Application of peanut micro2916 precursor and coding gene thereof in regulating and controlling aluminum resistance of peanut

Abstract

The invention discloses an application of a peanut micro2916 precursor and a coding gene thereof in regulating and controlling the aluminum resistance of peanuts. The inventor obtains a silent peanut plant of micro2916 by silencing mircoRNA2916 in the peanut, and the aluminum resistance of the silent peanut plant is higher than that of a wild type, which indicates that the micro2916 of the peanut negatively regulates the tolerance of the peanut to aluminum stress. Therefore, the peanut micro2916 precursor, the coding gene or the biological material can be used for regulating and controlling the aluminum resistance of the peanut, and the application of the invention has great value for breeding of peanut aluminum-resistant varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of peanut breeding, and in particular relates to application of a peanut micro2916 precursor and a coding gene thereof in regulating aluminum tolerance of peanut. Background Art

[0002] Aluminum is the main limiting factor for crop growth on acidic soils. Continuous application of fertilizers containing ammonia and ammonium compounds, industrial pollution, acid rain and nitrogen fixation by legumes have exacerbated aluminum toxicity. At present, 40-70% of the world's arable land is acidic soil, and about 21% of the land in my country is acidic soil, distributed in 15 provinces and regions in the south, with a total area of ​​20.3 million hectares. With environmental pollution becoming increasingly serious and the area of ​​acidic soils expanding, aluminum toxicity has become a global problem, and strengthening the research on the mechanism of plant aluminum tolerance has important practical significance.

[0003] Peanut is an oil and cash crop widely planted worldwide, rich in oil and protein. Acidic soil in the south is one of the important reasons for the low yield of peanuts. At present, the selection and breeding of peanut varieties is mainly carried out through conventional breeding methods, but the genetic basis of varieties bred by traditional methods is narrow, and the excellent traits of wild peanut resources are difficult to utilize due to factors such as hybrid incompatibility. With the rapid development of bioinformatics, genetics, modern molecular biology and the increasing maturity of transgenic technology, modern genetic engineering technology can introduce exogenous genes into plants, artificially select target trait genes, and achieve rapid variety improvement. Therefore, molecular breeding through modern genetic engineering technology has become one of the fast and effective ways to improve the aluminum tolerance of peanuts.

[0004] MircRNA is a type of small molecule widely found in plants, which can regulate target genes at different levels of transcription, post-transcription, translation, etc. At present, the research on mircoRNA mainly focuses on regulating plant growth and development, as well as the response to abiotic stress, but there are no reports on the research of mircoRNA2916, nor on the research on the response of mircoRNA to aluminum stress in peanut. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an application of a peanut micro2916 precursor and a coding gene thereof in regulating the aluminum tolerance of peanuts, which is of great value to the breeding of aluminum-tolerant peanut varieties.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The peanut micro2916 precursor has the base sequence of SEQ.ID.NO.1 in the sequence listing.

[0008] The gene encoding the above peanut micro2916 precursor.

[0009] Biological materials containing the above peanut micro2916 precursor or coding gene.

[0010] The above-mentioned biological materials are recombinant vectors, recombinant microorganisms, and transgenic plant cell lines.

[0011] The application of the above-mentioned peanut micro2916 precursor, encoding gene or biological material in regulating the aluminum tolerance of peanut.

[0012] Application of the above peanut micro2916 precursor, encoding gene or biological material in plant breeding.

[0013] Plant breeding is the development of transgenic plants that are tolerant to aluminum.

[0014] A method for cultivating aluminum-tolerant mutant peanuts, silencing mircoRNA2916 in peanuts through VIGS technology to obtain micro2916-silenced peanut plants.

[0015] The VIGS viral vector of the miRNA gene is constructed by connecting the miRNA gene-specific fragment to the VIGS viral backbone vector pTRV2 to obtain a recombinant viral vector; the miRNA gene-specific fragment (i.e., the mature sequence of micro2916) has the base sequence of SEQ.ID.NO.2 in the sequence table.

[0016] The inventors found in their research on peanuts that the aluminum tolerance of the micro2916-silenced peanut plants obtained by silencing mircoRNA2916 in peanuts was higher than that of the wild type, indicating that the micro2916 in peanuts negatively regulates the tolerance of peanuts to aluminum stress. Therefore, the aluminum tolerance of peanuts can be regulated by using the micro2916 precursor, encoding gene or biological material of peanuts, and the application of the present invention has great value in the breeding of aluminum-tolerant peanut varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram showing the expression pattern of peanut mircoRNA2916 under aluminum stress analyzed by qPCR.

[0018] Figure 2 This is a graph showing the growth phenotype of transgenic Arabidopsis, in which: A is the phenotype of transgenic Arabidopsis plants cultured on culture medium for 13 days, B is the phenotype of transgenic Arabidopsis plants cultured in soil for 37 days; C is the phenotype of transgenic Arabidopsis plants cultured in soil for 37 days; wherein WT represents wild-type Arabidopsis; OE1 and OE2 represent different strains of transgenic Arabidopsis, respectively.

[0019] Figure 3This is a graph showing the results of the aluminum tolerance analysis of transgenic Arabidopsis, in which: A. Root length elongation under aluminum stress; B. Hematoxylin staining of the root tip under aluminum stress; wherein WT represents wild-type Arabidopsis; OE1 and OE2 represent different strains of transgenic Arabidopsis, respectively.

[0020] Figure 4 These are the results of the growth phenotype and aluminum tolerance analysis of silent peanut plants, in which: A. Growth phenotype of silent peanut plants; B. qPCR analysis, where WT represents wild-type peanut, pTRV2 represents empty vector, pds represents positive control, and 1-10 represents peanut strains in the experimental group; C. Hematoxylin staining of root tips under aluminum stress. DETAILED DESCRIPTION

[0021] In order to explain how to implement the present invention, the following examples are provided. The experimental methods used in the examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified; and the quantitative tests are repeated three times, and the results are averaged.

[0022] 1. Discovery of mircoRNA2916 and cloning of its precursor sequence

[0023] 1. Discovery of mircoRNA2916

[0024] According to the microRNA predicted in the summary report of small RNA sequencing analysis of China Hangzhou Lianchuan Biological, microRNAs with significant differences under different aluminum treatment conditions were selected, and the screening of differentially expressed microRNAs was carried out with |log2(Foldchange)|>1 (i.e., two-fold difference) and p_value<=0.05 as the threshold value. It was found that peu-MIR2916-p5_1ss5AG (named according to the microRNA naming rules of China Hangzhou Lianchuan Biological) was treated with 100μmol / L aluminum for 24h, and the log2(Foldchange) value of Zhonghua No. 2 was 2.98 and the p_value was 0.014 when compared with no aluminum treatment, which met the conditions.

[0025] 2. Cloning of mircoRNA2916 precursor sequence

[0026] ① Primer design

[0027] Table 1 Primer sequences for PCR amplification of Pre-mircoRNA 2916 in Zhonghua No. 2

[0028]

[0029] ② Cloning of Pre-mircoRNA2916

[0030] Mature and plump peanut seeds of the cultivated variety Zhonghua No. 2 were selected and placed in the dark at 26°C for germination for 4 days. The seed coats of the sprouted peanuts were removed and cultured in Hoagland nutrient solution, with the nutrient solution replaced every 2 days. After the second true leaf was extracted from the peanut seedlings, they were pretreated in a solution containing 0.1mmol / L CaCl2 (pH4.2-4.3) for 24h. Three-leaf roots were taken, and the root RNA was extracted using the EastepSuper Total RNA Extraction Kit (LS1040, Promega, Beijing), and reverse transcribed using the HiScript III All-in-one RT SuperMix Perfect for qPCR Kit (R333, Novozyme, Nanjing). The obtained cDNA was used as a template, and the Pre-mircoRNA2916 primer was used for amplification, and sequencing was performed for detection.

[0031] cDNA fragment PCR amplification (50 μL system) and PCR amplification conditions are shown in Table 2.

[0032] Table 2 Reaction system and reaction conditions for PCR amplification of Pre-mircoRNA2916

[0033]

[0034]

[0035] Results: The precursor and coding sequence of mircoRNA2916 were successfully obtained.

[0036] 2. Analysis of the expression pattern of mircoRNA2916 under aluminum stress

[0037] Peanut Zhonghua No. 2 was used as the experimental material. The seeds were placed in coarse sand at 26°C for 4 days. The seed coats of the germinated peanuts were removed and cultured with Hoagland nutrient solution, with the nutrient solution replaced every 2 days. After the second true leaf of the peanut seedlings was extracted, they were pretreated in a solution containing 0.1 mmol / L CaCl2 (pH 4.2-4.3) for 24 hours.

[0038] 100 μmol / L aluminum was selected for treatment for 0, 4, 8, and 12 h, and roots of 0-1 cm were taken. The root RNA was extracted using the Eastep Super Total RNA Extraction Kit (LS1040, Promega, Beijing), and reverse transcribed using the HiScript III All-in-one RT SuperMix Perfect for qPCR Kit (R333, Novozymes, Nanjing). The obtained cDNA was used as a template, and the primers in Table 3 were used to detect the expression of mircoRNA2916 under aluminum treatment by qPCR.

[0039] Table 3 Primer sequences for qPCR identification of Pre-mircoRNA 2916 in Zhonghua No. 2

[0040]

[0041] like Figure 1 As shown, the results showed that under aluminum stress treatment, the expression level of Pre-mircoRNA2916 showed an upward trend and reached the highest level at 12h of aluminum stress treatment, indicating that the expression of Pre-mircoRNA2916 was positively induced by aluminum stress.

[0042] qPCR amplification conditions:

[0043] UBQ10R was used as the internal reference and cDNA of each sample was used as the template for qRT-PCR analysis. The reaction system was: 2×ChamQ Universal SYBR qPCR Master Mix 5μL, forward and reverse primers (10μmol / L) 0.5μL each, cDNA 1μL, ddH2O 3μL, total volume 10μL. The reaction procedure was: 95℃ pre-denaturation for 10min, 95℃ denaturation for 10s, 58℃ annealing for 30s, 95℃ extension for 10s, 40 cycles. Each treatment was repeated 3 times, using 2 -△△ The gene expression was analyzed by Ct method.

[0044] Results: In peanut, the expression of Pre-mircoRNA2916 was positively induced by aluminum stress.

[0045] 3. Acquisition of transgenic Arabidopsis and analysis of aluminum tolerance

[0046] 1. Construction of overexpression vector

[0047] Using the root tip cDNA of Zhonghua No. 2 as a template, the Pre-mircoRNA2916 primers in Table 1 were used for amplification to obtain the Pre-mircoRNA2916 sequence, and the pBI121-EGFP vector was double-digested with XhoⅠ and SaIⅠ restriction endonucleases to linearize the vector and obtain the recombinant expression vector pBI121-EGFP-mircoRNA2916, which was sequenced for detection. The reaction system and conditions are shown in Tables 4 and 5.

[0048] Table 4 Reaction system and reaction conditions for carrier linearization

[0049]

[0050] Table 5 Reaction system and reaction conditions for homologous recombination

[0051]

[0052] 2. Acquisition and identification of transgenic Arabidopsis

[0053] ① Agrobacterium containing the recombinant plasmid was cultured at 28°C with shaking at 180 rpm until OD 600 The cells were collected by centrifugation at 5000 rpm for 5 min and resuspended to an OD of 600 is 0.8.

[0054] ② The dried wild-type Arabidopsis seeds were sterilized with 75% ethanol for 5 minutes, 2% sodium hypochlorite for 5 minutes, sterilized with sterile ddH2O, and washed 3 times by shaking. The seeds were transferred to 1 / 2MS solid medium without resistance by pipette and sealed with sealing film. After culturing in a 22℃ light incubator with 16h light / 8h dark for one week, the seeds were transferred to the matrix for seedling growth and cultured to 4 weeks old for genetic transformation.

[0055] ③ Select 4-week-old Arabidopsis with good growth and a large number of flower buds, soak the inflorescence in the flower soaking solution for 1 minute, wrap it with plastic wrap, place it tilted and culture it in the dark for 24 hours, then restore it to normal light, continue to culture it for 3 days, then remove the plastic wrap, repeat the above operation 3 times. Collect the seeds when they are mature (T0 generation).

[0056] ④ Identify the correct T0 generation seedlings, culture them until the seeds are mature, and collect the seeds from individual plants. The seeds obtained are the T1 generation. T1 generation seeds continue to use Kan to screen resistant plants, culture them until they have 5-6 leaves, and identify the transcription level. The T1 generation seedlings whose expression level is significantly higher than that of the wild type are overexpression plants, and the seeds of the T2 generation are collected; T2 generation seeds are sown in Kan-resistant culture medium, most of which can germinate and grow normally, and the medium is cultured until seed formation, and the seeds collected are the T3 generation. At this time, the phenotype is stable and can be used for subsequent experimental research ( Figure 2 ).

[0057] Table 6 Primer sequences for PCR identification of transgenic plants

[0058]

[0059] Table 7 Reaction system and reaction conditions for PCR identification of transgenic plants

[0060]

[0061] 3. Analysis of aluminum tolerance in transgenic Arabidopsis thaliana expressing pre-mircoRNA2916

[0062] The concentration of aluminum treatment was screened. The three strains WT, OE1 and OE2 were germinated in 1 / 2MS medium for 2 days, and then transferred to solid 1 / 2MS medium containing 100mM AlCl3 for 15 days. The experiment was repeated 3 times, and it was found that the root growth of the overexpression strains OE1 and OE2 was significantly inhibited under aluminum stress ( Figure 3 A), hematoxylin staining was significantly darker, indicating that the aluminum tolerance of the overexpressing plants was weakened ( Figure 3 B).

[0063] Results: Transgenic Arabidopsis plants were successfully obtained, and it was found that overexpression of Pre-mircoRNA2916 reduced the plant's tolerance to aluminum stress.

[0064] IV. Obtaining peanut plants with silencing mircoRNA2916 and analyzing their aluminum tolerance

[0065] 1. Construction of STTM vector and acquisition of recombinant plasmid Agrobacterium

[0066] microRNA2916-STTM contains two miRNA binding sites that cannot be cut, connected by a spacer sequence in the middle (as shown in Table 8). The vector was constructed and transferred into Agrobacterium. The vector was pTRV2, and the restriction sites were EcoRⅠ and XhoⅠ.

[0067] Table 8 Primer sequences for PCR identification of transgenic plants

[0068]

[0069] Agrobacterium transformation and preparation of infection medium:

[0070] (1) The constructed pTRV2-R1 and pTRV2-R2 and the recombinant plasmid were transformed into Agrobacterium GV3101 competent cells by freeze-thaw method;

[0071] (2) Inoculate into 50 ml YEP liquid medium containing 25 mg / L rifampicin (Rif), 25 mg / L streptomycin (Str), and 100 mg / L kanamycin (Kana), and culture at 200 rpm and 28°C until OD 600 is 0.8;

[0072] (3) Centrifuge at 4800 rpm for 30 min to collect the cells, resuspend them in 30 ml of LB without antibiotics, centrifuge, and discard the supernatant.

[0073] (4) Resuspend the cells in 50 ml of LB liquid medium containing 10 mM / L MES, 10 mM / L MgCl2, and 200 μM / L AS.

[0074] (5) The bacterial solutions of PTRV2, PTRV2-2916, and PTRV2-AhPDS (positive control) were mixed with the bacterial solution of PTRV1 in equal amounts, incubated at room temperature for 3 h, and then used for seed infection in the next step.

[0075] 1. Obtaining and identifying peanut plants with silencing mircoRNA2916

[0076] ① Early VIGS method to obtain silent peanut plants

[0077] a. Infection of peanut seeds

[0078] Place the freshly peeled peanut seeds Zhonghua No. 2 in a clean bench, soak them in 75% alcohol for 30 seconds, sterilize them with 0.1% mercuric chloride for 5-8 minutes, wash them with sterile water for 4-5 times, remove the seed coat, make a cut on the back of the cotyledon, and soak them in OD 600 The seeds were immersed in a bacterial solution with a concentration of 0.8 for 120 minutes, and then the seeds were taken out and cultured on MS medium at 26°C in the dark for 2-3 days. After germination, the seeds were taken out for hydroponics and the phenotypes were observed.

[0079] b. Cultivation of Peanut Materials

[0080] The peanut materials were transferred to Hoagland culture medium for hydroponics. The culture medium was changed every 2 days. The culture conditions were 26±2℃ and light intensity was 30–50 μmol m -2 s -1 , photoperiod of 16 / 8h, and observe the phenotypic changes of plant leaves.

[0081] c. Early detection of silent peanuts

[0082] When the positive control showed yellow leaves, the peanut leaves of the experimental group were tested for the transcriptional expression of mircoRNA2916. New leaves were taken, and the root RNA was extracted using the Eastep Super Total RNA Extraction Kit (LS1040, Promega, Beijing), and reverse transcribed using the HiScript III All-in-one RT SuperMix Perfect for qPCR Kit (R333, Novozyme, Nanjing). The obtained cDNA was used as a template, and the primers (same as Table 3) were used to detect the expression of mircoRNA2916 by qPCR.

[0083] The specific reaction system is shown in the table below, and the reaction procedure is shown below.

[0084] qPCR amplification conditions:

[0085] UBQ10R was used as the internal reference and cDNA of each sample was used as the template for qRT-PCR analysis. The reaction system was: 2×ChamQ Universal SYBR qPCR Master Mix 5μL, forward and reverse primers (10μmol / L) 0.5μL each, cDNA 1μL, ddH2O 3μL, total volume 10μL. The reaction procedure was: 95℃ pre-denaturation for 10min, 95℃ denaturation for 10s, 58℃ annealing for 30s, 95℃ extension for 10s, 40 cycles. Each treatment was repeated 3 times, using 2- △△ The gene expression was analyzed by Ct method.

[0086] qPCR results Figure 4 As shown. The expression level of silencing mircoRNA2916 peanut strains at the transcriptional level was significantly lower than that of the control, and peanut plants with silencing mircoRNA2916 were successfully obtained ( Figure 4 A and B).

[0087] ②Analysis of aluminum tolerance in peanut plants with silenced Pre-mircoRNA2916

[0088] The three strains of WT, empty load and silenced peanut plants were pretreated in a solution containing 0.1mmol / L CaCl2 (pH4.2-4.3) for 24h and treated with 100μmol / L aluminum for 24h. The experiment was repeated 3 times. It was found that the root hematoxylin staining of the silenced peanut plants overexpressing the strains under aluminum stress conditions became significantly lighter, indicating that the aluminum tolerance of the silenced plants was enhanced ( Figure 4 C).

[0089] Results: Silenced peanut plants were successfully obtained, and silencing of mircoRNA2916 improved the tolerance of peanut to aluminum stress.

Claims

1. Peanut micro2916 precursor, characterized in that It has the base sequence of SEQ.ID.NO.1 in the sequence listing.

2. The gene encoding the peanut micro2916 precursor of claim 1.

3. A biological material containing the peanut micro2916 precursor of claim 1 or the encoding gene of claim 2.

4. The biomaterial according to claim 3, characterized in that It is a recombinant vector, a recombinant microorganism, and a transgenic plant cell line.

5. Use of the peanut micro2916 precursor according to claim 1, the encoding gene according to claim 2 or the biomaterial according to claim 3 in regulating aluminum tolerance of peanut.

6. Use of the peanut micro2916 precursor according to claim 1, the encoding gene according to claim 2 or the biological material according to claim 3 in plant breeding.

7. The use according to claim 6, characterized in that: The plant breeding is to cultivate transgenic plants with aluminum tolerance.

8. A method for cultivating aluminum-tolerant mutant peanuts, characterized in that Micro2916-silenced peanut plants were obtained by silencing mircoRNA2916 in peanut using VIGS technology.

9. A VIGS viral vector of a miRNA gene, characterized in that: The miRNA gene-specific fragment is connected to the VIGS virus backbone vector pTRV2 to construct a recombinant virus vector; the miRNA gene-specific fragment has the base sequence of SEQ.ID.NO.2 in the sequence table.

Images