Application of TaMYB1 gene to improve plant tolerance to Cd

Abstract

The application relates to the field of agricultural biotechnology, in particular to application of TaMYB1 gene for improving Cd tolerance of plants. The application takes yeast and wheat as research materials, adds different concentrations of Cd ions in a yeast culture medium to screen a yeast two-hybrid library of wheat root under Cd stress, obtains a wheat Cd stress response related gene TaMYB1, uses a virus-induced gene silencing technology to perform function verification and phenotype analysis on the related gene, and performs bioinformatics analysis on the TaMYB1, so as to lay a foundation for function research of the TaMYB1 responding to Cd stress and cultivation of a new wheat variety with low Cd accumulation.

Description

Technical Field

[0001] This application relates to the field of agricultural biotechnology, specifically to the application of the TaMYB1 gene in improving plant tolerance to Cd. Background Technology

[0002] Heavy metals are the main pollutants affecting the environmental quality of farmland soil. Excessive accumulation of cadmium (Cd) in plants can seriously affect their normal growth, leading to oxidative damage, inhibition of photosynthesis and respiration, and weakened absorption of mineral nutrients. This can cause leaves to turn yellow, root tips to turn brown, and growth to slow down, and in severe cases, even lead to plant death.

[0003] In recent years, Cd pollution has become increasingly serious in some major wheat-producing areas. Discovering Cd tolerance genes in plants and using plant breeding methods to cultivate low-Cd accumulation varieties or Cd-tolerant varieties is an important and sustainable means to achieve wheat food security, and has significant practical significance and application value.

[0004] Over long periods of evolution and development, plants have developed a series of transcriptional and post-translational regulatory networks in response to Cd stress, with transcription factors playing a crucial role. The MYB family is a key transcription factor involved in regulating various biological processes. Based on the number, location, and highly conserved domains of MYB transcription factors, the MYB family can be divided into four categories: 1R-MYB / MYB-related, R2R3-MYB, R1R2R3-MYB, and 4R-MYB.

[0005] Different members of the MYB family exhibit varying functions in different plants. For example, overexpression of the rice R2R3 type member OsMYB49 significantly increases Cd accumulation, while the Osmyb49 mutant reduces Cd accumulation. The rice R2R3-MYB protein OsMYB45 is induced by the heavy metal Cd, and the mutant Osmyb45 reduces rice's tolerance to Cd stress; the ramie 1R-MYB protein BnMYB2 positively regulates Cd tolerance and accumulation.

[0006] However, research on wheat MYB genes is limited, mainly focusing on responses to biotic stresses. For example, overexpression of the R2R3-MYB member TaRIM1 enhances wheat resistance to Rhizoctonia graminearum; overexpression of the R2R3-MYB member TaMYB391 positively regulates wheat resistance to stripe rust; and the MYB protein Tapipp2 is induced by fungi and upregulates salicylic acid and jasmonic acid levels. Currently, no research has been reported on the role of wheat MYB family members in responding to Cd stress. Summary of the Invention

[0007] The purpose of this invention is to provide an application of the TaMYB1 gene to improve plant tolerance to Cd.

[0008] Another object of the present invention is to provide a method for improving the plant's tolerance to Cd.

[0009] This invention provides the application of the TaMYB1 gene to improve plant tolerance to Cd, wherein the TaMYB1 gene encodes a protein with the amino acid sequence shown in SEQ ID No: 1.

[0010] SEQ ID No: 1

[0011] MCSMQAEHCQYMTEPEQMYHQQQQQFHDHRQHMSSRPSLSPENKFFMKGQGGAGAGGGG

[0012] DAGLILSTDAKPRLKWTPELHERFADAVKKLGGPDKATPKAIMRVMGIPGLTLYHLKSHLQKF

[0013] RLSKNLQAQANAVHAKNVYGFGTATDKACEGRGSPADHLNRETNTSRSMHINDALQMQIEVQ

[0014] RRLHEQIEVQRHLQLRIEAQGKYLHSVLEKAQEALGKQHVVAGLEAAEPTQRLPELASSVRRG

[0015] LLQNDGSADDSCLTASEDILSMGLSASATRRGCGAPFETSASASREEDGECYLFLGKPEGRREVRRDGCSGGAAFGTAAELDLSIGVVAASSRRRPDGGERLDLNGSGWN.

[0016]

[0017] According to the technical solution of the present invention, the plant's tolerance to Cd is improved by silencing the TaMYB1 gene in the plant.

[0018] According to the method for improving plant tolerance to Cd according to the present invention, the method includes the step of silencing the TaMYB1 gene in the plant, wherein the TaMYB1 gene encodes a protein with the amino acid sequence shown in SEQ ID No: 1.

[0019] According to the method for improving plant tolerance to Cd of the present invention, the nucleotide sequence of the TaMYB1 gene is shown in SEQ ID No: 2.

[0020] According to the method of improving plant tolerance to Cd of the present invention, the 529-728bp of the CDS region of the TaMYB1 gene is constructed into a viral vector by homologous recombination, and then the virus is inoculated into plants to silence the TaMYB1 gene.

[0021] This application screened wheat yeast strains resistant to Cd stress using *Saccharomyces cerevisiae*, extracted yeast plasmids, and obtained the wheat Cd stress response gene TaMYB1 through sequencing analysis. Virus-induced TaMYB1 gene silencing experiments showed that the growth of control plants was inhibited under Cd stress; however, the growth of TaMYB1 gene-silenced plants was significantly better than that of control plants, and the Cd content in the roots and leaves of TaMYB1 gene-silenced plants was significantly lower than that of control plants, indicating that the TaMYB1 gene positively regulates the wheat response to the heavy metal Cd. Furthermore, the silencing region (529bp-728bp) selected in this application for constructing the TaMYB1 gene silencing vector is an essential domain for TaMYB1 gene function.

[0022] This application examined the chlorophyll content, SOD, POD activity, and MDA content of TaMYB1 gene-silenced plants and control plants under normal growth conditions and Cd stress. TaMYB1 gene-silenced plants showed enhanced Cd tolerance; compared to control plants, their chlorophyll content, SOD and POD activity were significantly increased (P = 0.01), while their MDA content was significantly decreased (P = 0.01). This indicates that silencing the TaMYB1 gene reduced MDA content, enhanced the antioxidant enzyme system in wheat, and reduced Cd toxicity. In conclusion, this application lays the foundation for functional research on the wheat MYB family's response to heavy metal Cd stress. Future research will utilize gene editing technology to obtain TaMYB1-edited plants, providing a reference for the remediation of Cd-contaminated farmland soil. Attached Figure Description

[0023] Figure 1The following data are displayed: A: Phenotypic characteristics of wheat plants under Cd stress (the plant on the left is the control, and the plant on the right is under Cd stress); B: Root length of wheat plants under Cd stress; C: Aboveground length of wheat plants under Cd stress; D: Biomass of wheat plants under Cd stress.

[0024] Figure 2 The quality detection of the yeast two-hybrid library constructed from Cd-stressed wheat roots is shown in the figures: A: Total RNA from Cd-stressed wheat roots; B: High-quality cDNA obtained using SMART technology; C: cDNA purified by column chromatography; DE: Detection of yeast library insert fragments.

[0025] Figure 3 The growth of transformants is shown on media with different Cd concentrations: A: 0.002 mol / L CdCl2, B: 0.003 mol / L CdCl2, C: 0.004 mol / L CdCl2.

[0026] Figure 4 The growth curves of yeast colonies under Cd chloride conditions are shown. A: Growth curve of yeast under normal conditions; B: Growth curve of yeast in high Cd medium.

[0027] Figure 5 The silencing of the TaMYB1 gene enhances wheat's tolerance to Cd. A: Phenotypes of TaMYB1 gene-silenced and control plants under normal conditions and Cd stress. The two plants on the left are under normal treatment, and the two plants on the right are under Cd stress treatment. B: Expression level of TaMYB1 gene-silenced plants. C: Cd content of TaMYB1 gene-silenced and control plants after Cd stress. ** indicates a significant difference at the P=0.01 level.

[0028] Figure 6 The results show the physiological indicators of TaMYB1 gene silencing and control plants under normal conditions and Cd stress. A: chlorophyll content of wheat leaves, B: MDA content of wheat leaves, C: SOD activity of wheat leaves, D: POD activity of wheat leaves. ** indicates significant differences at the P=0.01 level. Detailed Implementation

[0029] Example 1

[0030] 1. Cd-stressed wild-type wheat

[0031] The plant material was Zhoumai 26. After the wheat seeds were disinfected, they were placed in an artificial climate chamber with 16 hours of light (25℃) / 8 hours of darkness (15℃) and 60% humidity. After hydroponics until the seedlings reached the one-leaf-one-heart stage, wheat seedlings with uniform growth were selected. Some seedlings were used as controls and cultured in Hoagland total nutrient solution, while others were treated with different concentrations of Cd in Hoagland total nutrient solution. After 10 days, the growth status of the wheat was observed, and the root length, stem length and biomass of the plants under different treatments were measured.

[0032] After Zhoumai 26 wheat seedlings were cultured in Hoagland total nutrient solution containing 40 mg / L Cd for 10 days, the results showed that the root length of Cd-stressed wheat plants was significantly different from that of control plants, while no significant changes were observed in the aboveground parts. The root length of Cd-stressed plants was significantly shorter than that of control plants, indicating that Cd stress significantly inhibited the growth of wheat root length. Figure 1 (A and B), and analysis of their biomass revealed that Cd stress reduced the biomass of wheat plants ( Figure 1 The above results indicate that cadmium stress mainly inhibits the development of wheat roots.

[0033] 2. Total RNA extraction and cDNA reverse transcription from wheat roots

[0034] Cd-treated wheat roots were taken, washed thoroughly with water, flash-frozen in liquid nitrogen, and then placed at -80°C. High-quality wheat RNA was extracted using TRIzol reagent (Invitrogen). A portion of the RNA was used to synthesize cDNA according to the cDNA reverse transcription kit instructions for gene cloning and real-time quantitative PCR.

[0035] High-quality RNA was extracted from Cd-stressed wheat roots using TRIzol reagent. Figure 2 A), and then high-purity cDNA was obtained by reverse transcription using SMART technology. Figure 2 B), and cDNA was purified by column chromatography. Figure 2 A two-hybrid cDNA library of yeast from Cd-stressed wheat roots was constructed. Clonal counting of the yeast library revealed a wheat cDNA library titer of 4.6 × 10⁻⁶. 7 cfu / mL. Thirty-two yeast clones were randomly selected for PCR detection, and all clones amplified bands. The length of the inserted fragments ranged from 300 bp to greater than 3 kbp, with a recombination rate of approximately 99%. Figure 2 The results (D, E) indicate that the constructed cDNA library is of good quality.

[0036] 3. Construction and screening of two-hybrid libraries of yeast from wheat roots under Cd stress

[0037] High-quality wheat RNA was collected after Cd stress, and a yeast two-hybrid library of Cd-stressed wheat roots was constructed using a yeast two-hybrid library construction kit. The quality of the library was then assessed. Simultaneously, the empty vector pGADT7 was transformed into wild-type yeast strain Y187 as a control for subsequent library screening.

[0038] Screening of yeast Y187 transformed with the pGADT7 empty vector using critical Cd concentrations revealed that the yeast could not grow on medium containing 0.002 mol / L Cd. Therefore, 0.002 mol / L Cd was used as the critical concentration for screening the yeast library. A total of 18 positive Cd-tolerant transformants were screened on media containing 0.002 mol / L, 0.003 mol / L, and 0.004 mol / L Cd. Figure 3 Yeast plasmids were extracted, sequenced, and compared, yielding four candidate genes. Five transformants encoded the transcription factor TaMYB1. TaMYB1 was selected as the target gene, and its function was verified. Furthermore, the growth curves of yeast transformed with pGADT7 and pGADT7-TaMYB1 were examined in normal and high-Cd (0.004 mol / L) media, respectively. The results showed that the growth rates of the two yeasts were comparable in normal media. Figure 4 (A). In high-Cd medium, the growth rate of the control strain transformed with pGADT7 was significantly inhibited, while the yeast strain transformed with pGADT7-TaMYB1 grew well, indicating that the expression of TaMYB1 improved the yeast's Cd tolerance. Figure 4 B).

[0039] 4. Screening of wheat genes related to Cd stress response and determination of growth curves

[0040] First, different concentrations of Cd were added to the yeast culture medium SD-Leu to screen for critical concentrations of yeast Y187 transformed with the pGADT7 empty vector. After screening to the critical concentration at which yeast transformed with the pGADT7 empty vector could not grow, a Cd-induced wheat root yeast two-hybrid library was plated on the medium with the critical Cd concentration and cultured at 30℃ for 3-5 days. Yeast plasmids were extracted using the glass bead liquid nitrogen method, and the plasmids were sequenced and sequence alignment analyzed. In addition, colonies of pGADT7 empty vector yeast and yeast colonies resistant to Cd stress were picked and placed in SD-Leu liquid medium supplemented with 0.004 mol / L Cd chloride. The culture was shaken at 30℃ and 220 rpm / min. The bacterial culture was sampled every 12 hours until 60 hours, and the OD was measured. 600 And record the data.

[0041] 5. Functional verification of Cd stress response genes

[0042] The obtained yeast plasmid was BLAST-aligned using NCBI to obtain the target gene. The CDS sequence of TaMYB1 (Accession No. XM_020305596.1) was downloaded, and virus-induced gene silencing (VIGS) primers were designed for PCR amplification. After purification of the PCR product, the viral vector pYL156 was constructed using a novoprotein (NR005) and sent to a biotechnology company for sequencing. The positive recombinant vector TRV:TaMYB1 and the empty pYL156 vector were transformed into Agrobacterium GV3101, and wheat seeds were infected.

[0043] Table 1 Primer sequences

[0044]

[0045] 6. Cd content detection in wheat

[0046] Wheat plants infected with Agrobacterium tumefaciens containing TRV:TaMYB1 recombinant plasmid and control plasmid for 16 days were irrigated with a 40 mg / L Cd solution. Ten days later, roots and leaves of control plants and TaMYB1 transiently silenced plants were collected, washed with clean water, blanched in an oven at 105℃ for 30 min, and dried at 80℃ to constant weight. 0.2 g of roots and leaves were sent to the Agricultural Product Quality Supervision and Testing Center of the Ministry of Agriculture and Rural Affairs for Cd content detection using inductively coupled plasma mass spectrometry.

[0047] 7. Detection of physiological indicators of wheat

[0048] Plants experience a surge in reactive oxygen species (ROS) under heavy metal stress, which damages plant cells. Through long-term evolution, plants have activated antioxidant systems to scavenge excess ROS and prevent toxic effects on plant cells. Meanwhile, MDA (membrane lipid peroxidation) is one of the main products of Cd stress-induced membrane lipid peroxidation in plants, which can exacerbate cell damage and serves as a reference indicator for studying resistance physiology. Studies have found that plants can resist oxidative damage caused by Cd stress by increasing SOD and POD activities.

[0049] Leaves from different lines of normally growing and Cd-treated control plants and TamYB1 silent plants were selected. Chlorophyll content was determined by acetone-ethanol extraction method, and malondialdehyde (MDA) content, SOD activity, and POD activity were determined according to the kit instructions.

[0050] 8. qRT-PCR

[0051] RNA was extracted from the remaining wheat roots and leaves (as described in section "7"), reverse transcribed into cDNA, diluted 10-20 times, and subjected to qRT-PCR to analyze the relative expression level of TaMYB1. The Actin gene (Accession No. KC775780) was used as an internal reference gene, and relative quantification was performed. -ΔΔCt Perform RNA transcriptome alteration analysis.

[0052] Homologous recombination was used to construct the 529-728 bp region of the TaMYB1 gene CDS into the TRV viral vector pYL156. Wheat was inoculated with TRV virus for 16 days, followed by 10 days of Cd stress treatment. Results showed that under normal growth conditions, both TaMYB1-silenced plants (TRV:TaMYB1) and control plants (TRV:00) grew normally. However, TRV:00 plants subjected to 40 mg / L Cd stress showed severely inhibited growth, stunted growth, significantly shortened total root length, and wilted, yellowing leaves. TRV:TaMYB1 plants, after Cd stress, grew well, exhibiting similar growth patterns to TaMYB1-silenced plants and the control under normal conditions. Figure 5 qRT-PCR results showed that, compared with the control, the expression level of TamyB1 in the roots and leaves of TRV:TaMYB1 plants was significantly decreased, indicating a good silencing effect. Figure 5 (B) The Cd content in the roots and leaves of TRV:00 and TRV:TaMYB1 plants was detected by inductively coupled plasma mass spectrometry. The results showed that the Cd content in the roots and leaves of TRV:00 plants was significantly higher than that in TRV:TaMYB1 plants, and the Cd content in the roots was much higher than that in the leaves. Figure 5 In addition, physiological indicators such as chlorophyll content, SOD (superoxide dismutase) activity, POD (peroxidase) activity, and MDA (malondialdehyde) content in the leaves of TRV:00 and TRV:TaMYB1 plants were measured. The results showed that after Cd stress, the chlorophyll content, SOD and POD activities of TRV:00 plants were significantly lower than those of TRV:TaMYB1 plants. Figure 6 (A, B, and C), while the MDA content of TRV:00 plants was significantly higher than that of TRV:TaMYB1 plants (A, B, and C). Figure 6 (D). This indicates that silencing TaMYB1 may resist Cd toxicity to plants by increasing chlorophyll content, SOD and POD activity, thereby reducing Cd content in the aboveground parts and roots of wheat plants.

[0053] The above embodiments are only used to explain the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. Through silent wheat TaMYB1 The application of genes to enhance wheat's tolerance to Cd is characterized by, The TaMYB1 The gene encodes a protein with the amino acid sequence shown in SEQ ID No:

1.

2. The application according to claim 1, characterized in that, The silence TaMYB1 The 529-728bp region of the gene's CDS.

3. The application according to claim 1, characterized in that, The TaMYB1 The nucleotide sequence of the gene is shown in SEQ ID No:

2.

4. A method for improving wheat's tolerance to Cd, characterized in that, The method includes silent wheat. TaMYB1 The steps of gene generation, wherein, TaMYB1 The gene encodes a protein with the amino acid sequence shown in SEQ ID No:

1.

5. The method for improving wheat's tolerance to Cd according to claim 4, characterized in that, Using homologous recombination to TaMYB1 The 529-728bp region of the gene's CDS region was constructed into a viral vector, and then the virus was inoculated into wheat to silence the virus. TaMYB1 Gene.

6. The method for improving wheat's tolerance to Cd according to claim 4, characterized in that, The TaMYB1 The nucleotide sequence of the gene is shown in SEQ ID No: 2.

Images