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

Abstract

The application provides application of a Lagerstroemia indica LiTCP14 gene in reducing leaf size of Lagerstroemia indica, and a coding sequence of the LiTCP14 gene is shown as SEQ ID NO:1. The application adopts a VIGS system to silence expression of the Lagerstroemia indica LiTCP14 gene, and a LiTCP14 gene silencing strain is obtained, and the LiTCP14 gene expression level and the leaf size of the LiTCP14 gene silencing strain are both significantly reduced compared with a wild type strain, which indicates that silencing expression of the Lagerstroemia indica LiTCP14 gene can obtain a small leaf Lagerstroemia indica, and has certain practical value in Lagerstroemia indica breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the LiTCP14 gene of Lagerstroemia indica in reducing the size of Lagerstroemia indica leaves. Background Technology

[0002] Crape myrtle (Lagerstroemia indica L.) is a deciduous small tree or shrub belonging to the genus Lagerstroemia L. and the family Lythraceae. There are approximately 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 exploration of its fragrance, color, shape, and plant characteristics.

[0003] Breeding small-leaved crape myrtle is a continuously focusing area of ​​research for breeders, primarily for the following reasons: 1. The smaller, more delicate leaves of small-leaved crape myrtle make the entire plant appear more compact and aesthetically pleasing, creating a delicate and refined atmosphere in landscaping and possessing high ornamental value; 2. The smaller leaf area of ​​small-leaved crape myrtle results in less water evaporation, helping to maintain internal water balance, thus better adapting to arid environments, reducing irrigation needs, and lowering maintenance costs; 3. Compared to crape myrtle varieties with larger leaves, the smaller leaf area of ​​small-leaved crape myrtle provides fewer hiding places for pests and diseases, and better ventilation and light penetration, which is unfavorable for the breeding and spread of pests and diseases, thereby reducing the probability of pest and disease occurrence and lowering control costs and difficulties. Therefore, developing genes that regulate the size of crape myrtle leaves and breeding small-leaved crape myrtle through genetic engineering is one of the hot topics of research in this field. Summary of the Invention

[0004] The technical problem to be solved by this invention is to develop genes that regulate the size of crape myrtle leaves and to cultivate small-leaved crape myrtle through genetic engineering.

[0005] The first aspect of this invention provides the application of a substance that inhibits the expression of the LiTCP14 gene in reducing the size of crape myrtle leaves, wherein the coding sequence of the LiTCP14 gene is shown in SEQ ID NO:1.

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

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

[0008] The pTRV2 gene target fragment was ligated with the LiTCP14 gene to obtain the recombinant viral plasmid pTRV2-LiTCP14;

[0009] The recombinant virus plasmid pTRV2-LiTCP14 is transformed into Agrobacterium to obtain a recombinant bacterial liquid;

[0010] The recombinant bacterial liquid and the bacterial liquid containing pTRV1 are used to infect a receptor Lagerstroemia indica, to obtain a Lagerstroemia indica with silenced LiTCP14 gene.

[0011] Further, the reduction of the size of the leaves of Lagerstroemia indica includes the reduction of the length and width of the leaves of Lagerstroemia indica.

[0012] Further, the Lagerstroemia indica is Lagerstroemia indica cv. Alba.

[0013] The second aspect of the present application provides a method for reducing the size of the leaves of Lagerstroemia indica, which inhibits the expression of a LiTCP14 gene of Lagerstroemia indica, the coding sequence of the LiTCP14 gene being shown as SEQ ID NO: 1.

[0014] The third aspect of the present application provides a method for cultivating Lagerstroemia indica, which inhibits the expression of a LiTCP14 gene of a receptor Lagerstroemia indica, to obtain a target Lagerstroemia indica with smaller leaves than the receptor Lagerstroemia indica.

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

[0016] Further, the inhibition of the expression of the LiTCP14 gene of Lagerstroemia indica is achieved by a virus-mediated gene silencing technology.

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

[0018] The pTRV2 is connected with a target fragment of the LiTCP14 gene to obtain a recombinant virus plasmid pTRV2-LiTCP14.

[0019] The recombinant virus plasmid pTRV2-LiTCP14 is transformed into Agrobacterium to obtain a recombinant bacterial liquid.

[0020] The recombinant bacterial liquid and the bacterial liquid containing pTRV1 are used to infect a receptor Lagerstroemia indica, to obtain a Lagerstroemia indica with silenced LiTCP14 gene.

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

[0022] The application provides application of a substance inhibiting expression of a Lagerstroemia indica LiTCP14 gene in reducing size of a leaf of the Lagerstroemia indica, and a coding sequence of the LiTCP14 gene is shown as SEQ ID NO:1. The application adopts a VIGS system to silence expression of the Lagerstroemia indica LiTCP14 gene, and a LiTCP14 gene silenced strain is obtained, and compared with a wild type strain, the leaf size is significantly reduced, and it is indicated that silencing expression of the Lagerstroemia indica LiTCP14 gene can obtain a small leaf Lagerstroemia indica, and has certain practical value in Lagerstroemia indica breeding. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a photo of leaves of the Lagerstroemia indica 'Japan White Flower' variety at different periods;

[0024] Figure 2 It is a photo of leaves of the Lagerstroemia indica 'Japan White Flower' variety at different periods; Figure 1 It is a photo of leaves of the Lagerstroemia indica 'Japan White Flower' variety at different periods;

[0025] Figure 3 It is a photo of leaves of the Lagerstroemia indica 'Japan White Flower' variety at different periods;

[0026] Figure 4 It is a photo of leaves of the Lagerstroemia indica 'Japan White Flower' variety at different periods;

[0027] Figure 5 It is a photo of leaves of the Lagerstroemia indica 'Japan White Flower' variety at different periods;

[0028] Figure 6 It is a photo of leaves of the Lagerstroemia indica 'Japan White Flower' variety at different periods; Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0030] Unless otherwise specified, the reagents and equipment used in this invention are conventional reagents and equipment in this technical field. Of course, the instruments and materials used in the embodiments are not limited to the examples listed herein, but are based on their ability to solve the technical problems of this invention and achieve the corresponding technical effects.

[0031] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual* (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise specified, all reagents used in the following examples are commercially available or publicly available. Various vectors known in the art, such as commercially available vectors including plasmids, may be used in the following examples.

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

[0033] Extraction Figure 1 The leaves of Lagerstroemia indica at different stages shown are named S1-S5. Total RNA was extracted from the leaves using an RNAplant kit (Aikerui Biotechnology Co., Ltd.), and the total RNA was reverse transcribed into cDNA using a reverse transcription kit (Aikerui Biotechnology Co., Ltd.). Based on the nucleotide sequence of the Lagerstroemia indica LiTCP14 gene, nucleotide primer sequences such as SEQ ID NO:2 and SEQ ID NO:3 were designed.

[0034] Using cDNA obtained from reverse transcription of crape myrtle leaves at different stages as templates, and employing primers with nucleotide sequences such as SEQ ID NO:2 and SEQ ID NO:3, the expression profiles of LiTCP14 at different stages were validated. The results are as follows: Figure 2 As shown.

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

[0036] Example 2, silencing of LiTCP14 gene of Lagerstroemia indica using VIGS system and identification

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

[0038] The vectors TRV2 and TRV1 are purchased from the plasmid vector bacterial strain cell gene preservation center.

[0039] The pTRV2 plasmid is extracted using a plasmid extraction kit, and the vector pTRV2 is double-enzyme cut using EcoR I and Xba I restriction endonucleases. After agarose gel electrophoresis, the gel is recovered to obtain a linearized vector. The primer with the nucleotide sequence as SEQ ID NO: 4 and SEQ ID NO: 5 is used to amplify the coding sequence of the LiTCP14 gene with the nucleotide sequence as SEQ ID NO: 1, and the amplification product is used as the LiTCP14 interference fragment. The linearized vector and the LiTCP14 interference fragment are connected using a seamless cloning enzyme to obtain a connection product. The connection product is introduced into E. coli DH5a competent cells (purchased from Beijing Chengke Biological Technology Co., Ltd.), and the E. coli is cultured overnight at 37°C. After that, positive monoclonal colonies are picked and expanded. The primers with the nucleotide sequences as SEQ ID NO: 6-7 are designed according to the Lagerstroemia indica LiTCP14 gene sequence, and PCR identification and extraction of positive plasmids are performed for sequencing verification, as shown in Figure 3 The connection of the recombinant virus vector is successful, and the vector is named pTRV2-LiTCP14.

[0040] The pTRV2-LiTCP14 vector is transformed into Agrobacterium GV3101 pSoup p19 (purchased from Shanghai Weidi Biological Technology Co., Ltd.), and pTRV2-LiTCP14 Agrobacterium liquid is obtained by culture. At the same time, pTRV2-GFP with a GFP label is set as a control group, and pTRV2-GFP is transformed into Agrobacterium GV3101 pSoup p19 to obtain pTRV2-GFP Agrobacterium liquid by culture. In addition, the pTRV1 vector is transformed into Agrobacterium GV3101 pSoup p19 to obtain pTRV1 Agrobacterium liquid.

[0041] Take the positive identification of the correct Agrobacterium pTRV2-LiTCP14 and control empty pTRV2-GFP, draw a line on RFP (rifampicin) and KAN (kanamycin) double resistance LB solid medium, inverted in 28℃ incubator dark culture 2 days; using a sterile pipette tip scrape down the long strip of bacterial plaque, and evenly spread on new RFP and KAN double resistance LB solid medium, inverted in 28℃ incubator dark culture 1 day; using a sterile pipette tip scrape down all bacterial plaque, using concentration of 1M MgCl2 solution resuspended to OD600 1.0-1.2. pTRV1 Agrobacterium is activated by the same method and resuspended to the same concentration. pTRV2-LiTCP14 / GFP Agrobacterium suspension and equal volume of pTRV1 Agrobacterium suspension mixed, 1:100 volume ratio of 1M 2-morpholinoethanesulfonic acid (MES) solution (pH = 5.6) was added, 1:1000 volume ratio of 100μM acetyl-syringone (AS) solution was added, mixed evenly, placed in 28℃ incubator dark culture 2-3h, complete the preparation of the infection solution.

[0042] Take wild type of white flower Bauhinia variety 'Japan white flower', and get the irradiation type after 60Co-γ ray irradiation, named 'Yuxi', cut the 'Japan white flower' and 'Yuxi' into cutting branches and keep the bud points for standby.

[0043] Divide 20-30mL of the infection solution into 50mL centrifuge tubes which are autoclaved, invert the cutting branches in the infection solution, the infection solution covers all the wounds, use vacuum negative pressure method to infect the cutting branches, repeat 3 times; after washing with water, place in room temperature dark culture for 2d, after the culture ends, place in the plant incubator, change water every 1-2d, the culture conditions are light intensity 5000LUX, humidity 60%, temperature 26℃, 16h light 8h dark alternation.

[0044] 2, phenotype identification

[0045] Observe the phenotype after the cutting branches are cultured in the plant incubator for 7d, 14d and 21d, the observation results are shown in Figure 4 , and the leaf width and length are counted, measure the widest part of three same parts of leaves in each group and take the average as the leaf width, measure the longest part of three same parts of leaves in each group and take the average as the leaf length, the results are shown in Figure 5 .

[0046] According to Figures 4-5 , whether it is wild type 'Japan white flower' or irradiation type 'Yuxi', after being infected by pTRV2-LiTCP14 Agrobacterium infection solution, it is found that the leaf growth is weaker than that of Bauhinia branches infected by pTRV2-GFP Agrobacterium infection solution. It can be seen that Bauhinia LiTCP14 gene has a promoting effect on the leaf growth of Bauhinia.

[0047] 3. Silencing efficiency detection

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

[0049] The detection results are shown in Table 1. Figure 6 As shown in Table 1, the LiTCP14 expression level in the Lagerstroemia indica leaves after being infected by the pTRV2-LiTCP14 Agrobacterium infection solution is significantly lower than that of the control group pTRV2-GFP, which indicates that silencing the expression of the Lagerstroemia indica LiTCP14 gene can reduce the size of the Lagerstroemia indica leaves, thereby providing a gene source for cultivating small-leaf Lagerstroemia indica.

[0050] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method of reducing leaf size in a Lagerstroemia indica plant, comprising, Inhibition of mangrove LiTCP14 Expression of the gene, which is LiTCP14 The coding sequence of the gene is shown in SEQ ID NO:

1.

2. The method of claim 1, wherein, Reducing the size of a Lagerstroemia indica leaf includes reducing the length and width of a Lagerstroemia indica leaf.

3. The method according to claim 1 or 2, characterized in that, The Lagerstroemia indica is Lagerstroemia indica cv. Alba.

4. The method of claim 1, wherein, Inhibition of bauhinia purpurea LiTCP14 Gene expression was achieved by virus-mediated gene silencing techniques.

5. The method of claim 4, wherein, Inhibition of bengal LiTCP14 Gene expression includes: The pTRV2 is connected with the gene target fragment to obtain a recombinant virus plasmid pTRV2- LiTCP14 The gene target fragment is connected with the pTRV2 to obtain a recombinant virus plasmid pTRV2- LiTCP14 ; The recombinant virus plasmid pTRV2- LiTCP14 is transformed into Agrobacterium to obtain a recombinant bacterial solution; The recombinant bacteria liquid and the bacteria liquid containing pTRV1 are used to infect the receptor Lagerstroemia indica, and a Lagerstroemia indica with gene silencing is obtained LiTCP14 The Lagerstroemia indica with gene silencing is obtained 6. A method for cultivating crape myrtle, characterized in that, Inhibition of receptor Lagerstroemia indica LiTCP14 The expression of the genes results in the objective Lagerstroemia indica with leaf size smaller than that of the receptor Lagerstroemia indica; The LiTCP14 The coding sequence of the gene is shown in SEQ ID NO:

1.

7. The method of claim 6, wherein, Inhibition of bauhinia purpurea LiTCP14 Gene expression was achieved by virus-mediated gene silencing techniques.

8. The method of claim 7, wherein, Inhibition of bengal LiTCP14 Gene expression includes: The pTRV2 was connected with the gene objective fragment to obtain the recombinant virus plasmid pTRV2- LiTCP14 The gene objective fragment was connected with the pTRV2 to obtain the recombinant virus plasmid pTRV2- LiTCP14 ; The recombinant virus plasmid pTRV2- LiTCP14 is transformed into Agrobacterium to obtain a recombinant bacterial solution; The recombinant bacteria liquid and the bacteria liquid containing pTRV1 are used to infect the receptor Lagerstroemia indica, and Lagerstroemia indica with gene silencing is obtained LiTCP14 Lagerstroemia indica with gene silencing.

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