Apple stress resistance gene mdpp13-like-1 and application thereof

Abstract

An apple stress-resistant gene MdRPP13-like-1 and its applications, the MdRPP13-like-1 The nucleotide sequence of the gene is shown in SEQ.ID.NO.1. Using transgenic technology driven by the 35S strong promoter, the gene will... MdRPP13-like-1 Gene overexpression vectors were transferred into apples to obtain transgenic apples. This invention is the first to improve plant resistance to continuous cropping using plant genetic engineering technology. A complete coding segment of a disease resistance-related gene was isolated and cloned from apple rootstock, and the function of this gene was verified. Its function was ultimately used to discover the optimal use of [the specific technology / method / approach]. MdRPP13-like-1 MdRPP13-like-1 Transgenic plants that were overexpressed in apples showed significantly improved resistance to continuous cropping.

Description

Technical Field

[0001] This invention relates to a stress-resistance gene and its application, specifically an apple stress-resistance-related gene. MdRPP13-like-1 And its applications. Background Technology

[0002] Continuous cropping obstacle in apple orchards is a common disease that occurs when re-establishing orchards after the clearing of old ones. It leads to slow growth in young apple trees, inhibits root development, reduces yield, and can even cause the death of the entire tree, shortening the lifespan of the new orchard. In recent years, with the optimization of apple tree varieties and the increasing frequency of orchard renewal, continuous cropping obstacle has become more prominent. The main factors contributing to continuous cropping obstacle in older apple orchards include the presence of Fusarium spp. and Mosporum spp. fungi in the soil, soil organic matter content, and phlorizin content. Due to the numerous factors causing continuous cropping obstacle, variations in planting areas and orchard conditions, and the existence of many uncertainties even within the same region, resistant rootstocks are generally considered the most promising method for controlling continuous cropping obstacle. However, due to the complexity of fruit tree genetic backgrounds and the fact that stress resistance traits are generally controlled by multiple genes, the screening of genes for resistance to continuous cropping in apples is relatively lagging. Therefore, it is urgent to research a stress-resistance gene in fruit trees that can play a key role in improving the resistance of apples under continuous cropping stress. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide an apple replanting resistance gene. MdRPP13-like-1 And its application in transgenic apples resistant to continuous cropping obstacles.

[0004] The technical solution adopted by this invention to solve its technical problem is that an apple replant resistance gene is a DNA fragment of the complete coding region of a replant resistance-related gene isolated and cloned from apple resistant rootstocks, and named... MdRPP13-like-1 Its nucleotide sequence is shown in SEQ.ID.NO.1, and its protein amino acid sequence is shown in SEQ.ID.NO.2.

[0005] The technical solution adopted by the present invention to further solve its technical problem is an apple MdRPP13-like-1 The gene preparation method includes the following steps: (1) RNA extraction and reverse transcription from the roots of apple-resistant rootstock No. 6; (2) Obtaining the full-length cDNA sequence: Based on the cDNA sequence found in apples using GDR... MdRPP13-like-1 Gene sequence, design specific primers MdRPP13-like-1 -F, MdRPP13-like-1 -R, and then PCR amplification was performed using the cDNA synthesized from the root system of tobacco rootstock 6 via reverse transcription as a template to obtain the full-length cDNA sequence; among which, MdRPP13-like-1 The -F sequence is shown in SEQ ID NO. 3. MdRPP13-like-1 -R sequences are shown in SEQ. ID. NO.4; (3) The PCR products were recovered, ligated into vectors, transformed, and sequenced. MdRPP13-like-1 Gene, MdRPP13- like-1 The gene's open reading frame (ORF) is 3786 bp and encodes 1262 amino acids.

[0006] Furthermore, in step (3), the PCR amplification reaction system is as follows: 10×PCR contains buffer (containing Mg) 2+ 2.5μl 2.5 mM / L dNTP 2.0 μl, 10μM / L MdRPP13-like-1 1.5 μl of forward primer, 10μM / l MdRPP13-like-1 1.5 μl of reverse primer Template cDNA 2.0 μl, LA Taq enzyme 0.3 μl, Add ddH2O to a final volume of 25.0 μl; The PCR reaction program was 94℃ pre-denaturation for 5 min; the cycling parameters were 94℃ pre-denaturation for 3 min; 94℃ denaturation for 45 sec; 56.5℃ annealing for 1 min; 72℃ extension for 2 min, 35 cycles; 72℃ extension for 10 min.

[0007] The technical solution adopted by this invention to further solve its technical problem is an apple stress-resistance gene. MdRPP13- like-1 In the application of genetically modified apples, the transgenic technology, driven by a strong promoter (cauliflower mosaic virus 35S promoter), will... MdRPP13-like-1 Gene overexpression vectors were transferred into apples to obtain transgenic apple plants. Experiments have shown that overexpression... MdRPP13-like-1 Transgenic apples showed significantly less damage under stress from the Fusarium moniliforme strain MR5 compared to wild-type apples, indicating that... MdRPP13-like-1 Genes play an important role in plant resistance to continuous cropping stress.

[0008] In summary, this invention is the first to improve the ability of plants to withstand continuous cropping and other beneficial production traits through plant genetic engineering technology. A DNA fragment of the complete coding region of the stress-resistance-related gene was isolated and cloned from the root system of apple resistant rootstock, and the function of the gene was verified. Using its function, it was finally found that the ability of transgenic plants to withstand continuous cropping was significantly improved after overexpression. Attached Figure Description

[0009] Figure 1 For stress-resistant genes MdRPP13-like-1 The effect of overexpression on replant tolerance in apple trees was investigated. Wild-type and transgenic apple trees with uniform growth were treated with the apple replant tolerance fungus MR5. After 14 days of treatment, plant growth was observed, and various phenotypic indices were measured. (A) represents WT and... MdRPP13-like-1 -OE apple plants MdRPP13-like-1 level of expression 。 (B) Effects of control (WT) and transgenic lines OE1-OE3 on aboveground plant height (B), root length (C), aboveground fresh weight (D), root fresh weight (E), and root area (F) after 14 days of stress from the replanting strain MR5.

[0010] Figure 2 The effects of MdRPP13-like-1 gene overexpression on physiological indicators in apple plants under 14 days of continuous cropping stress were investigated. (A) shows the H2O2 content in the aboveground parts of the three transgenic lines; (B) shows the O2 content in the aboveground parts of the three transgenic lines. - (C) is the MDA content in the aboveground parts of the three transgenic plant lines; (E) is the H2O2 content in the roots of the three transgenic plant lines; (F) is the O2 content in the roots and aboveground parts of the three transgenic plant lines. - Generation rate; (G) MDA content in the roots of the three transgenic lines; (H) MdCAT gene expression determined by qRT-PCR; (I) MdSOD gene expression determined by qRT-PCR; (J) MdPOD gene expression determined by qRT-PCR. Detailed Implementation

[0011] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0012] Example 1: Apple MdRPP13-like-1 Cloning of genes

[0013] 1. Total RNA extraction using the CTAB method: Total RNA was extracted from plants using the RNAprep Pure Plant Plus Kit (DP441) from Tiangen Biotech. The procedure is as follows: (1) Take 0.05 g of plant material and grind it into powder quickly in an RNase-free mortar with liquid nitrogen, then transfer it to a 1.5 mL RNase-free centrifuge tube; (2) Add 500 μL of lysis buffer SL and immediately vortex vigorously to mix, then centrifuge at 12,000 rpm for 2 min; (3) Transfer the supernatant to a filter CS, centrifuge at 12,000 rpm for 2 min, and carefully aspirate the supernatant from the collection tube into a new 1.5 mL RNase-Free centrifuge tube. Avoid contact between the pipette tip and the cell debris in the collection tube as much as possible. (4) Slowly add 0.4 times the volume of the supernatant of anhydrous ethanol, mix well (a precipitate may appear at this time), transfer the obtained solution and precipitate together into the adsorption column CR3, centrifuge at 12,000 rpm for 15 seconds, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube. (5) Add 350 μL of protein removal solution RW1, centrifuge at 12,000 rpm for 15 seconds, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube; (6) Add 80 μL of DNase I working solution and let stand at room temperature for 15 min; (7) Add 350 μL of protein removal solution RW1, centrifuge at 12,000 rpm for 15 seconds, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube; (8) Add 500 μL of washing buffer RW, centrifuge at 12,000 rpm for 15 seconds, discard the waste liquid in the collection tube, put the adsorption column CR3 back into the collection tube, and repeat once; (10) Centrifuge at 12,000 rpm for 2 min, place the adsorption column into a new RNase-Free centrifuge tube, add 40 μL of RNase-Free ddH2O to the center of the adsorption membrane, place at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min to obtain the RNA solution.

[0014] The obtained RNA solution was added to the adsorption column CR3, incubated at room temperature for 2 min, centrifuged at 12000 rpm for 1 min to obtain the RNA solution, and stored at -80 ℃ for later use.

[0015] 2. Synthesis of the first strand of reverse-transcribed cDNA First-strand cDNA synthesis was performed using the FastQuant RT Kit (with gDNsae) (KR106) from Tiangen Biotech, as follows: All the following operations were performed on ice. To ensure the accuracy of the reaction solution preparation, each reaction was first prepared as a mix and then dispensed into individual reaction tubes.

[0016] (1) Prepare the mixture for the reaction system to remove genomic DNA as follows, centrifuge briefly, and incubate at 42 °C for 3 min; gDNA removal reaction system (2) Prepare the mixture according to the following reverse transcription reaction system, and add it to the reaction solution of the gDNA removal step, and mix thoroughly; reverse transcription reaction system Composition Usage 10×Fast RT Buffer 2 μL RT Enzyme Mix 1 μL FQ-RT Primer Mix 2 μL <![CDATA[RNase-Free ddH2O]]> 5 μL (3) Incubate at 42 ℃ for 15 min; (4) After incubating at 95 °C for 3 min, place on ice. The resulting cDNA solution can be used for subsequent experiments or stored in a -20 °C refrigerator.

[0017] two, MdRPP13-like-1 Full-length gene sequence obtained According to the apples found by the NCBI MdRPP13-like-1 Gene sequence, design specific primers MdRPP13-like- 1 -F, MdRPP13-like-1 -R, and then PCR amplification was performed using apple rootstock cDNA synthesized via reverse transcription as a template to obtain the full-length cDNA sequence; among which, MdRPP13-like-1 The -F sequence is shown in SEQ ID NO. 3. MdRPP13-like-1 -R sequences are shown in SEQ. ID. NO.4; The PCR reaction system and program parameters are as follows: (1) PCR reaction system: PCR reaction system Composition Usage Template 2.0μL 10×PCR Buffer 2.5 μL dNTPs (2.5mM) 2 μL Primer-F 1.5 μL Primer-R 1.5μL LA Taq DNA polymerase 0.3μL <![CDATA[RNase-Free ddH2O]]> Up to 25 μL (2) PCR reaction program parameters: 94 °C pre-denaturation for 3 min; 94 °C denaturation for 45 sec; 56.5 °C annealing for 1 min, 72 °C extension for 2 min, 35 cycles; 72 °C extension for 10 min.

[0018] (3) Take 5 μL of PCR product, add 1 μL of bromophenol blue indicator and mix well. Then, use 1.0% agarose gel for electrophoresis detection. The amplified band size is about 3800bp. Perform gel excision and recovery (recovery is performed according to the Takara "Agarose GelDNA Purification Kit").

[0019] (4) The target sequence was ligated into a vector (3.0 μl of the PCR product was ligated into the pMD18-T vector, following the instructions for the pMD18-T Vector), transformed (the ligation product was transformed into competent E. coli DH5α cells, and cultured upside down at 37°C for 12-20 hours on LB agar plates coated with IPTG / X-Gal; white colonies were picked and cultured overnight in LB liquid medium), and sequenced (1 ml of the shaken bacterial culture was placed in a 1.5 ml centrifuge tube, and the sequence was determined at Shanghai Sangon Biotech Co., Ltd.). MdRPP13-like-1 Gene.

[0020] 1. Take leaves from healthy, aseptic apple tissue culture seedlings (GL3), remove the midrib, cut into 1cm x 1cm cubes, and inoculate them onto apple adventitious bud induction medium (MS + 6-BA 3.0 mg / L + NAA 0.2 mg / L + ... Pre-cultured on a medium containing 30 g / L sucrose and 5.0 mg / L agar for 2 days. When the OD value of Agrobacterium EHA105 containing the recombinant plasmid was reached 0.5-0.6, the bacterial suspension was diluted 10-fold. The pre-cultured leaves were then immersed in the bacterial suspension for 10 minutes. The leaves were removed, blotted dry with sterile filter paper, and returned to the co-culture medium (MS + 2.0 mg / L TDZ + 0.5 mg / L NAA + 30 g / L sucrose + 5 g / L glucose + 0.5 g / L LCH + 5.0 mg / L agar). The leaves were incubated in the dark at 25°C with the underside down for 2-3 days. Finally, the leaves were transferred to an adventitious bud delayed induction medium containing 250 mg / L cephalosporin and 250 mg / L termethin (MS + 2.0 mg / L TDZ + 0.5 mg / L NAA). Leaves were placed under dark conditions with the underside of the sucrose (30 g / L sucrose + 5.0 mg / L agar) for 2-3 days. Then, they were transferred to an adventitious bud induction medium (MS + TDZ 2.0 mg / L + NAA 0.5 mg / L + 25 mg / L kanamycin + 30 g / L sucrose + 5.0 mg / L agar) containing 250 mg / L termethin and 250 mg / L cephalosporin, with the underside of the leaves facing upwards, and placed in the dark for 30 days. Afterward, they were transferred to a proliferation medium (MS + 6-BA 1.0 mg / L + NAA 0.2 mg / L + 0.5 mg / L GA3 + 30 g / L sucrose + 5.0 mg / L agar) at 25°C, 2000 lx, with a 16h / 8h light / dark cycle for proliferation culture. The medium was changed periodically until adventitious buds emerged. Positive transgenic plants were obtained by PCR detection, and phenotypic analysis was performed.

[0021] Example 3: MdRPP13-like-1 Verification of the gene's resistance to continuous cropping in genetically modified apples MdRPP13-like-1 Effects of gene overexpression in apples on apple replant resistance phenotype Fifty vigorous wild-type and three different transgenic apple seedlings were selected and transplanted into a seedling substrate. After slightly trimming the roots by 0.5 cm, the seedlings were soaked in MR5 spore suspension for 2 hours before being planted in a sterile substrate. 20 mL of spore suspension was then applied to the rhizosphere. The remaining 50 seedlings served as controls and were irrigated with an equal volume of PDB medium. The plants were grown in a tissue culture chamber at 24±2℃ with a photoperiod of 16 h·d. −1 The light intensity was 1000 lx. Irrigation was done via bottom watering, maintaining soil moisture content at 60%. Two weeks later, five plants of similar size and growth status were randomly selected from each treatment group. Physiological indicators of the aboveground and underground parts were measured. For the aboveground leaves, the 3rd to 5th leaves of the diseased plants were tested (from top to bottom). After the aboveground indicators were measured, the corresponding root systems were taken for relevant indicator testing.

[0022] Depend on Figure 1 It was found that under continuous cropping fungal stress, plant growth was inhibited after 14 days of treatment compared to the control. MdRPP13-like-1-OE alleviated the phenotype of continuous cropping fungal stress compared to WT. Under MR5 stress treatment, the plant height, root length, and fresh weight of WT plants were significantly lower than those of transgenic plants. Furthermore, this study also measured root area and volume. Under stress, the root area and volume of MdRPP13-like-1-OE were similar to those of the control, while the root area and volume of WT were significantly smaller. These results indicate that MdRPP13-like-1-OE enhances the tolerance of apple plants to continuous cropping fungal stress (MR5).

[0023] MdRPP13-like-1 The effects of gene overexpression in apples on apple physiological indicators Physiological parameters of transgenic plants and controls under MR5 stress were measured, and the expression of resistance defense enzyme genes was determined. Figure 2 It can be seen that under MR5 stress, the transgenic plants exhibit increased activity of defensive enzymes and decreased MDA content, showing better growth than the wild type.

[0024] In conclusion, MdRPP13-like-1 The results of the validation of the stress resistance function of the gene in apples fully demonstrate that MdRPP13- like-1 The gene is a stress-related gene, and its overexpression can improve the plant's resistance to continuous cropping.

Claims

1. An apple replanting resistance gene MdRPP13-like-1 Its characteristics are, The MdRPP13-like-1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The apple according to claim 1 MdRPP13-like-1 Genes, characterized by, The MdRPP13-like-1 The amino acid sequence encoded by the nucleotide sequence of the gene is shown in SEQ. ID. NO.

2.

3. An apple as described in claim 1 or 2 MdRPP13-like-1 A method for preparing genes, characterized in that, Includes the following steps: (1) RNA was extracted from the roots of apple rootstocks resistant to continuous cropping and reverse transcribed; (2) Obtaining the full-length cDNA sequence: Based on the information found in apples by NCBI... MdRPP13-like-1 Gene sequence, design specific primers MdRPP13-like-1 -F, MdRPP13-like-1 -R, and then PCR amplification was performed using the reverse-transcribed apple rootstock genomic cDNA as a template to obtain the full-length cDNA sequence; among which, MdRPP13-like-1 The -F sequence is shown in SEQ ID NO.

3. MdRPP13-like-1 -R sequences are shown in SEQ. ID. NO.4; (3) The PCR products are recovered, ligated into vectors, transformed, and sequenced to obtain the results. MdRPP13-like-1 Gene.

4. The apple according to claim 3 MdRPP13-like-1 A method for preparing genes, characterized in that, In step (3), the PCR amplification reaction system is as follows: 10×PCR buffer (containing Mg) 2+ 2.5μl 2.5 mM / L dNTP 2.0 μl, 10 μM / L primers MdRPP13-like-1 -F 1.5μl, 10 μM / L primers MdRPP13-like-1 -R 1.5μl, 2.0 μl of cDNA template LA Taq enzyme 0.3 μl, Add ddH2O up to 25.0 μl; The PCR reaction program was 94℃ pre-denaturation for 5 min; the cycling parameters were 94℃ pre-denaturation for 3 min; 94℃ denaturation for 45 sec; 56.5℃ annealing for 1 min; 72℃ extension for 2 min, 35 cycles; 72℃ extension for 10 min.

5. The apple stress-resistance gene according to claim 1 or 2 MdRPP13-like-1 Application in genetically modified apples.

Images