Biological method for improving tolerance of sorghum to cadmium stress and application thereof

Abstract

The application discloses a biological method for improving the cadmium stress tolerance of sorghum, identifies and clones a cadmium-induced expansin gene SbEXPA11 from a first sorghum variety plant, and transforms the overexpressed SbEXPA11 into a second sorghum variety plant to obtain a third sorghum variety with the gene, which has the biological property of cadmium stress tolerance. The SbEXPA11 overexpression in the sorghum enhances the cadmium tolerance of the sorghum and promotes the long-distance transportation of cadmium from the sorghum root to the stem.

Description

Technical Field

[0001] This invention relates to biological gene technology, specifically a biological method for improving the tolerance of sorghum to cadmium stress and its application. Background Technology

[0002] In recent decades, widespread industrial and human activities have caused severe heavy metal pollution in soils globally. Cadmium (Cd) eventually enters the food chain and ends up in plants, animals, and humans, ultimately endangering human health. Cadmium is a non-essential nutrient for plant growth and development, but it is harmful to plant cells. For example, cadmium can disrupt the electron transport chain, producing reactive oxygen species (ROS) and directly affecting plant photosynthesis. Furthermore, cadmium inhibits the absorption of essential nutrients and enzyme activity.

[0003] Sorghum (Sorghum bicolor L.) is an important multi-purpose crop used for food, feed, sugar, and lignocellulose bioenergy. Cadmium has a significant negative impact on the yield and quality of sorghum. After accumulating to a certain level, cadmium in sorghum will enter the food chain and ultimately affect human health. Therefore, there is an urgent need in current production practices for a method or technology to improve the tolerance of sorghum to cadmium stress. Summary of the Invention

[0004] (I) Analysis of the technical problems to be solved

[0005] To improve sorghum's tolerance to cadmium stress, the applicant believes it is necessary to identify key gene fragments that can control sorghum's tolerance to cadmium stress and to modify these gene fragments to improve sorghum's tolerance to cadmium stress.

[0006] Expansins play important roles in cell division and elongation, as well as in various abiotic stress responses. They are ubiquitous in plants and constitute a superfamily, divided into four subfamilies: α-expansins (EXPA), β-expansins (EXPB), expansin-like a (EXLA), and expansin-like B (EXLB) (Noh et al. 2013). Expansins belong to a large gene family, and the functions of each member are not fully understood.

[0007] Current technology indicates that there are 58 genes encoding swelling proteins in rice. In Arabidopsis, 38 swelling protein or swelling protein-like sequences have been identified. Genome-wide analysis has identified 241 swelling protein genes in the wheat genome. Furthermore, in Arabidopsis, the AtEXP7 and AtEXP18 genes have been found to be involved in root hair formation, and the HvEXPB gene is closely related to root hair formation.

[0008] This application, based on extensive preliminary experiments, discovered the mechanism of action of the SbEXPA11 gene in the cadmium stress response of sorghum, and obtained transgenic sorghum plants containing the SbEXPA11 gene. The SbEXPA11 gene was upregulated in the transcriptome sequencing of cadmium-treated sorghum. The full-length SbEXPA11 cDNA is 735 bp, encoding a polypeptide of 244 amino acid residues. Multiple sequence alignment of EXPA11 from sorghum, barley, maize, rice, and wheat showed (e.g.) Figure 1 As shown in the figure, these proteins are very similar in sequence. Multiple alignment analysis revealed significant variations in the N-terminal signal peptide region of EXPA11; however, EXPA11 exhibits remarkable similarity in the structural domains of the swelling protein EG45 and the swelling-like protein CBD. This finding provides a new pathway for the efficient enrichment of heavy metals from the environment.

[0009] (II) Technical Solution

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] A biological method for improving the tolerance of sorghum to cadmium stress, characterized by:

[0012] A cadmium-induced swelling protein gene, SbEXPA11, was identified and cloned from the first sorghum variety. After overexpression, it was transformed into the second sorghum variety to obtain the transgenic third sorghum variety, which possesses the biological properties of tolerance to cadmium stress.

[0013] Furthermore, the first sorghum variety is the Hongyingzi sorghum variety, the second sorghum variety is a wild-type (WT) variety, and the third sorghum variety is a transgenic variety that overexpresses the protein gene SbEXPA11.

[0014] Furthermore, the first sorghum variety and the second sorghum variety are the same sorghum variety, both being wild-type (WT) varieties.

[0015] Furthermore, the overexpression process is as follows: an overexpression vector is constructed, the open reading frame (ORF) of the SbEXPA11 gene is amplified and cloned into the binary vector pCambia3201 between the restriction sites EcoRI and HindIII, and the overexpression vector is introduced into wild-type (WT) varieties using the Agrobacterium-mediated sorghum transformation steps in the prior art, specifically following the operation described in previous studies (Che et al. 2018).

[0016] Furthermore, in the method for identifying the cadmium-induced swelling protein gene SbEX PA11 from the first sorghum variety, the primers used to amplify SbEXPA11 were designed based on sequences downloaded from the Gramene database (https: / / ensembl.gramene.org) (as shown in Table 1); using cDNA from red cherry sorghum seedlings grown under normal conditions as a template, the full-length SbEXPA11 ORF was amplified; then the PCR product was cloned into the pMD18-T vector, and at least three single colonies were selected for sequencing (Shanghai Sangon, China); the protein sequence was aligned using DNAMAN software (version 6.0, Lynnon Biosoft, Quebec, Canada).

[0017]

[0018] Table 1

[0019] Furthermore, the steps for extracting and detecting cDNA from the above-mentioned red cherry sorghum seedlings were as follows: total RNA was extracted from the roots and shoots of the seedlings using an RNA extraction kit (Beijing Tiangen, China); first-strand cDNA was synthesized using a cDNA synthesis kit (Beijing Tiangen, China); gene expression levels were detected using SYBR Green (Beijing Tiangen, China) on an Applied Biosystems 7900 quantitative PCR instrument; qPCR data were normalized using the 2-ΔΔCt method; and the expression of β-actin was used as an internal control (Livak and Schmittgen, 2001).

[0020] Another objective of this invention is to apply the biological method for improving the tolerance of sorghum to cadmium stress to sorghum cultivation in soil that has already been contaminated with cadmium.

[0021] Beneficial effects

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) Overexpression of SbEXPA11 in sorghum enhanced tolerance to cadmium and promoted long-distance cadmium transport from root to stem. Overexpression of SbEXPA11 in sorghum plants increased cadmium accumulation and reduced cadmium toxicity. Therefore, transformation of SbEXPA11 in sorghum improved the adaptability of cadmium-contaminated soil to phytoremediation.

[0024] (2) Overexpression of SbEXPA11 can alter the physiological and biochemical indicators of transgenic sorghum. Under cadmium stress, the activities of CAT, POD and SOD in transgenic plants are significantly higher than those in wild-type (WT) plants, while malondialdehyde (MDA) accumulates less in transgenic plants. Compared with wild-type plants, transgenic sorghum plants have a stronger ability to scavenge reactive oxygen species (ROS) and reduce cadmium-induced cell membrane damage. Attached Figure Description

[0025] Figure 1 Amino acid sequence alignment results of EXPA11 from sorghum, corn, rice, and wheat.

[0026] Figure 2 SbEXPA11 gene expression under different concentrations of cadmium treatment.

[0027] Figure 3 Expression of the SbEXPA11 gene at different time points under 50 μM cadmium treatment. Different letters at the top of the column indicate significant differences (p<0.05).

[0028] Figure 4 Growth phenotypes of WT and SbEXPA11 transgenic plants under hydroponic conditions.

[0029] Figure 5 and Figure 6 Cadmium content in the aboveground parts and roots of SbEXPA11 transgenic plants under hydroponic conditions, with asterisks indicating significant differences (p<0.05).

[0030] Figure 7 Growth phenotypes of WT and SbEXPA11 transgenic plants under pot conditions.

[0031] Figure 8-11 Under hydroponic conditions, the content of MDA, and the activities of SOD, POD and CAT in WT and SbEXPA11 transgenic plants were compared. Asterisks indicate significant differences (p<0.05).

[0032] Figure 12 : Identification of transgenic lines using PCR technology.

[0033] Figure 13 The relative expression levels of SbEXPA11 in transgenic and wild-type plants, with asterisks indicating significant differences (p<0.05). Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Please see Figure 1-13 The technical solution of this application is described in detail.

[0036] Example 1: Synthesis steps of transgenic varieties overexpressing the protein gene SbEXPA11

[0037] S1: Using cDNA from red cherry sorghum seedlings grown under normal conditions as a template, the full-length SbEXPA11ORF was amplified; next, the SbEXPA11 ORF was identified, cloned into the pMD18-T vector, and at least 3 single colonies were selected for sequencing; finally, the protein sequence was aligned using DNAMAN software (version 6.0, Lynnon Biosoft, Quebec, Canada). If the alignment was successful, it was the desired cadmium-induced swelling protein gene SbEXPA11.

[0038] S2: Overexpress the swelling protein gene SbEXPA11 obtained in step S1, and transform the overexpressed swelling protein gene SbEXPA11 into wild-type (WT) sorghum plants. The overexpression process is as follows: construct an overexpression vector, and use the Agrobacterium-mediated sorghum transformation step in the existing technology to amplify and clone the open reading frame (ORF) of the SbEXPA11 gene into the binary vector pCambia3201 between the restriction sites EcoR I and Hind III, and finally obtain a transgenic variety that overexpresses the protein gene SbEXPA11.

[0039] Example 2: Screening and identification of 3 sorghum lines overexpressing the protein gene SbEXPA11

[0040] To understand the function of the SbEXPA11 gene, transgenic sorghum plants overexpressing SbEXPA11 were bred based on the method in Example 1. Four independent transgenic lines were obtained, and positive transgenic plants were identified by genomic PCR and RT-qPCR. Figure 12 ).

[0041] After PCR screening, we identified four positive transgenic sorghum plants. Figure 7Then, RT-qPCR was used to determine gene expression in transgenic plants. Subsequent experiments were conducted using three independent T3 transgenic plants; the highest SbEXPA11 expression levels were observed in plants OE1 and OE2. Figure 13 ).

[0042] Example 3: Identification of cadmium stress tolerance in transgenic varieties overexpressing the SbEXPA11 protein gene

[0043] In this embodiment, a control group and an experimental group were set up. The control group was wild-type (WT) seeds, and the experimental group was transgenic sorghum seeds.

[0044] (1) Preparation of experimental materials

[0045] In the hydroponic experiment, wild-type (WT) seeds and transgenic sorghum seeds were germinated on filter paper in a dark and humid environment at 30°C. The germinated seeds were then transferred to a hydroponic incubator filled with Hoagland's solution, which was changed every 3 days. Seedlings approximately 3 weeks old were then treated with 0 or 50 μM Cd (in the form of CdCl2·5H2O) in Hoagland's solution for 3 days, and root and shoot tissues were collected as needed.

[0046] The pot experiment was conducted under greenhouse conditions. 10 kg of potting soil was mixed with 100 mg of Cd (in the form of CdCl2·5H2O). The control group was only watered with potting soil. Seedlings approximately 3 weeks old were planted in flowerpots, and 2 L of Hoagland's nutrient solution was applied every 10 days. Photographs were taken after 30 days of cultivation (e.g., ...). Figure 4 (As shown).

[0047] (2) Determination of cadmium and malondialdehyde content and SOD, CAT and POD activity

[0048] According to Zhang et al. (2019), cadmium content was measured by inductively coupled plasma mass spectrometry. The MDA content and antioxidant enzyme activities, including SOD, CAT, and POD, in sorghum were determined using a kit from the Nanjing Jiancheng Biotechnology Institute, China.

[0049] (3) Statistical analysis

[0050] Statistical analysis was performed using one-way ANOVA, followed by Tukey's post-hoc test. Data are expressed as mean ± standard error of three replicates. A p-value less than 0.05 was considered statistically significant.

[0051] (4) Results

[0052] Treatment with different concentrations of cadmium increased SbEXPA11 expression, with the highest expression level observed at 50 μM cadmium induction. Figure 2The expression level of SbEXPA11 increased after treatment with different concentrations of cadmium, and its expression level was highest under 50 μM cadmium induction. Figure 2 Furthermore, SbEXPA11 expression reached its highest level at 12 h after treatment with 50 μM cadmium. Figure 3 ).

[0053] (5) Results Analysis

[0054] Firstly, overexpression of SbEXPA11 enhanced long-distance cadmium transport: cadmium content in the aboveground parts and roots of sorghum was determined in a 50 μM cadmium solution. The cadmium content in the aboveground parts of transgenic sorghum was significantly higher than that in wild-type (WT) plants. Figure 5 Furthermore, the cadmium accumulation in the roots of transgenic sorghum was significantly higher than that in wild-type (WT) plants. Figure 6 When transgenic plants were cultured in a cadmium-containing nutrient solution, they exhibited significant cadmium sensitivity. In contrast, transgenic plants were less affected by cadmium stress damage. Figure 7 These results indicate that SbEXPA11 overexpression in sorghum enhances cadmium tolerance and promotes long-distance cadmium transport from sorghum roots to stems.

[0055] Secondly, overexpression of SbEXPA11 altered the physiological and biochemical indicators of transgenic sorghum: under cadmium stress, the activities of CAT, POD, and SOD in transgenic plants were significantly higher than those in wild-type (WT) plants, while malondialdehyde (MDA) accumulation was lower in transgenic plants. Figure 8-11 The results showed that, compared with wild-type plants, transgenic sorghum plants had a stronger ability to scavenge reactive oxygen species (ROS) and reduced cadmium-induced cell membrane damage.

[0056] It should be noted that the methods and steps not described in detail in the above embodiments (such as: cloning technology for introducing exogenous genes into receptors, PCR amplification methods, cDNA synthesis methods, etc.) are all common methods and techniques in the existing biotechnology field.

[0057] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A biological method for improving the tolerance of sorghum to cadmium stress, characterized in that: a cadmium-induced expansin gene SbEXPA11 is identified and cloned from the first sorghum variety plant, and after overexpression and transformation into the second sorghum variety plant, a third sorghum variety is obtained, which has the biological property of tolerance to cadmium stress; the first sorghum variety is Hongyingzizi sorghum variety, the second sorghum variety is wild type variety, and the third sorghum variety is a transgenic variety overexpressing the protein gene SbEXPA11.

2. The biological method of increasing tolerance of sorghum to cadmium stress as claimed in claim 1, wherein, The overexpression process is: constructing an overexpression vector, amplifying and cloning the open reading frame of the SbEXPA11 gene into the binary vector pCambia3201 between the restriction sites EcoRI and HindIII.

3. The biological method of increasing tolerance of sorghum to cadmium stress as claimed in claim 2, wherein: In the process of identifying the cadmium-induced expansin gene SbEXPA11 from the first sorghum variety plant, the primers for amplifying SbEXPA11 are designed according to the sequence downloaded from the Gramene database; the cDNA of Hongyingzizi sorghum seedlings grown under normal conditions is used as the template to amplify the full-length SbEXPA11 ORF; then the PCR product is cloned into the pMD18-T vector, and at least 3 single colonies are picked for sequencing; the protein sequence is aligned using DNAMAN software.

4. The biological method of increasing tolerance of sorghum to cadmium stress as claimed in claim 3, wherein: The extraction and detection steps of the above-mentioned cDNA of Hongyingzizi sorghum seedlings are: total RNA is extracted from the roots and shoots of plant seedlings using an RNA extraction kit, first-strand cDNA synthesis is performed using a cDNA synthesis kit, gene expression levels are detected using SYBR Green on an Applied Biosystems 7900 quantitative PCR instrument, qPCR data is normalized using the 2-ΔΔCt method, and the expression of β-actin is used as an internal control.

5. Use of a biological method for increasing the tolerance of sorghum to cadmium stress, characterized in that: The biological method for improving the tolerance of sorghum to cadmium stress according to any one of claims 1-4 is applied to the planting of sorghum in soil that has been contaminated by cadmium.

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