Application of CmPHL1 transcription factor in citrus leaf oil cell formation inhibition and volatile regulation

Abstract

The application belongs to the technical field of biology, and discloses application of CmPHL1 transcription factor in inhibition of citrus leaf oil cell formation and regulation of volatile substances. In view of the problem of insufficient research on existing citrus oil cell formation and essential oil synthesis, a pGBI-CmPHL1 overexpression vector is constructed, and positive strains are obtained by means of Agrobacterium-mediated transformation of citrus. Experiments prove that overexpression of CmPHL1 can significantly reduce the number of oil cells and the content of monoterpenes, sesquiterpenes and other volatile substances. CmPHL1 targets the molecular regulation network of oil cell development, accurately regulates and is heritable, provides a new gene target for citrus quality improvement and breeding, is in line with the direction of green agriculture, and has important economic and ecological benefits.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a CmPHL1 transcription factor in the inhibition of oil cell formation and regulation of volatiles in citrus leaves. Background Technology

[0002] Citrus fruits (Citrus) are evergreen small trees or shrubs belonging to the genus Citrus in the family Rutaceae. Their fruits have a unique flavor, with sweet and sour flesh and a bitter peel. Citrus oil cells (also called oil glands or oil dots) are secretory structures unique to the peel and leaves of citrus fruits, typically appearing as tiny pits or granular protrusions. Their main function is to synthesize, store, and release volatile aromatic oils. These volatile oils (mainly terpenes, sesquiterpenes, alcohols, aldehydes, and other volatile substances) release a rich, spicy aroma.

[0003] Oil cells are structurally fragile and prone to rupture under stress. After necrosis, they are susceptible to fungal infection, leading to yellowing and wilting of leaves and impacting plant health. Reducing the number of oil cells can mitigate these risks. However, traditional chemical methods (such as spraying essential oil synthesis inhibitors) or physical means are insufficient to precisely and specifically regulate oil cell development when reducing this complex trait, and may also lead to non-target effects or environmental residues. Traditional breeding relies on natural variation, has a long selection cycle, and struggles to accurately control the complex trait of oil cell reduction.

[0004] CmPHL1, as an important regulatory factor, not only participates in the synthesis of carotenoids in fruits but also significantly affects the number of oil cells and the relative content of volatile substances in citrus leaves. Previous studies on citrus oil cells have focused primarily on their structure and physiological functions, with limited research on their formation and essential oil synthesis. This invention utilizes transcriptome sequencing of the 'Guanxi' pomelo and its bud variants to screen and functionally identify the transcription factor CmPHL1, systematically elucidating its biological function in citrus oil cells, aiming to provide new gene targets and theoretical basis for citrus quality improvement and breeding. Summary of the Invention

[0005] Based on the above, the purpose of this invention is to propose the application of CmPHL1 transcription factor in the inhibition of oil cell formation and regulation of volatiles in citrus leaves.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention proposes the use of the CmPHL1 transcription factor and its encoding gene in any of the following: (1) Application in negative regulation of oil cell formation in citrus leaves; (2) Application in negatively regulating the content of volatile substances in citrus leaves; (3) Application in citrus breeding with reduced oil cell count; (4) Application in cultivating citrus fruits with reduced oil cell count; The nucleotide sequence of the CmPHL1 gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the CmPHL1 gene is shown in SEQ ID NO.2.

[0007] Furthermore, the volatile substances include monoterpenes, sesquiterpenes, and aldehydes.

[0008] In a second aspect, the present invention provides a recombinant vector containing the CmPHL1 gene and the use of a host bacterium containing the recombinant vector in any of the following: (1) Application in negative regulation of oil cell formation in citrus leaves; (2) Application in negatively regulating the content of volatile substances in citrus leaves; (3) Application in citrus breeding with reduced oil cell count; (4) Application in cultivating citrus fruits with reduced oil cell count; The nucleotide sequence of the CmPHL1 gene is shown in SEQ ID NO.1.

[0009] Furthermore, the volatile substances include monoterpenes, sesquiterpenes, and aldehydes.

[0010] In a third aspect, the present invention proposes a breeding method for citrus with reduced oil cell count, comprising: (1) Construct an expression vector containing the CmPHL1 gene, the nucleotide sequence of which is shown in SEQ ID NO.1; (2) Introduce the carrier into citrus fruits; (3) Obtain transgenic citrus fruits overexpressing CmPHL1.

[0011] Compared with the prior art, the present invention has the following significant advantages: This invention has experimentally demonstrated that overexpression of CmPHL1 can achieve precise intervention in the trait of "oil cell number," avoiding the blindness and broad-spectrum side effects of traditional methods, and providing a new gene target for citrus quality improvement and breeding. By integrating CmPHL1 into the citrus genome through genetic transformation, its regulatory effect is continuous and heritable. Once a stable transgenic line is obtained or a similar effect is introduced through gene editing, its oil cell reduction phenotype can exist stably throughout the plant's life cycle and be passed on to offspring through reproduction, which has significant economic and ecological benefits. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0013] Figure 1 The procedure for the stable transformation of CmPHL1-OE in citrus is as follows: A: sterile seedling sowing; B: explant culture; C: epicotyl co-culture; D: callus induction; E: bud elongation culture; F: grafting.

[0014] Figure 2 To identify positive CmPHL1-OE transgenic citrus lines by PCR; where M: 2000 plus Marker; +: positive control; -: negative control; 1~9 represent 9 independent transgenic candidate citrus lines; primers: pGBI-CmPHL1-F, pGBI-CmPHL1-R.

[0015] Figure 3 To investigate the effect of CmPHL1 overexpression on the number of oil cells in citrus leaves.

[0016] Figure 4 To determine the number of oil cells per unit area in citrus leaves that overexpress CmPHL1.

[0017] Figure 5 To determine the volatile content in citrus leaves overexpressing CmPHL1. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be particularly noted that the following embodiments are for illustrative purposes only and do not limit the scope of the invention. Similarly, the following embodiments are only some, not all, embodiments of the present invention, and all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1: Construction of Citrus Genetic Transformation Vector (1) Cloning of the target gene Using cDNA from 'Zhangyi' Guanxi pomelo pulp as a template, primers were designed using Snapgene software. The primer sequences are shown in Table 1 below. The CmPHL1 gene was amplified using a high-fidelity enzyme, and the PCR system is shown in Table 2 below. After agarose gel electrophoresis, the PCR products were excised and recovered using the Tiangen DNA Recovery Kit according to the kit instructions. Finally, the DNA concentration and purity were measured, and the DNA was stored at -20℃ for subsequent experiments. The nucleotide sequence of the CmPHL1 gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the CmPHL1 gene is shown in SEQ ID NO.2.

[0020] Table 1 PCR Primers

[0021] Table 2 PCR reaction system

[0022] (2) pGBI vector double digestion The pGBI vector was double-digested using NEB endonucleases SacI and XbaI, as shown in Table 3 below. After the reaction, the samples were analyzed by agarose gel electrophoresis and then recovered from the gel.

[0023] Table 3. Vector double enzyme digestion reaction system

[0024] (3) Constructing the pGBI-CmPHL1 overexpression vector The purified transcription factors were recombined with the linearized pGBI vector using DNA ligase, and the reaction system is shown in Table 4 below. After the reaction, the vector was transformed into competent E. coli cells. The pGBI-CmPHL1 plasmid was extracted using the Tiangen Plasmid Mini-Prep Kit.

[0025] Table 4 Seamless Cloning Reaction System

[0026] Example 2: Agrobacterium-competent transformation Add 1.0 μL of the target plasmid to 100 μL of EHA105 competent cells. After ice bath, liquid nitrogen treatment, heat shock at 37°C, and another ice bath, the cells were revived in antibiotic-free LB liquid medium. After centrifugation, the cells were plated on LB agar plates containing Kans and Rif antibodies and incubated at 28°C for 2-3 days. Single colonies were picked for shake culture, and positive clones were verified by colony PCR.

[0027] Example 3: Agrobacterium infection and transformation of citrus (1) Explant preparation: Take late orange seeds, remove the seed coat after surface disinfection, and inoculate them on MS medium. First, culture in the dark for 15-20 days, then culture in the light for 3-5 days to obtain sterile seedling stem segments for later use.

[0028] (2) Agrobacterium infection: Activate recombinant Agrobacterium containing pGBI-CmPHL1 and expand the culture until the OD600 value of the bacterial solution is 0.5. Collect the bacterial cells and resuspend them in the activation solution. Soak the pre-cultured explants (cut into 1.5 cm pieces) in the bacterial solution for 10-15 min.

[0029] (3) Co-culture: After the infected explants are dried, they are transferred to co-culture medium and cultured in the dark for 3 days.

[0030] (4) Screening culture: After co-culture, the explants were transferred to a culture medium containing screening antibiotics and cultured in the dark until callus tissue was generated at the cut. Then, they were cultured in light with a photoperiod of 16 h / day.

[0031] (5) Bud elongation culture: After the adventitious buds generated at the cut site are initially confirmed to be positive by fluorescence detection (GFP), they are transferred to the bud elongation medium and cultured under the same light and temperature conditions.

[0032] (6) Grafting: Select healthy positive buds as scions and graft them onto one-year-old trifoliate orange rootstock. Routine management promotes survival and growth.

[0033] Example 4: Detection of transgenic positive lines Leaf DNA was extracted and subjected to PCR experiments to detect whether the target gene CmPHL1 was successfully transferred into the transgenic plant; leaf DNA was extracted and subjected to PCR amplification to detect whether the target gene was introduced; leaf RNA was extracted, reversed into cDNA, and subjected to qPCR experiments to detect the relative expression level of the transferred target gene CmPHL1.

[0034] Example 5: Determination of volatiles in citrus leaves 0.5 g of citrus leaf powder was added to 1 mL of saturated NaCl solution and 20 μL of 3-hexanone (internal standard), and placed in a 20 mL headspace vial for storage at -20℃. Before detection, the sample was brought to room temperature and dried, then placed in a 40℃ incubator for 30 min. Solid-phase microextraction (SPME, fiber tip: 50 / 30 μm DVB / CAR / PDMS) was performed at 40℃ for 60 min, followed by desorption at 250℃ in splitless mode for 5 min. The sample loading order was blank control (6 mL of saturated NaCl), alkane standard mixture (10 μL C8-C20, final concentration 10 mg / L; 6 mL of saturated NaCl), and the sample to be tested. GC-MS analysis was performed using a DB-5MS column, helium carrier gas flow rate of 1 mL / min, electron impact ion source (250℃, 70 eV), and mass scan range of 30–500 m / z. The specific temperature gradient is shown in Table 5 below.

[0035] Table 5. GC-MS column temperature gradient

[0036] Results and Analysis 1. Transgenic citrus cultivation and positive plant selection: Tissue culture seedlings of Late Jin Orange were cultured in the dark, and stem segments were cut and infected with Agrobacterium tumefaciens overexpression vector CmPHL1-OE. The specific procedure is as follows: Figure 1 Positive buds were initially screened by observing green fluorescence. These buds were then grafted onto one-year-old trifoliate orange trees and numbered for cultivation. Once new leaves emerged, the leaves were cut to extract DNA for final identification. Figure 2 These are PCR identification results of some strains, proving that they are positive transgenic strains overexpressing CmPHL1.

[0037] 2. Oil cell number determination: To investigate the effect of CmPHL1 on the number of oil cells in citrus leaves, the control group (EV) was compared with plants overexpressing CmPHL1. Morphological observation showed that the number of oil cells in the leaves of the overexpressing line was significantly less than that in the control group. Figure 3 As shown (scale bar = 50 mm), the oil cell distribution in the leaves of the overexpression lines (#1~#4) was significantly sparser, and the number was significantly less than that of the control lines (EV-1 to EV-3). Statistical analysis of the number of oil cells per unit area showed that overexpression of CmPHL1 significantly reduced the oil cell density in leaves. Compared with EV, the number of oil cells in all four overexpression lines (#1~#4) was significantly reduced, indicating that overexpression of CmPHL1 can inhibit the development of oil cells in citrus leaves.

[0038] 3. Determination of volatile matter content: Volatile matter spectroscopy analysis showed ( Figure 5 Compared to EV, the relative total volatile matter content of the four overexpression lines (#1~#4) decreased significantly, with a particularly pronounced decrease in the content of monoterpenes, the main components of citrus aroma. Sesquiterpenes showed a more significant reduction in #3 and #4. Furthermore, the content of aldehyde volatiles also showed a significant decreasing trend. These results indicate that CmPHLI inhibits the biosynthesis of terpenes volatiles by suppressing the development of oil cells in citrus leaves.

[0039] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made based on the content of the present invention specification and drawings, or direct or indirect application in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. The application of the CmPHL1 transcription factor and its encoding gene in any of the following: (1) Application in negative regulation of oil cell formation in citrus leaves; (2) Application in negatively regulating the content of volatile substances in citrus leaves; (3) Application in citrus breeding with reduced oil cell count; (4) Application in cultivating citrus fruits with reduced oil cell count; in, The nucleotide sequence of the CmPHL1 gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the CmPHL1 gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The volatile substances include monoterpenes, sesquiterpenes, and aldehydes.

3. The application of recombinant vectors containing the CmPHL1 gene and host bacteria containing such recombinant vectors in any of the following: (1) Application in negative regulation of oil cell formation in citrus leaves; (2) Application in negatively regulating the content of volatile substances in citrus leaves; (3) Application in citrus breeding with reduced oil cell count; (4) Application in cultivating citrus fruits with reduced oil cell count; in, The nucleotide sequence of the CmPHL1 gene is shown in SEQ ID NO.

1.

4. The application according to claim 1, characterized in that, The volatile substances include monoterpenes, sesquiterpenes, and aldehydes.

5. A breeding method for citrus with reduced oil cell count, characterized in that, include: (1) Construct an expression vector containing the CmPHL1 gene, the nucleotide sequence of which is shown in SEQ ID NO.1; (2) Introduce the carrier into citrus fruits; (3) Obtain transgenic citrus fruits overexpressing CmPHL1.

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