Activity regulation and control method of pear self-incompatible gene PpS-RNase promoter

Abstract

The invention discloses an activity regulation and control method of a pear self-incompatible gene PpS-RNase promoter, and belongs to the field of plant genetic engineering. According to the invention, a 1284bp promoter sequence of the pear PpS-RNase gene is determined, the fact that the activity of the pear PpS-RNase gene promoter is induced by brassinolide is verified in an arabidopsis thaliana stable transformation system, and the expression of the pear PpS-RNase gene can be obviously reduced by inhibiting the synthesis of endogenous brassinolide; a new thought is provided for solving the problem of self-incompatibility and intergeneric cross breeding of the pear PpS-RNase gene mediation.

Description

Technical Field

[0001] The invention relates to an activity regulation method for a pear self-incompatibility gene PpS-RNase promoter, and belongs to the field of plant genetic engineering. Background Art

[0002] Pear is a plant of the genus Pyrus in the subfamily Apple of the Rosaceae family. Pear is a typical self-incompatible gametophyte fruit tree. Pollination trees or assisted pollination must be configured to ensure production, which greatly increases the difficulty and cost of pear orchard management. Inventing a method for self-fertile pear germplasm innovation, changing the traditional cultivation mode that pollination trees must be configured in pear orchards, and simplifying production management are important research directions in pear production. In addition, distant hybridization is an important way to innovate germplasm and can enrich the excellent genes of species. Pears are closely related to plants of the genus Apple. Apple has abundant genes such as storage resistance and red skin. Transferring these genes into the pear genome will help obtain better pear varieties. Inter-generic hybridization between pears and apples is difficult, which is manifested as inter-generic hybridization incompatibility. The inter-generic hybridization incompatibility between pears and apples is mainly manifested in low hybrid fruit setting rate and difficult survival of seedlings obtained from hybridization. Recent studies have found that inhibiting self-incompatibility reactions can achieve distant hybridization of Brassica plants. Therefore, inhibiting the self-incompatibility reaction of pear and avoiding the death of pollen tubes in intergeneric hybridization are effective ways to improve the success rate of intergeneric hybridization.

[0003] PpS-RNase is the pistil-determining factor of self-incompatibility in pear and the key factor leading to the death of incompatible pollen tubes. PpS-RNase protein has ribonuclease activity. When the pollen tube passes through the style guiding tissue, PpS-RNase can indiscriminately enter the self-or non-self pollen tube and inhibit the growth of the pollen tube by degrading the rRNA in the pollen. Studies have shown that the growth performance of pollen tubes in hybrid incompatibility and self-incompatibility is similar. The genes that control self-incompatibility also play a role in controlling interspecific hybrid incompatibility. Therefore, regulatory measures to break self-incompatibility are also effective in overcoming interspecific hybrid incompatibility. At present, there are few reports on the regulatory measures of PpS-RNase gene in pear. Summary of the invention

[0004] Purpose of the invention: The first purpose of the invention is to provide a method for regulating the activity of the promoter of the pear self-incompatibility gene PpS-RNase. The second purpose of the invention is to provide a method and application for overcoming the pear self-incompatibility.

[0005] Technical solution: The present invention provides a method for regulating the activity of a pear self-incompatibility gene PpS-RNase promoter, wherein the method is to reduce the activity of the PpS-RNase promoter by inhibiting the synthesis of endogenous brassinolide.

[0006] Furthermore, the nucleotide sequence of the pear self-incompatibility gene PpS-RNase promoter is shown in SEQ ID NO.1.

[0007] Furthermore, the primer sequences for identifying the pear self-incompatibility gene PpS-RNase are shown in SEQ ID NOs. 2-3.

[0008] The invention also provides a method for overcoming the self-incompatibility of pears, and the self-incompatibility of pears is overcome by reducing the brassinolide content in the pear style.

[0009] The method for reducing the brassinolide content in the pear style comprises removing the pear style, placing the style in deionized water containing a brassinolide synthesis inhibitor, and culturing at room temperature for 2 days.

[0010] The style is 5 to 1 day before flowering.

[0011] The brassinolide synthesis inhibitor is brassinolide.

[0012] The concentration of brassinolide is 2.5-10 μM.

[0013] The present invention also provides the use of the pear style treated by the method in pear breeding.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention determines the 1284bp promoter sequence of the pear PpS-RNase gene, and verifies in the Arabidopsis stable transformation system that the activity of the pear PpS-RNase gene promoter is induced by brassinolide, and inhibiting the synthesis of endogenous brassinolide can significantly reduce the expression of the pear PpS-RNase gene, providing a new idea for solving the pear self-incompatibility mediated by the pear PpS-RNase gene and inter-generic hybrid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The relative expression analysis of endogenous brassinolide (A) and PpS-RNase gene at different developmental stages of pear style (B);

[0016] Figure 2 is the analysis of the relative expression of PpS-RNase gene after treatment with different endogenous brassinolide synthesis inhibitors;

[0017] Figure 3 It is a schematic diagram of the cis-acting element in the promoter of the PpS-RNase gene that responds to brassinolide;

[0018] Figure 4 is a schematic diagram of the reporter gene vector;

[0019] Figure 5This is the result of GUS protein staining of the PpS-RNase gene promoter in Arabidopsis. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0021] The plant materials used in the present invention are as follows: the pear is the “Cui Guan” sand pear variety, and the Arabidopsis thaliana is the Columbia wild-type Arabidopsis thaliana. The plant materials used are all stored in the fruit tree experimental base of Yangzhou University.

[0022] Example 1

[0023] Extraction of total DNA, total RNA and cDNA synthesis from pear stigma: Total DNA of pear was extracted by CTAB method. Total RNA of pear was extracted by RNA simple Total RNA Kit (Tiangen, Beijing). Total RNA of pear was converted into cDNA by HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing).

[0024] Example 2

[0025] Real-time quantitative PCR reaction: The PpS-RNase gene sequence was obtained from the genome database of white pear. Based on the sequence of the PpS-RNase gene of pear, a pair of real-time fluorescence quantitative PCR primers were designed using Primer 5.0 software: forward primer: 5'-ACATGGCTCCTGTGGGTATC-3' (SEQ ID NO.2), reverse primer: 5'-GTCCGGTTCAATCTTCGCCT-3' (SEQ ID NO.3). The cDNA prepared by the method in Example 1 was used as a template and the SsoAdvanced TM GreenSupermix (Shanghai Bio-Rad Company) was used to perform fluorescence quantitative PCR experiments on the CFX96 Real-time PCR system to detect the expression level of the pear PpS-RNase gene at different developmental stages of the style (5 days and 1 day before flowering), and the Agilent1200 high performance liquid chromatography was used to detect the changes in the brassinolide content in the corresponding period. In addition, the expression level of the PpS-RNase gene after treatment with different concentrations of brassinolide synthesis inhibitors (brassinolide, Shanghai Aladdin Biochemical Technology Co., Ltd., B152405, concentrations of 2.5μM, 5μM and 10μM) was also detected. The method for treating with brassinolide synthesis inhibitors is: break the flowers 5 days before flowering at the pedicel, soak them in the inhibitor solution, and culture them at room temperature for 2 days. PCR detection system: total 20μl, 10μl Green Supermix, 0.2 μl cDNA, 0.5 μl primers (10 μM), 9.3 μl sterile water. Program: 95°C for 3 min, 95°C for 10 s, 60°C for 30 s, 40 cycles of the second and third steps.

[0026] The results are as follows Figure 1 and 2 As shown, it was shown that the expression of PpS-RNase gene was induced by brassinolide, and inhibiting the synthesis of brassinolide reduced its expression level.

[0027] Example 3

[0028]

[0029] Sequence analysis: The PpS-RNase promoter is 1284 bp upstream of the transcription start site of the PpS-RNase gene. The sequence of the PpS-RNase gene promoter was obtained by sequencing, and the cis-acting elements were analyzed by plantPAN4.0. The results showed that the PpS-RNase gene promoter contained multiple E-box elements ( Figure 3 ), which is the binding element of BRZ1, an important transcription factor induced by brassinolide signal.

[0030] Example 4

[0031] The PpS-RNase gene promoter was connected to the pCAMBIA-2301 vector (Shanghai Zeye Biotechnology Co., Ltd.) using homologous recombination technology. The primers used included forward primer: 5'-CAGTCTCAGAAGACCAAAGGGCAATAACCCTTCTCAGCCGA-3' (SEQ ID NO.6), reverse primer: 5'-ATCTACCATGGTCAAGAGTCCCCCGCCCCGTAATCCCTAGTGAAT-3' (SEQ ID NO.7), PCR amplification reaction: 50 μL system: 25 μl 2×Phanta Max Master Mix (Nuoweizan, Nanjing), 2 μl forward primer, 2 μl reverse primer, 1 μl DNA template, 20 μl sterile water. Then placed in a PCR instrument: 95°C 3min; 95°C 15s; 57°C 15s; 72°C 60s, 35 cycles; 72°C 5min. pPpS-RNase was obtained. pro Recombinant vector ( Figure 4 ), and the recombinant vector was transformed into Agrobacterium GV3101. The transformed Agrobacterium was shaken on a shaker at 30°C, and the cells were collected by centrifugation. The cells were resuspended in infection solution (1 / 2MS medium, 5% sucrose, 0.05% Silwet-77, pH7.0) to an OD600 of 0.8, and the Arabidopsis style was immersed in the resuspension solution for 1 minute. After the infection, it was placed on absorbent paper and gently wiped off the excess bacterial solution, turned upside down, and cultured normally in the dark for 24 hours.

[0032] Identification of transgenic Arabidopsis, hormone treatment and GUS protein staining: DNA was extracted from the transformed Arabidopsis, and PCR amplification was performed using primers of SEQ ID NO.4 and SEQ ID NO.5. PCR amplification reaction: 25 μL system: 12.5 μl 2×PhantaMax Master Mix (Novozymes, Nanjing), 1 μl forward primer, 1 μl reverse primer, 0.5 μl DNA template, 10 μl sterile water. Then placed in a PCR instrument: 95℃3min; 95℃15s; 57℃15s; 72℃60s, 35 cycles; 72℃5min. After obtaining positive plants, Arabidopsis containing the PpS-RNase gene promoter after about 20 days of normal culture was treated with 5 μM brassinolide and stained with GUS staining solution. The control was wild Arabidopsis ( Figure 5 ). The results showed that brassinolide induced the increase of PpS-RNase gene promoter activity.

Claims

1. A method for regulating the activity of the pear self-incompatibility gene PpS-RNase promoter, characterized in that: The method is to reduce the activity of the PpS-RNase promoter by inhibiting the synthesis of endogenous brassinolide.

2. The method for regulating the activity of the pear self-incompatibility gene PpS-RNase promoter according to claim 1, characterized in that: The nucleotide sequence of the pear self-incompatibility gene PpS-RNase promoter is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The primer sequences for identifying the pear self-incompatibility gene PpS-RNase are shown in SEQ ID NOs. 2-3.

4. A method for overcoming self-incompatibility of pear, characterized in that: Overcoming pear self-incompatibility by reducing the brassinolide content in the pear style.

5. The method according to claim 4, characterized in that The method for reducing the brassinolide content in the pear style comprises removing the pear style, placing the style in deionized water containing a brassinolide synthesis inhibitor, and culturing at room temperature for 2 days.

6. The method according to claim 5, characterized in that The style is 5 to 1 day before flowering.

7. The method according to claim 5, characterized in that The brassinolide synthesis inhibitor is brassinolide.

8. The method according to claim 7, characterized in that The concentration of brassinolide is 2.5-10 μM.

9. Use of the pear style treated by the method according to any one of claims 4 to 8 in pear breeding.

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