Method for regulating activity of a pyrus self-incompatible gene pps-rnase promoter

Abstract

The application discloses a method for regulating activity of a pear self-incompatibility gene PpS-RNase promoter and belongs to the field of plant genetic engineering. The application determines a 1284bp promoter sequence of the pear PpS-RNase gene, and verifies that the activity of the pear PpS-RNase gene promoter is induced by brassinosteroid in a stable transformation system of Arabidopsis thaliana, and the synthesis of endogenous brassinosteroid can be inhibited to significantly reduce the expression of the pear PpS-RNase gene, thereby providing a new idea for solving pear self-incompatibility mediated by the pear PpS-RNase gene and interspecific hybrid breeding.

Description

Technical Field

[0001] This invention relates to a method for regulating the activity of the PpS-RNase promoter, a self-incompatible gene in pear, and belongs to the field of plant genetic engineering. Background Technology

[0002] Pears belong to the genus *Pyrus* of the subfamily Maloideae in the family Rosaceae. Pears are typical gametophyte-incompatible fruit trees, requiring pollinator trees or assisted pollination to ensure yield, significantly increasing the difficulty and cost of pear orchard management. Developing innovative methods for self-fertile pear germplasm, changing the traditional cultivation model that necessitates pollinator trees in pear orchards, and simplifying production management are important research directions in pear production. Furthermore, distant hybridization is an important pathway for germplasm innovation, enriching the superior genes of species. Pears are closely related to plants in the genus *Malus*. Apples possess abundant genes for storage tolerance and red skin; transferring these genes into the pear genome can help obtain better pear varieties. Intergeneric hybridization between pears and apples is difficult, exhibiting incompatibility. This incompatibility is mainly manifested in low fruit set rates and poor survival rates of seedlings obtained from hybridization. Recent studies have found that suppressing self-incompatibility can achieve distant hybridization in plants in the genus *Brassica*. Therefore, suppressing the self-incompatibility reaction in pears and avoiding pollen tube death during intergeneric hybridization are effective ways to improve the success rate of intergeneric hybridization.

[0003] PpS-RNase is a key factor in determining self-incompatibility in pear pistils and is a crucial factor leading to the death of incompatible pollen tubes. The PpS-RNase protein possesses ribonuclease activity; when pollen tubes pass through the style guiding tissue, PpS-RNase can indiscriminately enter both self and non-self pollen tubes, inhibiting pollen tube growth by degrading rRNA in the pollen. Studies have shown that the growth behavior of pollen tubes in hybrid incompatibility and self-incompatibility is similar. Genes controlling self-incompatibility also play a role in controlling interspecific hybrid incompatibility; therefore, regulatory measures to overcome self-incompatibility are also effective in overcoming interspecific hybrid incompatibility. Currently, research on the regulatory mechanisms of the PpS-RNase gene in pear is scarce. Summary of the Invention

[0004] Objectives of this invention: The first objective of this invention is to provide a method for regulating the activity of the PpS-RNase promoter, a gene associated with pear self-incompatibility. The second objective of this invention is to provide a method and its application for overcoming pear self-incompatibility.

[0005] Technical solution: The present invention provides a method for regulating the activity of the self-incompatible gene PpS-RNase promoter in pear, wherein the method reduces 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 NO.2-3.

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

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

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

[0011] The brassinolide synthesis inhibitor is brassinoazole.

[0012] The concentration of brassinosteroids is 2.5–10 μM.

[0013] The present invention also provides the application of pear styles treated by the above method in pear breeding.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention determined the 1284bp promoter sequence of the pear PpS-RNase gene and verified in the Arabidopsis thaliana stable transformation system that the activity of the pear PpS-RNase gene promoter is induced by brassinolide. Inhibiting the synthesis of endogenous brassinolide can significantly reduce the expression of the pear PpS-RNase gene, providing a new approach to solving the self-incompatibility of pear mediated by the pear PpS-RNase gene and intergeneric hybridization breeding. Attached Figure Description

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

[0016] Figure 2 This study analyzed the relative expression levels of the PpS-RNase gene after treatment with different endogenous brassinolide synthesis inhibitors.

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

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

[0019] Figure 5These are the results of GUS protein staining in the PpS-RNase gene promoter in Arabidopsis thaliana. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0021] The plant materials used in this invention are: the pear is the "Cuiguan" sand pear variety, and the Arabidopsis is the Colombian wild type Arabidopsis. All plant materials used are preserved at the Yangzhou University Fruit Tree Experiment Base.

[0022] Example 1

[0023] Total DNA, total RNA, and cDNA synthesis from pear blossom columns: Total DNA was extracted from pear using the CTAB method. Total RNA was extracted from pear using the RNA Simple Total RNA Kit (Beijing Tiangen). The total pear RNA was converted into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Vazyme).

[0024] Example 2

[0025] Real-time quantitative PCR reaction: The PpS-RNase gene sequence was obtained from the pear genome database. Based on the pear PpS-RNase gene sequence, a pair of real-time 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). Using the cDNA prepared in Example 1 as a template, the reaction was performed using SsoAdvanced... TM GreenSupermix (Shanghai Bio-Rad) conducted quantitative real-time PCR experiments on a CFX96 Real-time PCR system to detect the expression level of the pear PpS-RNase gene at different developmental stages of the pistil (5 days and 1 day before flowering), and used Agilent 1200 high-performance liquid chromatography to detect changes in brassinolide content at the corresponding stages. In addition, the expression level of the PpS-RNase gene was detected after treatment with different concentrations of brassinolide synthesis inhibitors (brassinolide, Shanghai Aladdin Biochemical Technology Co., Ltd., B152405, at concentrations of 2.5 μM, 5 μM, and 10 μM). The brassinolide synthesis inhibitor treatment method was as follows: flowers 5 days before flowering were broken at the pedicel and immersed in the inhibitor solution, then cultured at room temperature for 2 days. PCR detection system: total 20 μl, 10 μl Green Supermix, 0.2 μl cDNA, 0.5 μl primers (10 μM), 9.3 μl sterile water. Program: 95℃ for 3 min, 95℃ for 10 s, 60℃ for 30 s, run the second and third steps 40 times.

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

[0027] Example 3

[0028]

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

[0030] Example 4

[0031] The PpS-RNase gene promoter was ligated into the pCAMBIA-2301 vector (Shanghai Zeye Biotechnology Co., Ltd.) using homologous recombination technology. Primers used included a forward primer: 5'-CAGTCTCAGAAGACCAAAGGGCAATAACCCTTCTCAGCCGA-3' (SEQ ID NO.6) and a reverse primer: 5'-ATCTACCATGGTCAAGAGTCCCCCGCCCCGTAATCCCTAGTGAAT-3' (SEQ ID NO.7). PCR amplification was performed in a 50 μL system: 25 μL 2×Phanta Max Master Mix (Novazia, Nanjing), 2 μL forward primer, 2 μL reverse primer, 1 μL DNA template, and 20 μL sterile water. The system was then placed in a PCR instrument for the following cycles: 95℃ for 3 min; 95℃ for 15 s; 57℃ for 15 s; 72℃ for 60 s, 35 cycles; 72℃ for 5 min. pPpS-RNase was obtained. pro Recombinant vector ( Figure 4 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 medium (1 / 2 MS medium, 5% sucrose, 0.05% Silwet-77, pH 7.0) until the OD600 reached 0.8. Arabidopsis styles were then immersed in the resuspension for 1 min. After infection, excess bacterial solution was gently patted off onto absorbent paper, and the cells were incubated upside down in the dark for 24 hours before normal incubation.

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

Claims

1. A method for overcoming self-incompatibility in pears, characterized in that, By reducing the brassinolide content in the pear style, PpS-RNase The activation of the promoter is used to overcome pear self-incompatibility. PpS-RNase The nucleotide sequence of the promoter is shown in SEQ ID NO.1; The method for reducing the brassinolide content in the pear style includes removing the pear style and placing it in deionized water containing a brassinolide synthesis inhibitor, and culturing it at room temperature for 2 days. The brassinolide synthesis inhibitor is brassinoazole.

2. The method according to claim 1, characterized in that, The style is 5-1 days before flowering.

3. The method according to claim 1, characterized in that, The concentration of brassinosteroids is 2.5~10 μM.

4. The pear style treated by any one of claims 1 to 3 is used in pear breeding to overcome self-incompatibility.

Images