness.

[0044] 2. Phenotypic identification

[0045] The cuttings were cultured in the plant incubator for 7 days, 14 days and 21 days before phenotypic observation. Figure 4 As shown, the leaf width and length were statistically analyzed. The widest parts of three leaves at the same position were measured in each group and the average value was taken as the leaf width. The longest parts of three leaves at the same position were measured in each group and the average value was taken as the leaf length. The results are shown in Figure 5 shown.

[0046] according to Figure 4-5 It can be seen that the leaf growth of both the wild-type 'Japanese White Flower' and the irradiated 'Yuwei' after being infected with the pTRV2-LiTCP14 Agrobacterium infection solution was weaker than that of the crape myrtle branches infected with the pTRV2-GFP Agrobacterium infection solution. This shows that the crape myrtle LiTCP14 gene has a promoting effect on the leaf growth of crape myrtle.

[0047] 3. Silencing efficiency test

[0048] After the branches of Crape Myrtle infected with pTRV2-LiTCP14 Agrobacterium infection solution and pTRV2-GFP Agrobacterium infection solution were grown for 21 days, the leaves at the young leaf stage (S1) were taken, and the total RNA of the leaves was extracted using an RNA kit. cDNA was synthesized using a reverse transcription kit, and the silencing efficiency of the LiTCP14 gene was detected by real-time fluorescence quantitative qRT-PCR. The nucleotide sequences used were shown in SEQ ID NO:2-3, and the internal reference primers used were EF-1α-F (SEQ ID NO:8) and EF-1α-R (SEQ ID NO:9).

[0049] Test results such as Figure 6 As shown, the expression level of LiTCP14 in the leaves of Lagerstroemia indica infected with pTRV2-LiTCP14 Agrobacterium infection solution was significantly lower than that in the control group pTRV2-GFP, indicating that silencing the expression of the Lagerstroemia indica LiTCP14 gene can reduce the leaf size of Lagerstroemia indica and provide a gene source for the cultivation of small-leaf Lagerstroemia.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a substance that inhibits the expression of the Lagerstroemia indica LiTCP14 gene in reducing the size of Lagerstroemia indica leaves, characterized in that: The coding sequence of the LiTCP14 gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: Inhibition of LiTCP14 gene expression in Lagerstroemia indica was achieved by virus-mediated gene silencing technology.

3. The method according to claim 2, characterized in that Inhibition of LiTCP14 gene expression in Lagerstroemia indica includes: The pTRV2 and the target fragment of the LiTCP14 gene were connected to obtain the recombinant viral plasmid pTRV2-LiTCP14; Transforming the recombinant viral plasmid pTRV2-LiTCP14 into Agrobacterium to obtain a recombinant bacterial solution; The recombinant bacterial solution and the bacterial solution containing pTRV1 are used to infect the recipient crape myrtle to obtain the target crape myrtle with LiTCP14 gene silenced.

4. The use according to claim 1, characterized in that: Reducing crape myrtle leaf size involves reducing both the length and width of the crape myrtle leaves.

5. The use according to any one of claims 1 to 4, characterized in that: The crape myrtle is a white-flowered crape myrtle.

6. A method for reducing the leaf size of Lagerstroemia indica, characterized in that: The expression of the Lagerstroemia indica LiTCP14 gene is inhibited, and the coding sequence of the LiTCP14 gene is shown in SEQ ID NO:

1.

7. A method for cultivating Lagerstroemia indica, characterized in that: Inhibiting the expression of the receptor crape myrtle LiTCP14 gene to obtain the target crape myrtle with a leaf size smaller than that of the receptor crape myrtle; The coding sequence of the LiTCP14 gene is shown in SEQ ID NO:

1.

8. The method according to claim 6 or 7, characterized in that: Inhibition of LiTCP14 gene expression in Lagerstroemia indica was achieved by virus-mediated gene silencing technology.

9. The method according to claim 8, characterized in that Inhibition of LiTCP14 gene expression in Lagerstroemia indica includes: The pTRV2 and the target fragment of the LiTCP14 gene were connected to obtain the recombinant viral plasmid pTRV2-LiTCP14; The recombinant viral plasmid pTRV2-LiTCP14 is transformed into Agrobacterium to obtain a recombinant bacterial solution; The recombinant bacterial solution and the bacterial solution containing pTRV1 are used to infect the recipient crape myrtle to obtain the target crape myrtle with LiTCP14 gene silenced.

10. The substance for inhibiting the expression of the Lagerstroemia indica LiTCP14 gene according to claim 1.

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