Application of pea PsCESA gene in improving aluminum toxicity stress resistance of plants

Abstract

The invention discloses application of a pea PsCESA gene in improving aluminum toxicity stress resistance of plants. The invention also discloses application of the pea PsCESA gene in promoting plant growth and screening aluminum-resistant varieties of peas, and a kit for screening aluminum-resistant peas. The pea PsCESA gene provided by the invention can improve the aluminum toxicity stress resistance of plants, can be applied to screening aluminum-resistant pea varieties, and plays an important role in plant improved breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to application of pea PsCESA gene in improving plant resistance to aluminum toxicity stress. Background Art

[0002] Pea (Pisum sativum L.) is an annual or biennial herbaceous plant of the genus pea in the Fabaceae family. It prefers cool, humid climates and has the characteristics of cold resistance, drought resistance, and barrenness resistance. It has a wide geographical distribution and is a perennial twining herb belonging to the Fabaceae family, but is usually cultivated as an annual crop in agricultural practice. Native to the Mediterranean coast and West Asia, pea is one of the earliest crops domesticated by humans and has a long history of cultivation. It has not only become an important crop widely planted around the world due to its rich nutritional value, but also occupies an important position in genetic research and is a key experimental material for Mendel to discover the law of inheritance. Globally, pea is not only an important food crop, but also has a significant effect on improving soil structure and increasing soil fertility. Due to the presence of symbiotic nitrogen-fixing bacteria in the roots, pea helps to reduce the use of chemical fertilizers, which meets the needs of sustainable agricultural development. Analyzing the molecular genetic mechanism of pea seed size and shape and exploring key genes will not only be conducive to the exploration and innovation of excellent pea germplasm resources, but also have important guiding significance for pea production and breeding.

[0003] The structure and composition of pea cell walls are important components of its cell biological characteristics, and cell wall genes involve a series of genes related to cell wall synthesis, modification and degradation. These genes encode a variety of enzymes and other proteins, which are involved in regulating the biosynthesis of cellulose, hemicellulose, pectin and structural proteins, the main components of the cell wall. In plants, the cell wall not only provides mechanical support for cells, but also plays an important role in intercellular signal transduction, defense response, cell expansion and morphological construction. For peas, studying cell wall-related genes helps to understand the mechanisms of pea growth and development, and the mechanisms of coping with environmental stresses (such as pathogen infection or abiotic stress), and can provide a theoretical basis for improving crop quality and stress resistance.

[0004] The key gene family involved in the synthesis and modification of pea cell walls is the cellulose synthase (CesA) gene. This type of gene is responsible for catalyzing the synthesis of cellulose microfibrils, which is one of the main structural components of the cell wall. Cellulose synthase genes encode a key class of enzymes that are responsible for catalyzing the synthesis of cellulose microfibrils in plant cell walls. Cellulose is one of the most abundant organic polymers on earth and the main structural component of plant cell walls, which gives the plant body morphological stability and mechanical strength. Reduced cell wall cellulose content can lead to root tip deformation and inhibited root growth. The number and sequence of CesA genes in different species vary, reflecting the evolution of plant adaptability to their respective living environments. For example, there are ten known CesA gene members in Arabidopsis thaliana, and there are corresponding homologous genes in pea. Each member may be expressed in specific tissues or developmental stages and participate in different physiological processes.

[0005] Aluminum in acidic soils poisons plant growth and reduces crop yields. About 50% of cultivated land worldwide is acidic soil. Normally, aluminum exists in the soil in a bound form, such as aluminum oxide and aluminosilicate, and bound aluminum does not affect plant growth. When the soil solution pH is <5.5, aluminum mainly exists in the form of trivalent aluminum ions and causes serious toxicity to plant growth. The toxic effect of aluminum (Al) on plants is first manifested in the inhibition of root growth. Related studies have shown that aluminum first accumulates at the root tip, so the root tip is considered to be the initial toxic site of aluminum toxicity, and the cell wall of the plant root tip is the main binding site for aluminum ions. The binding of aluminum to the cell wall will cause the cell wall to harden, thereby reducing the ductility of the plant cell wall and inhibiting the elongation of the root. When the roots are subjected to aluminum stress, the elongation of the main axis is inhibited, the number of lateral roots decreases and becomes shorter and thicker, and the root system as a whole becomes coral-like. In severe cases, the roots turn brown and the root crown structure falls off. Plants under aluminum stress are generally shorter, and are often accompanied by symptoms such as reduced leaf area, curled and yellowed leaves, purple stems, and decreased photosynthesis efficiency. Summary of the invention

[0006] The purpose of the present invention is to provide an application of pea PsCESA gene in improving plant resistance to aluminum toxicity stress in view of the above technical problems to be solved.

[0007] In order to achieve the above invention objectives, the present invention provides the use of pea PsCESA gene in improving plant resistance to aluminum toxicity stress.

[0008] As a preferred embodiment, the nucleotide sequence of the pea PsCESA gene is shown as SEQ ID NO.1.

[0009] As a preferred embodiment, the amino acid sequence of the protein expressed by the pea PsCESA gene is shown in SEQ ID NO:2.

[0010] As a preferred embodiment, the plant is pea.

[0011] On the other hand, the present invention also provides the use of pea PsCESA gene in promoting plant growth.

[0012] On the other hand, the present invention also provides the use of the pea PsCESA gene in screening pea aluminum-resistant varieties.

[0013] On the other hand, the present invention also provides a kit for screening aluminum-tolerant peas, comprising primers for amplifying the pea PsCESA gene.

[0014] Compared with the prior art, the present invention finds that the pea PsCESA gene can improve plant resistance to aluminum toxicity stress, can be used to screen aluminum-tolerant pea varieties, and plays an important role in plant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The cellulose content at aluminum treatment concentrations of 0 μM, 15 μM, and 30 μM is shown.

[0016] Figure 2 The expression results of different aluminum treatment times and different aluminum-resistant varieties are shown.

[0017] Figure 3 Quantitative expression differences at an aluminum treatment concentration of 15 μM are shown. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention, rather than to limit the scope of the present invention.

[0019] Example 1: Pea seed cultivation

[0020] Pea seeds were soaked in 7.5% sodium hypochlorite (AR) solution for 30 minutes, and then rinsed with sterile ultrapure water for 6 times, each rinse lasting 5 minutes to remove floating and swollen seeds. After sterilization, they were shaded at 24°C and soaked for 12 hours (0.5mmol·L-1CaCl2). After 12 hours, they were transferred to the aerosol culture box and cultured for 2 days at 24°C with shade and aerosol. When the roots grew to about 3-4 cm in length, they were transferred to the hydroponic solution. After 24 hours of culture, the roots were cut and the 2-3 mm root segment at the root tip of the main root was cut off to promote the growth of lateral roots. When the maximum lateral root length reached 1-2 cm, low acid treatment was carried out and the nutrient solution was changed to 0.5 mM KCl, 0.1 mM CaCl2, pH 4.5, overnight.

[0021] After 12 hours of low-acid treatment, pea seedlings were grown under 0 μM AlCl3, 15 μM AlCl3, and 30 μM AlCl3 (the concentration of AlCl3 in the nutrient solution) for 3 hours and 12 hours respectively. Seedlings with relatively consistent growth were selected for sampling and measurement at 1 cm from the pea lateral roots.

[0022] The pea varieties are as follows: Medium Bowl No. 6; Onward; Sima.

[0023] Example 2: RNA extraction and reverse transcription to synthesize cDNA

[0024] 1. RNA extraction

[0025] According to the instructions of Eastep super total RNA extraction kit, RNA was extracted from 1 cm of pea root tip.

[0026] 2. Reverse transcription of RNA to synthesize cDNA

[0027] Adjust the RNA concentration to 500 ng / μL and follow the TAKARA Prime Script TM Reagent Kit with gDNAEraser (Perfect Real Time) kit instructions for reverse transcription.

[0028] After reverse transcription, the cDNA concentration was determined and diluted 5 times according to the sample concentration.

[0029] 3. Amplification of target sequence

[0030] Based on the pea PsCESA gene sequence LOC127138457 (SEQ ID NO: 1, the amino acid sequence of the protein expressed is shown in SEQ ID NO: 2) on GenBank, its specific PCR primers were designed and synthesized, PCR amplification was performed on pea genomic DNA, and fluorescence quantitative PCR experiments were performed. The results are shown in Figure 3 shown.

[0031] The primer sequences are as follows:

[0032] Forward primer: 5'-GCACGGCTTGTTATTCTCG-3' (SEQ ID NO: 3);

[0033] Reverse primer: 5'-GAAAGGCGGTCAAGGTAGG-3' (SEQ ID NO: 4).

[0034] The reaction system for PCR amplification is 20 μL, including: 2 μL pea genomic DNA template (concentration of 100-200 ng / μL), 10 μL PCR universal premix, 0.8 μL forward primer (concentration of 0.4 μM), 0.8 μL reverse primer (concentration of 0.4 μM), and ddH2O to make up to 20 μL. The experiment preferably uses TAKARATB Green Premix Ex Taq II reaction system.

[0035] The amplification program of PCR amplification was as follows: pre-denaturation at 95°C for 30 s, one cycle; denaturation at 95°C for 5 s, annealing at 60°C for 30 s, 40 cycles; extension at 95°C for 5 s, 60°C for 1 min, 95°C for 10 s, one cycle; cooling at 50°C for 30 s, one cycle.

[0036] The PCR products were verified by 1% agarose gel electrophoresis, purified, recovered and sequenced.

[0037] The PCR amplification reaction product was sequenced to obtain the pea PsCESA gene nucleotide sequence as shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the pea PsCESA gene was shown in SEQ ID NO.2.

[0038] Example 3: Transcriptome data analysis

[0039] The transcriptome data analysis process is as follows:

[0040] The raw data (raw reads) were filtered using the FASTX-Toolkit tool of the FASTQ Quality Filter software (http: / / hannonlab.cshl.edu / fastxtoolkit / ). The specific steps are as follows: (1) remove reads with adapters; (2) remove reads with an N ratio of more than 10%; (3) remove low-quality reads; after all the invalid reads are removed, clean sequences (clean reads) are obtained, and all subsequent bioinformatics analyses are based on clean reads. The clean reads were aligned with the pea whole genome sequence using Tophat 2 software. After sequence alignment with the reference genome, mapped data was obtained, and then FPKM (Fragments Per Kilobases per Million fragments mapped) was converted. FPKM was used as an indicator to measure gene expression levels.

[0041] FPKM=cDNA / [Fragments Mapped Fragments(Millions)×Transcript length(Kb)]

[0042] In the formula, cDNAFragments represents the number of fragments mapped to a certain transcript, that is, the number of double-end reads; Mapped Fragments (Millions) represents the total number of fragments mapped to the transcript.

[0043] Differential expression analysis was performed to identify genes that were significantly expressed under different conditions. Differentially expressed genes (DEGs) are genes or transcripts that are significantly differentially expressed under two different conditions. |log2FC|≥1 and the corrected p-value of the difference (p-value<0.05) were used as the criteria for differential gene screening, where FC represents the ratio of expression levels between samples with two different treatments.

[0044] First, GOseq software was used to annotate all differentially expressed genes to the GO (Gene Ontology) database (http: / / www.geneontology.org / ), and the number of genes for each GO term (including molecular function, cellular composition, and biological process) was calculated. Then, the GO terms that were significantly enriched compared with the genomic background were found, and the threshold P-value was set to ≤ 0.05. Those that met this condition could be considered as significantly enriched GO terms, thereby determining the main functions of the differentially expressed genes in the biological system and the main biological processes they participated in.

[0045] Example 4: Cellulose content determination and gene expression analysis

[0046] The cell wall cellulose content of the Zhongwan No. 6 pea seedlings treated with aluminum at concentrations of 0 μM (negative control), 15 μM, and 30 μM in Example 1 was measured respectively.

[0047] The determination method is as follows: First, extract the crude cell wall, add 50mmol / L Na2Co3 (containing 20mmol / LCDTA) solution to the crude cell wall, extract at room temperature for 24h, centrifuge at 12000r for 10min, and the obtained supernatant is the pectin extract. Then add 2ml 4mol / L KOH (containing 0.1% NaBH4) to the precipitate and extract for 24h, centrifuge at 12000r for 10min, the remaining precipitate is cellulose, dry in a 50℃ oven, add 200μL 72% concentrated sulfuric acid to the cellulose precipitate and digest for 1 hour, dilute ten times with DEPC water, and hydrolyze in boiling water. The cellulose content is determined by the modified phenol-sulfuric acid method. That is, after hydrolysis, add concentrated sulfuric acid, then add 5% phenol, place in an 80℃ water bath for 15min, stand in an ice water bath for 30min, and measure the absorbance at 490nm.

[0048] Figure 1 The cellulose content of pea cell walls in pea variety Zhongwan No. 6 when treated with aluminum at concentrations of 0μM, 15μM, and 30μM. Figure 1 As shown, after being subjected to aluminum toxicity stress, the cellulose content increased with the increase of aluminum treatment concentration, indicating that this pea variety can still grow under 15μM and 30μM aluminum stress, showing high aluminum tolerance and being an aluminum-tolerant variety.

[0049] Figure 2 The results of PsCESA gene expression in different aluminum treatment times (3 hours, 12 hours) and different aluminum-resistant varieties (aluminum-resistant variety Onward, aluminum-sensitive variety Sima) with an aluminum treatment concentration of 15 μM are shown. The negative control was not treated with aluminum. Figure 2 As shown in the figure, compared with the control group without aluminum treatment, the expression level of the Onward aluminum-treated group increased, and the expression level decreased with time (A). The one with higher expression level is the aluminum-tolerant variety Onward (B), and the one without expression is the sensitive variety Sima (C). After aluminum poisoning, the aluminum-tolerant variety has a higher expression level and a significant expression effect, while the sensitive variety has no obvious expression. It can be seen that pea varieties resistant to aluminum toxicity stress can be screened by detecting the expression level of the pea PsCESA gene.

[0050] Figure 3 The results of PCR quantitative expression analysis showed differences in PCR quantitative expression when the aluminum treatment concentration was 15 μmol / L. Figure 3 As shown, the quantitative results showed that the expression was obvious.

Claims

1. Application of pea PsCESA gene in improving plant resistance to aluminum toxicity stress.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the pea PsCESA gene is shown in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that: The amino acid sequence of the protein expressed by the pea PsCESA gene is shown in SEQ ID NO:

2.

4. The use according to any one of claims 1 to 3, characterized in that: The plant is pea.

5. Application of pea PsCESA gene in promoting plant growth.

6. The use according to claim 5, characterized in that: The nucleotide sequence of the pea PsCESA gene is shown in SEQ ID NO:

1.

7. Application of pea PsCESA gene in screening pea aluminum-resistant varieties.

8. The use according to claim 7, characterized in that: The nucleotide sequence of the pea PsCESA gene is shown in SEQ ID NO:

1.

9. A kit for screening aluminum-resistant peas, characterized in that: The kit includes primers for amplifying the pea PsCESA gene.

10. The kit according to claim 9, characterized in that The nucleotide sequence of the pea PsCESA gene is shown in SEQ ID NO.1.

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