Plant growth-promoting rhizobacteria specific primer pair, kit and application

Abstract

The invention discloses a plant growth-promoting bacterium specific primer pair, a kit and application. The plant growth-promoting bacterium specific primer pair is composed of an upstream primer P463-F as shown in SEQ ID NO. 1 and a downstream primer P463-R2 as shown in SEQ ID NO. 2. According to the present invention, the plant growth-promoting bacterium specific primer pair is used for identifying the plant growth-promoting bacterium Penicillium sp.PQxj3, such that the survival state of the Penicillium sp.PQxj3 in the soil can be monitored in real time, the application conditions of the Penicillium sp.PQxj3 in different environments, different crops and different soils can be optimized, and the optimal effect of plant growth promotion can be achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and in particular to a plant growth-promoting rhizobacteria specific primer pair, a kit and application thereof. BACKGROUND

[0002] In agricultural production, chemical fertilizers and pesticides are often used to ensure the normal growth and development of crops, which often changes or even pollutes the soil composition. Plant growth-promoting microorganisms (PGPM) are a class of beneficial bacteria or fungi that can directly promote plant growth, resist pests and diseases, and repair soil through various mechanisms, and have very good application prospects. Among them, some plant growth-promoting microorganisms mainly colonize in the rhizosphere of plants, and their physical, chemical and biological properties are affected by plant root growth and activity, so these plant growth-promoting microorganisms are also called plant growth-promoting rhizobacteria (PGPR). As a green, efficient and pollution-free fertilizer and soil repair agent, PGPR can colonize in the rhizosphere or root surface of plants and promote plant growth and resist diseases through various mechanisms, and is an important development direction for realizing ecological environment protection and green agricultural development at the same time. Therefore, the related research on the colonization of PGPR in the rhizosphere of plants has a positive significance for subsequent analysis of the growth-promoting mechanism of PGPR and further improvement of the growth-promoting efficiency of PGPR. Among them, specific identification of target PGPR in soil and real-time dynamic monitoring of the survival state of PGPR in soil are the key points of PGPR colonization research. SUMMARY

[0003] Therefore, the present application provides a plant growth-promoting rhizobacteria specific primer pair, a kit for identifying plant growth-promoting rhizobacteria, and application and a method for identifying plant growth-promoting rhizobacteria.

[0004] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: The plant growth-promoting rhizobacteria specific primer pair provided by the present application is composed of an upstream primer P463-F as shown in SEQ ID NO. 1 and a downstream primer P463-R2 as shown in SEQ ID NO. 2. The sequence shown in SEQ ID NO. 1 is 5'-ACGAGGCTCCTGGAAGTATGG-3', and the sequence shown in SEQ ID NO. 2 is 5'-ACGTCCTGTTGCGTGTTGACG-3'.

[0005] This invention also proposes a kit for identifying plant rhizosphere growth-promoting bacteria, comprising a specific primer pair of plant rhizosphere growth-promoting bacteria composed of P463-F and P463-R2, for the specific identification of plant rhizosphere growth-promoting bacteria. Penicillium sp. PQxj3.

[0006] This invention also proposes the application of specific primer pairs or kits for identifying plant rhizosphere growth-promoting bacteria in the identification of plant rhizosphere growth-promoting bacteria. In practical applications, these kits can be used for the specific identification of plant rhizosphere growth-promoting bacteria. Penicillium sp. PQxj3, for Penicillium sp. This research provides a foundation for studying the optimal dosage of PQxj3 under different plant and environmental conditions, and is beneficial to... Penicillium sp. Large-scale application of PQxj3.

[0007] The present invention also proposes a method for identifying plant rhizosphere growth-promoting bacteria, the method comprising the following steps: using the genomic DNA of the biological sample to be tested as a template, PCR amplification is performed using the plant rhizosphere growth-promoting bacteria-specific primer pair of the present invention, obtaining PCR products, and electrophoresis detection of the PCR products; If the PCR product contains a 246 bp target DNA fragment (base sequence as shown in SEQ ID NO. 3), the tested biological sample is a plant rhizosphere growth-promoting bacterium or contains plant rhizosphere growth-promoting bacteria; conversely, if the PCR product does not contain the target DNA fragment, the biological sample is not a plant rhizosphere growth-promoting bacterium or does not contain plant rhizosphere growth-promoting bacteria. The PCR amplification program is as follows: pre-denaturation 95℃, 3 min; denaturation 94℃, 30 s; annealing 60℃, 30 s; 72℃, 15 s (30 cycles); final extension 72℃, 10 min.

[0008] Compared with the prior art, the plant rhizosphere growth-promoting bacteria-specific primer pair of the present invention is only effective against plant root growth-promoting bacteria. Penicillium sp. A specific band was amplified in the genome of PQxj3, while no amplification product was obtained in other strains (such as Penicillium baicalensis microbial preparations). These results demonstrate that the specific primer pair for plant rhizosphere growth-promoting bacteria of this invention can be used to identify plant rhizosphere growth-promoting bacteria. Penicillium sp. PQxj3, and thus real-time monitoring Penicillium sp. The survival status of PQxj3 in the soil, thereby optimizing Penicillium sp. PQxj3 achieves optimal plant growth promotion under different application conditions in different environments, crops, and soils. Attached Figure Description

[0009] Figure 1 This is the present invention. Penicillium sp. Genome loop of PQxj3. Figure 1The outermost circle is genome size; the second and third circles are CDS on the plus and minus strands; the fourth and fifth circles are rRNA and tRNA on the plus and minus strands, rRNA is green, and tRNA is pink; the sixth circle is GC content; and the innermost circle is GC skew, positive values are indicated in yellow, and negative values are indicated in purple.

[0010] Figure 2 is the position of gene00463 in Penicillium sp. PQxj3.

[0011] Figure 3 is an electrophoretic banding diagram after amplification of the primer pair P463-F and P463-R2 in the present application. Figure 3 M is Marker, 1 is test group 2, 2 is comparison group, 3 is test group 1 (PQxj3 treatment for 20 days of tobacco rhizosphere soil), 4 is blank tobacco rhizosphere soil without adding any microbial agent, and 5 is blank ddH2O control. Penicillium sp. M is Marker, 1 is test group 2, 2 is comparison group, 3 is test group 1 (PQxj3 treatment for 20 days of tobacco rhizosphere soil), 4 is blank tobacco rhizosphere soil without adding any microbial agent, and 5 is blank ddH2O control.

[0012] Figure 4 is an electrophoretic banding diagram after amplification of the primer pair P463-F3 and P463-R3 in the present application. Figure 4 M is Marker, 1 is test group 2, 2 is comparison group, 3 is test group 1 (PQxj3 treatment for 20 days of tobacco rhizosphere soil), 4 is blank tobacco rhizosphere soil without adding any microbial agent, and 5 is blank ddH2O control. Penicillium sp. M is Marker, 1 is test group 2, 2 is comparison group, 3 is test group 1 (PQxj3 treatment for 20 days of tobacco rhizosphere soil), 4 is blank tobacco rhizosphere soil without adding any microbial agent, and 5 is blank ddH2O control.

[0013] Figure 5 is a standard curve of gene00463 in Example 3 of the present application.

[0014] Figure 6 is the qPCR detection result after PQxj3 treatment for 20 days in test group 1 of the present application. Penicillium sp. PQxj3 treatment for 20 days. DETAILED DESCRIPTION

[0015] The present application will be further described in conjunction with specific embodiments. The embodiments given herein are only to illustrate the present application, and are not intended to limit the scope of the present application.

[0016] It should be noted that the experimental methods in the following examples are all conventional methods unless otherwise specified; and the materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0017] It should be noted that the plant growth-promoting rhizobacteria in the following examples is a Penicillium fungus with significant growth-promoting effect on tobacco, named PQxj3. Penicillium sp.PQxj3, which is preserved in the China General Microbiological Culture Collection Center, has a preservation number of CGMCC NO: 40147 and a preservation date of March 17, 2022. The public can obtain the strain from the China General Microbiological Culture Collection Center.

[0018] It should be noted that the PDA culture medium of the present application is divided into test tubes for sterilization to make a slant culture medium, and the strain Penicillium sp. PQxj3 is inoculated in PDA culture medium (preferably solid); the CTAB method is used to extract the whole genome DNA of the strain Penicillium sp. The whole genome DNA of PQxj3; the whole genome of soil is extracted by using a D5625-01-E.Z.N.A. Soil DNA Kit kit, and the extraction is performed according to the kit instructions.

[0019] Example 1 Design of plant growth-promoting rhizobacteria specific primer pairs according to the present application I, Penicillium sp. Whole genome data of PQxj3 The whole genome DNA of PQxj3 is extracted by using the CTAB method Penicillium sp. The whole genome DNA of PQxj3 is sequenced by Shanghai Meiji Biomedicine Technology Co., Ltd., and the whole genome sequencing data (gene accession number: PRJNA1309989) result is as shown in Figure 1 The results show that Penicillium sp. The genome size of PQxj3 is 37454497 bp, the assembly Scaffolds is 10, the GC content is 49.2%, the BUSCO software is used to evaluate the genome integrity, which is 98.4%, indicating that the integrity of the genome assembly is good; the total number of non-coding RNA is 233, 191 transport RNAs are predicted, and 42 ribosomal RNAs are predicted; the coding gene prediction result shows that the total number of predicted genes is 10150, the coding sequence (CDS) of the predicted coding protein in the genome is 9917, the total number of exons is 33633, and the total number of introns is 23716.

[0020] II, the strain Penicillium sp.The predicted gene sequence of PQxj3 was functionally aligned with COG, Pfam, GO, KEGG, Swiss-Prot, and NR databases to realize amino acid sequence prediction, and the results are shown in Table 1. As shown in Table 1, a total of 9589 functional genes were obtained by multi-database joint annotation, and the annotation coverage showed a gradient distribution as follows: the number of genes annotated by NR database was the highest (9578, accounting for 99.88%), followed by Pfam (7408, 77.25%), Swiss-Prot (7080, 73.83%), KEGG (6678, 69.64%), and GO database (6521, 68.00%), and the number of genes annotated by COG database was relatively low (4036, accounting for 42.08% of the total).

[0021] Table 1 Strain Penicillium sp. Genome database annotation of PQxj3 Three, Penicillium sp. Design of specific primer pairs for PQxj3 genome According to Penicillium sp. According to the genome prediction and whole genome functional annotation of PQxj3, there are a total of 9917 coding sequences (CDS) encoding proteins, and 9589 genes are annotated by the six databases in Table 1, and 328 genes are not annotated. Among the 328 unannotated genes, 279 genes have a length of less than 1000 bp, 37 genes have a length of 1000-2000 bp, 8 genes have a length of 2000-5000 bp, and 4 genes have a length of more than 5000 bp. When designing specific primers, genes with a length of less than 1000 bp and more than 5000 bp are excluded, and the Penicillium sp. The base sites different from other Penicillium gene sequences of PQxj3 are used as the positions of the primer pairs to realize specific detection of Penicillium sp. PQxj3. The results show that there is a gene with a length of 1374 bp (take gene00463 as an example, the gene sequence is shown as SEQ ID NO. 7), and the unannotated genes with the same sequence as gene00463 sequence appear 12 times repeatedly, which are shown in Figure 2 Therefore, in order to more sensitively detect the presence of Penicillium sp. PQxj3 in soil and ensure the specificity of detection, the gene00463 of the present application is used as the primer design site for primer design.

[0022] Primers for the gene00463 gene sequence were designed using Primer Premier 5 (version 5.00) with the following parameters: sequence length between 18 and 30 nt; product length between 100 and 300 bp; annealing temperature between 55 and 65℃; and GC content between 40% and 60%. Suitable primers were screened and designed. The designed primer sequences and corresponding target fragments are shown in Table 2.

[0023] Among them, the sequence number of the target fragments corresponding to P463-F and P463-R2 is SEQ ID NO.3, and the sequence number of the target fragments corresponding to P463-F3 and P463-R3 is SEQ ID NO.4, as shown in Table 2.

[0024] Table 2 Penicillium sp.PQxj3 specific primer sequence and target fragment Example 2: Specificity verification analysis of the primer pairs in Example 1. The first step is to culture the slant culture Penicillium strain PQxj3 was inoculated into Erlenmeyer flasks containing wheat bran solid medium and cultured at 28°C for 7 days. During the culture process, initial static incubation allowed for abundant mycelial growth, followed by appropriate shaking to ensure even distribution of the mycelium. Finally, static incubation was repeated until sufficient mycelium and spores were produced, yielding the desired strain. Penicillium sp. PQxj3 microbial agent (hereinafter referred to as "PQxj3 microbial agent"); Among them, the bran solid culture medium (placed in a 250ml Erlenmeyer flask): weigh 40 g of fine bran, add 32 mL of distilled water, control the moisture content of the bran to 80%, and sterilize at 121℃ for 20 min. The second step is to mix water, nutrient soil and vermiculite in a mass ratio of 3:2:1 to obtain tobacco potting soil. Divide the tobacco potting soil into 10cm×10cm plastic pots, sow tobacco seeds in each plastic pot, and cultivate in a 28℃ cultivation room until the tobacco has 4-6 leaves. Tobacco plants with uniform growth were selected and divided into two groups: one group (experimental group 1, with 1 g of PQxj3 inoculant added to the root system of each tobacco plant); and the other group (blank soil group, i.e., without any added microbial inoculant). A commercially available *Penicillium baicalensis* strain was used as a control group. The soil was inoculated onto PDA medium. Penicillium sp.PQxj3 strain was used as experimental group 2, and ddH2O was used as the blank group; The third step was to obtain whole-genome DNA by sampling. Whole-genome DNA was extracted from the control group and experimental group 2 using the CTAB method. For experimental group 1 and the blank soil group, soil around the roots of tobacco was taken and whole-genome DNA of microorganisms in each soil group was extracted using the D5625-01-EZNA SoilDNA Kit (following the instructions of the kit). Two pairs of primers designed in Example 1 were used to amplify the whole genomic DNA of each group by PCR. The PCR products were then analyzed by electrophoresis on a 0.8% agarose gel to detect whether the PCR products contained bands of the target fragment. The results are shown in [Figure 1]. Figures 3-4 The PCR amplification reaction system and PCR amplification program are shown in Tables 3-4, respectively. Table 3 PCR reaction system Table 4 PCR reaction procedure Depend on Figures 3-4 It can be seen that both primer pairs can amplify Penicillium sp. The target band of the PQxj3 genome was detected, but primer pairs P463-F3 and P463-R3 amplified nonspecifically in the blank group. Furthermore, using primer pairs P463-F and P463-R2, the target band was still present in experimental group 1 after 20 days, while no target band was observed in the untreated blank soil group after 20 days. Neither the *Penicillium baicalensis* strain nor the blank ddH2O group amplified the corresponding band. These results demonstrate that the primer pairs P463-F and P463-R2 of this invention can be used to specifically identify *P. baicalensis* in soil. Penicillium sp. PQxj3 can be used to optimize its application conditions in different environments, crops, and soils to achieve the best effect in promoting plant growth.

[0025] Example 3: Soil for Tobacco Potted Plants Penicillium sp. qPCR detection of PQxj3 This invention proposes a method for preparing soil for tobacco potted plants. Penicillium sp. The qPCR detection method for PQxj3 is based on Penicillium sp. After amplification of the target fragment using PQxj3, a standard curve was obtained. Based on the standard curve, the target fragment in tobacco potted soil was purified. Penicillium sp. The qPCR detection of PQxj3 includes the following: The first step is to Penicillium sp . After PCR amplification of the target-specific gene PQxj3, the target gene fragment was recovered by gel extraction using the SanPrepColumn DNA Gel Extraction Kit (following the instructions). The target fragment was cloned into a plasmid vector using the One step ZTOPO-Blunt / TA Zero Background Fast Cloning Kit (following the instructions), and then transformed into E. coli DH5α to amplify the plasmid. Plasmids were extracted using the SanPrep Column Plasmid Mini-Preps Kit (following the instructions). The extracted plasmids were then diluted 10-fold to 10⁻⁶. 8 -10 2 copiea / μL was used to obtain the standard plasmid; The plasmid was prepared according to the formula Y (copiea / μL) = [DNA concentration X (ng / μL) × 10]. -9 [DNA length (bp) × 660] × 6.02 × 10 23 Convert the copy number to the standard plasmid, perform qPCR test, and then plot the standard curve. The qPCR reaction system was prepared according to the instructions for 2×HQ SYBR qPCR Mix (High ROX) (see Table 5), and the qPCR reaction procedure is shown in Table 6. Table 5 qPCR reaction system Table 6 qPCR reaction procedure A standard curve was plotted based on the qPCR test results (Ct) and copy number of the standard plasmid. The R-squared value of the standard curve was... 2 A value of 0.999 indicates a good linear relationship; see details below. Figure 5 ; The second step involved obtaining the whole-genome DNA from the tobacco potted soil. qPCR was performed according to Tables 4 and 5 to obtain the Ct values ​​from the tobacco potted soil. The copy number of the target gene in the actual sample could be obtained from the standard curve plotted in the first step. The results are shown in [Table 4]. Figure 6 .Depend on Figure 6 It can be seen that, according to Penicillium sp. The target gene in the soil of tobacco potted plants treated with PQxj3 still had a copy number as high as 10 after 20 days. 8 / g, while almost no copy number was detected in the blank tobacco soil (CK). The results indicate that P463-F and P463-R2 of this invention can specifically detect / g in soil. Penicillium sp. PQxj3 and can also... Penicillium sp. Quantitative analysis of PQxj3.

[0026] In summary, the P463-F and P463-R2 primer pairs of this invention only in... Penicillium sp.The specific band appears in the amplification of the genome of PQxj3, and no amplification product is obtained in other strains (such as Penicillium bilaiae microbial preparation). The results prove that the specific primer of the plant growth promoting rhizobacteria of the application can be used for identifying Penicillium sp. PQxj3, and further real-time monitoring the survival state of the PQxj3 strain in the soil, optimizing the application conditions of PQxj3 in different environments, different crops and different soils, and achieving the optimal effect of promoting the growth of tobacco.

Claims

1. A specific primer pair for plant rhizosphere growth-promoting bacteria, characterized in that: It consists of upstream primer P463-F as shown in SEQ ID NO.1 and downstream primer P463-R2 as shown in SEQ ID NO.

2.

2. A method for identifying plant rhizosphere growth-promoting bacteria Penicillium sp. The PQxj3 reagent kit is characterized by: Includes the plant rhizosphere growth-promoting bacteria specific primer pair as described in claim 1.

3. The application of the specific primer pair for plant rhizosphere growth-promoting bacteria according to claim 1 or the kit according to claim 2 in the identification of plant rhizosphere growth-promoting bacteria, wherein the plant rhizosphere growth-promoting bacteria are... Penicillium sp. PQxj3.

4. A method for identifying plant rhizosphere growth-promoting bacteria, characterized in that: The plant rhizosphere growth-promoting bacteria are Penicillium sp. PQxj3; The method includes the following: using the genomic DNA of the biological sample to be tested as a template, PCR amplification is performed using the plant rhizosphere growth-promoting bacteria specific primer pair as described in claim 1, PCR products are obtained, and the PCR products are detected by electrophoresis; If the PCR product contains a 246 bp target DNA fragment, the biological sample to be tested is a plant rhizosphere growth-promoting bacterium or contains plant rhizosphere growth-promoting bacteria; conversely, if the PCR product does not contain the target DNA fragment, the biological sample is not a plant rhizosphere growth-promoting bacterium or does not contain plant rhizosphere growth-promoting bacteria.

5. The method for identifying plant rhizosphere growth-promoting bacteria according to claim 4, characterized in that: The base sequence of the target DNA fragment is shown in SEQ ID NO.3.

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