A method for rapid induction of citrus juice sacs in vitro lignification and application thereof

Abstract

The application discloses a controllable, rapid and highly-repetitive in-vitro lignification induction method of citrus juice sacs, and provides application of the method in screening of substances for inhibiting lignification / granulation regulation and evaluation of lignification sensitivity of different varieties or tissues. The method comprises the following steps: step 1, taking citrus fruit locule tissues to obtain juice sac samples; and step 2, culturing the juice sac samples obtained in step 1 in a mineral element treatment liquid for 6-72 hours. The mineral element treatment liquid comprises one or more of CaCl2 solution, MnSO4 solution, Fe-EDTA solution and ZnSO4 solution. Compared with the prior method which relies on field observation or long-period storage observation, the method has the advantages of rapid induction speed, high repeatability, low sample requirement, and can be used for screening of substances for inhibiting lignification / granulation regulation and evaluation of lignification sensitivity of varieties / tissues.

Description

Technical Field

[0001] This invention belongs to the field of fruit quality regulation and physiological disorder mechanism research, specifically involving a method for rapidly constructing a juice cell lignification model by in vitro induction of citrus fruit juice cells with mineral element solution, and the application of this model in screening regulators and evaluating sensitivity. Background Technology

[0002] Citrus fruits (such as pomelos and tartaries) are prone to physiological disorders such as juice cell granulation and textural deterioration during development or storage, one of the typical characteristics of which is cell wall thickening and enhanced lignin deposition. Existing research and evaluation methods largely rely on field fertilization / environmental differences or long-term storage observations, which suffer from problems such as long cycles, significant environmental interference, insufficient reproducibility, and difficulty in high-throughput screening. Therefore, there is an urgent need for an in vitro model method that can rapidly and stably induce and quantitatively assess juice cell lignification under controlled conditions for mechanistic research and screening of regulatory strategies. Summary of the Invention

[0003] This invention provides a controllable, rapid, and highly reproducible method for inducing lignification in citrus juice cells in vitro, and provides its application in screening substances that inhibit lignification / granulation and evaluating the lignification sensitivity of different varieties or tissues.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A rapid induction method for in vitro lignification of citrus juice cells includes the following steps: Step 1: Collect juice vesicle tissue from citrus fruit segments to obtain juice vesicle samples; Step 2: Place the sac sample from Step 1 in a mineral element treatment solution for in vitro culture for 6–72 hours; the mineral element treatment solution includes one or more of CaCl2 solution, MnSO4 solution, Fe-EDTA solution and ZnSO4 solution.

[0006] Furthermore, the concentration of the CaCl2 solution is 0.5–50 mM, the concentration of the MnSO4 solution is 0.5–20 mM, the concentration of the Fe-EDTA solution is 50–500 μM, and the concentration of the ZnSO4 solution is 50–1000 μM.

[0007] Furthermore, the concentration of the CaCl2 solution is 25 mM, the concentration of the MnSO4 solution is 10 mM, the concentration of the Fe-EDTA solution is 300 μM, and the concentration of the ZnSO4 solution is 500 μM.

[0008] Furthermore, the in vitro culture in step 2 is carried out at 20–30 °C; the culture system is a sterile water system or a culture system containing basic salts.

[0009] Furthermore, step 3 is included: detecting changes in the expression of lignin synthesis-related marker genes during 6–24 hours of culture, or detecting changes in sap lignin content during 24–72 hours of culture.

[0010] Furthermore, in step 3, the sampling time for gene expression detection is 12 h after culture, and the sampling time for lignin content detection is 48 h after culture. The marker genes include lignin synthesis-related marker genes and ion homeostasis / signal-related marker genes. The lignin synthesis-related marker genes include 4CL1, LAC5, PAL2, PAL3, and PAL5; the ion homeostasis / signal-related marker genes include CML3, NRAMP1, ZIP5, HIPP05, and YSL3.

[0011] Furthermore, in step 1, the juice cell sample is dried in a 60 ℃ forced-air drying oven until constant weight.

[0012] The above-mentioned rapid induction method for in vitro lignification of citrus juice cells is applied to the rapid screening of candidate regulators that inhibit lignification / granulation. Specifically, the test substance and the mineral element treatment solution are applied together to the juice cell sample, and the inhibitory effect of the test substance on the upregulation of marker gene expression and / or the increase in lignin content is used as the indicator for screening candidate regulators.

[0013] The above-mentioned rapid induction method for lignification of citrus juice cells in vitro is applied to evaluate the lignification sensitivity of citrus varieties or tissues. Specifically, after rapid induction of lignification of different test samples in vitro, the expression intensity of marker genes or the intensity of lignin accumulation of the corresponding induction products of different test samples are compared to determine the differences in lignification sensitivity of different test samples.

[0014] A kit for constructing a rapid induction model of citrus juice cell lignification in vitro includes a mineral element treatment solution and a primer set for marker gene detection or a reagent for lignin detection. The mineral element treatment solution includes one or more of CaCl2 solution, MnSO4 solution, Fe-EDTA solution, and ZnSO4 solution. The marker genes include lignin synthesis-related marker genes and ion homeostasis / signal-related marker genes. The lignin synthesis-related marker genes include 4CL1, LAC5, PAL2, PAL3, and PAL5; the ion homeostasis / signal-related marker genes include CML3, NRAMP1, ZIP5, HIPP05, and YSL3.

[0015] The beneficial effects of this invention are as follows:

[0016] The induction method of this invention can significantly upregulate lignin biosynthesis-related genes such as 4CL1, LAC5, and PAL2 / 3 / 5 in citrus fruits and increase lignin content in juice sacs. ZnSO4 treatment can induce the expression of transport genes such as ZIP5, but has a weaker effect on lignin content. Compared with existing methods that rely on field or long-term storage observation, this invention has the advantages of rapid induction, high reproducibility, low sample requirements, and can be used to screen regulatory substances that inhibit lignification / granulation and assess the lignification sensitivity of varieties / tissues. The specific advantages are described below:

[0017] 1. Fast speed: A clear molecular response signal can be obtained within 12 hours, and the difference in lignin accumulation can be obtained within 24–72 hours;

[0018] 2. High controllability and repeatability: The in vitro system reduces interference from field environment and individual fruit differences;

[0019] 3. Wide range of applications: It can be used for rapid screening of candidate regulators that inhibit lignification / granulation, and can also be used to compare the lignification sensitivity of different varieties and tissues;

[0020] 4. Clear indicator system: 4CL1, LAC5, PAL2 / 3 / 5, etc. can be used as marker genes for lignin synthesis, and combined with lignin content to form a "molecular-phenotype" dual indicator.

[0021] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0022] Figure 1 : Changes in the relative expression levels of cellular marker genes after in vitro treatment with solutions containing different mineral elements;

[0023] Figure 2 Graph showing the changes in lignin content in sap cells after in vitro treatment with solutions of different mineral elements; Figure 3 Photographs of the experimental process. Detailed Implementation

[0024] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0025] This invention employs a mineral element solution (including at least one of CaCl2, MnSO4, Fe-EDTA, and ZnSO4) to treat sap sac tissue in vitro. By detecting changes in the expression of lignin synthesis-related marker genes within 6–24 h and changes in lignin content within 24–72 h, rapid induction and quantitative evaluation of the sap sac lignification process are achieved. Preferred concentrations are: CaCl2 25 mM, MnSO4 10 mM, Fe-EDTA 300 μM, and ZnSO4 500 μM; preferred sampling times are: 12 h for qPCR and 48 h for lignin content determination.

[0026] Example 1

[0027] Fruits of the Sanhong Honey Pomelo variety with uniform maturity were selected, and the segments were removed to separate the juice vesicle tissue. CaCl2 treatment solution (25 mM), MnSO4 treatment solution (10 mM), Fe-EDTA treatment solution (300 μM), and ZnSO4 treatment solution (500 μM) were prepared separately, using sterile water or a basal culture system as solvents; an equal volume of sterile water was used as a control (CK). The juice vesicle samples were placed in the above treatment solutions and cultured in vitro at 25 ℃.

[0028] After 12 h of in vitro culture, samples were collected, RNA was extracted, and cDNA was obtained by reverse transcription. The relative expression levels of marker genes were detected using real-time quantitative PCR. The results showed that CaCl2 and MnSO4 treatment significantly upregulated lignin synthesis-related genes 4CL1, LAC5, and PAL2 / 3 / 5, accompanied by increased expression of CaCl2 and MnSO4. 2+ The expression of signal-related genes such as CML3 and ion transport-related genes such as NRAMP1 was enhanced; ZnSO4 treatment induced the expression of transport genes such as ZIP5; Fe-EDTA treatment had a certain inducing effect on some lignin synthesis genes. This demonstrates that mineral element treatment can trigger a response in the molecular network related to sap cell lignification (corresponding to...) within a short period of time. Figure 1 ).

[0029] After 48 h of in vitro culture, samples were collected, and the sap cells were dried or freeze-dried to constant weight. The lignin content was then determined using a quantitative lignin method. Results showed that the lignin content in the CaCl2, MnSO4, and Fe-EDTA treatment groups was significantly higher than the control, while the lignin content in the ZnSO4 treatment group showed no significant difference (corresponding to...). Figure 2 The above results demonstrate that the method of the present invention can stably induce and quantitatively evaluate sap cell lignification within a short period of time.

[0030] The above description provides examples of the preferred embodiments of the present invention. Parts not detailed herein are common knowledge to those skilled in the art. The scope of protection of the present invention is determined by the claims, and any equivalent modifications based on the technical teachings of the present invention are also within the scope of protection of the present invention.

Claims

1. A method for rapid induction of lignification in citrus juice cells in vitro, characterized in that, Includes the following steps: Step 1: Collect juice vesicle tissue from citrus fruit segments to obtain juice vesicle samples; Step 2: Place the sac sample from Step 1 in a mineral element treatment solution for in vitro culture for 6–72 hours; the mineral element treatment solution includes one or more of CaCl2 solution, MnSO4 solution, Fe-EDTA solution and ZnSO4 solution.

2. The method for rapid induction of citrus juice cell lignification in vitro according to claim 1, characterized in that, The concentration of the CaCl2 solution is 0.5–50 mM, the concentration of the MnSO4 solution is 0.5–20 mM, the concentration of the Fe-EDTA solution is 50–500 μM, and the concentration of the ZnSO4 solution is 50–1000 μM.

3. The method for rapid induction of citrus juice cell lignification in vitro according to claim 2, characterized in that, The concentration of the CaCl2 solution was 25 mM, the concentration of the MnSO4 solution was 10 mM, the concentration of the Fe-EDTA solution was 300 μM, and the concentration of the ZnSO4 solution was 500 μM.

4. The method for rapid induction of citrus juice cell lignification in vitro according to claim 1, characterized in that, The in vitro culture in step 2 is carried out at 20–30 ℃; the culture system is a sterile water system or a culture system containing basic salts.

5. The method for rapid induction of citrus juice cell lignification in vitro according to claim 1, characterized in that, It also includes step 3: detecting changes in the expression of lignin synthesis-related marker genes during 6–24 hours of culture, or detecting changes in sap lignin content during 24–72 hours of culture.

6. The method for rapid induction of citrus juice cell lignification in vitro according to claim 5, characterized in that, In step 3, the sampling time for gene expression detection is 12 h after culture, and the sampling time for lignin content detection is 48 h after culture. The marker genes include lignin synthesis-related marker genes and ion homeostasis / signal-related marker genes. The lignin synthesis-related marker genes include... 4CL1, LAC5, PAL2, PAL3 and PAL5 The ion homeostasis / signal-related marker genes include CML3, NRAMP1, ZIP5, HIPP05 and YSL3 .

7. The method for rapid induction of citrus juice cell lignification in vitro according to claim 1, characterized in that, In step 1, the juice cell sample is dried in a 60 ℃ forced-air drying oven until constant weight.

8. The application of the rapid induction method for in vitro lignification of citrus juice cells according to any one of claims 1-7 in the rapid screening of candidate regulators that inhibit lignification / granulation, characterized in that, Specifically, the test substance and the mineral element treatment solution are applied together to the juice cell sample, and the inhibitory effect of the test substance on the upregulation of marker gene expression and / or the increase in lignin content is used as an indicator for screening candidate regulators.

9. The application of the rapid induction method for in vitro lignification of citrus juice cells according to any one of claims 1-7 in evaluating the lignification sensitivity of citrus varieties or tissues, characterized in that, Specifically, after rapidly inducing lignification in citrus juice cells in different test samples, the expression intensity of marker genes or the intensity of lignin accumulation of the corresponding induction products of different test samples were compared to determine the differences in lignification sensitivity among different test samples.

10. A kit for constructing a rapid induction model of citrus juice cell lignification in vitro, characterized in that, The system includes a mineral element treatment solution and a primer set for marker gene detection or a reagent for lignin detection. The mineral element treatment solution includes one or more of CaCl2 solution, MnSO4 solution, Fe-EDTA solution, and ZnSO4 solution. The marker genes include lignin synthesis-related marker genes and ion homeostasis / signal-related marker genes. The lignin synthesis-related marker genes include... 4CL1, LAC5, PAL2, PAL3 and PAL5 The ion homeostasis / signal-related marker genes include CML3, NRAMP1 ZIP5, HIPP05 and YSL3 .

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