Application of TaZFP2 gene to improve plant tolerance to Cd

Abstract

The application relates to the field of agricultural biotechnology, in particular to application of TaZFP2 gene for improving Cd tolerance of plants. The application takes yeast and wheat as research materials, selects a wheat root system yeast two-hybrid library under Cd stress by adding different concentrations of Cd ions in a yeast culture medium, obtains a wheat Cd stress response related gene TaZFP2, uses a virus-induced gene silencing technology to perform function verification and phenotype analysis on the related gene, and performs bioinformatics analysis on the TaZFP2, so as to lay a foundation for function research of the TaZFP2 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 TaZFP2 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] Zinc finger transcription factors respond to and regulate the growth and development of eukaryotes, as well as biotic and abiotic stress responses, at the transcriptional level. The Dof subfamily of transcription factors contains a highly conserved Dof domain of 52 amino acid residues at its N-terminus, and four highly conserved cysteine ​​(Cys) residues (CX2CX21CX2C) that interact with Zn. 2+ The covalent binding between them is a hallmark of Dof transcription factors; however, the amino acid sequences of their C-terminal transcriptional regulatory domains vary considerably, which may be the reason for the different response mechanisms of plants to various abiotic stresses. As plant-specific transcription factors, DoF genes play important roles in plant cell signal transduction, hormone signaling pathways, guard cell differentiation, fatty acid metabolism, flower drop, nitrogen assimilation, flowering time regulation, seed germination, and biotic and abiotic stresses. For example, OsDOF24 delays leaf senescence by inhibiting the activity of genes related to jasmonic acid synthesis, while AtDOF2.1 plays an active role in jasmonic acid-induced leaf senescence through a positive feedback transcription loop of MYC2-DOF2.1-MYC2. In Arabidopsis, overexpression of pear PbDOF9.2 leads to delayed flowering time, and similarly, overexpression of alfalfa MtCDFd1-1 and tomato SlCDF3 leads to delayed flowering. Overexpression of grape VvDOF3 enhances plant resistance to powdery mildew, and overexpression of birch BpDOF4, BpDOF11, and BpDOF17 genes improves drought resistance by enhancing reactive oxygen species scavenging capacity. OsDOF11 regulates nitrogen metabolism by affecting sugar transport and distribution, thereby coordinating carbon and nitrogen balance to maintain plant life processes. However, there is limited research on the biological functions of wheat Dof transcription factors in the heavy metal stress regulatory network. Summary of the Invention

[0005] The purpose of this invention is to provide an application of the TaZFP2 gene to improve plant tolerance to Cd.

[0006] Another object of the present invention is to provide a method for improving the plant's tolerance to Cd.

[0007] This invention provides the application of the TaZFP2 gene to improve plant tolerance to Cd, wherein the TaZFP2 gene encodes a protein with the amino acid sequence shown in SEQ ID No: 1.

[0008] SEQ ID No: 1

[0009] MDAAQWHQGLGLVKPMEEMIMAGNPNPPNPNGNPNPQPAPPSGAEAQRAPVPGPPAGGAG

[0010] AAAGTGSTERKAARPQKEKAINCPRCNSTNTKFCYYNNYSLQQPRYFCKTCRRYWTEGGSLR

[0011] NVPVGGGSRKNKRSSSSVVSSAAGAVSTSGAASGTVPVGGMAAKNPKLMHEGAHDLNLAFP

[0012] HHHGRVLHPSEFAAFPSLESSSVCNPGGTMAANGAGGGRGMGAFSAMELLRSTGCYVPMPQV

[0013] QLGMPPEYAAAGFALGEFRMPLQHQQHHQQQQQQHQQHQQQVQNMLGFSLDTGGGGDAG

[0014] GYGAGLQGAQESATGRMLFPFEDLKPGTNAGGGGASGGDQFEHSKDQGGGGGGGHETLGFWNNSMIGNGSSNDAGGGGGGGGGSW.

[0015] According to the technical solution of the present invention, the nucleotide sequence of the TaZFP2 gene is shown in SEQ ID No: 2.

[0016] SEQ ID No: 2

[0017] ATGGATGCGGCCCAGTGGCACCAGGGGCTAGGACTCGTGAAGCCCATGGAGGAGATGATCA

[0018] TGGCTGGAAACCCAAATCCTAATCCTAATCCTAATGGGAACCCGAACCCACAGCCGGCGCC

[0019] ACCGTCTGGCGCAGAAGCCCAGAGGGCTCCCGTTCCTGGCCCGCCGGCAGGAGGAGCTGG

[0020] CGCGGCGGCCGGGACGGGCTCCACCGAGCGGAAGGCGGCGCGGCCGCAGAAGGAGAAGG

[0021] CAATCAACTGCCCGAGGTGCAACTCCACCAACACCAAGTTCTGCTACTACAACAACTACAG

[0022] CCTCCAGCAGCCGCGCTACTTCTGCAAGACGTGCCGCCGCTACTGGACCGAGGGCGGCTCG

[0023] CTCCGGAACGTGCCCGTCGGCGGCGGCTCACGGAAGAACAAGAGATCGTCGTCGTCGGTG

[0024] GTGTCGTCCGCGGCGGGCGCCGTCTCCACCTCCGGGGCGGCGTCCGGGACGGTCCCGGTC

[0025] GGCGGGATGGCGGCCAAGAACCCGAAGCTGATGCACGAGGGCGCGCACGACCTCAACCTG

[0026] GCGTTCCCGCACCACCACGGCCGCGTCTTGCACCCGTCGGAGTTCGCGGCGTTCCCTAGCC

[0027] TGGAGAGCAGCAGCGTCTGCAACCCGGGAGGCACCATGGCGGCCAACGGCGCGGGTGGC

[0028] GGGAGGGGCATGGGCGCGTTTTCGGCGATGGAGCTGCTGAGGAGCACCGGCTGCTACGTA

[0029] CCGATGCCGCAGGTGCAGCTCGGGATGCCGCCTGAGTACGCAGCCGGGATTCGCGCTTG

[0030] GCGAGTTCCGCATGCCGCTGCAGCATCAGCAACACCATCAGCAGCAGCAGCAGCAGCAAC

[0031] ACCAGCAACACCAGCAGCAGGTTCAGAACATGCTCGGGTTCTCTTTGGACACGGGAGGAG

[0032] GTGGCGACGCCGGGGGATACGGCGCTGGATTGCAGGGGGCGCAGGAGAGCGCAACGGGA

[0033] AGGATGCTGTTCCCGTTCGAGGACTTGAAGCCGGGTACGAACGCGGGTGGAGGAGGTGCA

[0034] AGCGGCGGCGATCAGTTTGAGCACAGCAAAGACCAAGGCGGCGGTGGCGGCCGGCGGCCAT

[0035] GAGACCCTGGGGTTCTGGAATAACAGCATGATCGGGAATGGCAGCAGCAATGACGCCGGCGGTGGCGGTGGCGGCGGCGGTGGTTCGTGGTAG.

[0036] According to the technical solution of the present invention, the plant's tolerance to Cd is improved by silencing the TaZFP2 gene in the plant.

[0037] According to the method for improving plant tolerance to Cd according to the present invention, the method includes the step of silencing the TaZFP2 gene in the plant, wherein the TaZFP2 gene encodes a protein with the amino acid sequence shown in SEQ ID No: 1.

[0038] According to the method for improving plant tolerance to Cd of the present invention, the nucleotide sequence of the TaZFP2 gene is shown in SEQ ID No: 2.

[0039] According to the method of improving plant tolerance to Cd of the present invention, the 482bp-681bp region of the CDS region of the TaZFP2 gene is constructed into a viral vector by homologous recombination, and then the virus is inoculated into the plant to silence the TaZFP2 gene.

[0040] This application screened wheat yeast strains resistant to Cd stress using *Saccharomyces cerevisiae*, extracted yeast plasmids, and obtained the wheat Cd stress response gene TaZFP2 through sequencing analysis. Virus-induced TaZFP2 gene silencing experiments showed that the growth of control plants was inhibited under Cd stress; however, the growth of TaZFP2 gene-silenced plants was significantly better than that of control plants, and the Cd content in the roots and leaves of TaZFP2 gene-silenced plants was significantly lower than that of control plants, indicating that the TaZFP2 gene positively regulates the wheat response to the heavy metal Cd. Furthermore, the silencing region (482-681 bp) selected in the construction of the TaZFP2 gene silencing vector coincides with the location of the Dof domain, suggesting that the Dof domain is essential for the function of the TaZFP2 gene. Further point mutation technology will be used to determine its function in future studies.

[0041] This application examined the chlorophyll content, SOD, POD activity, and MDA content of TaZFP2 gene-silenced plants and control plants under normal growth conditions and Cd stress. Similar to previous studies, TaZFP2 gene-silenced plants exhibited enhanced Cd tolerance. Compared to control plants, their chlorophyll content, SOD and POD activity were significantly increased (P = 0.01), while MDA content was significantly decreased (P = 0.01). These results suggest that TaZFP2 gene silencing reduces MDA content, enhances the antioxidant enzyme system in wheat, and reduces Cd toxicity. In conclusion, this application lays the foundation for functional research on wheat ZFP family Dof transcription factors in response to heavy metal Cd stress. Future research will utilize gene editing technology to obtain TaZFP2-edited plants, providing a reference for the remediation of Cd-contaminated farmland soil. Attached Figure Description

[0042] Figure 1 The 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] Figure 5 The silencing of the TaZFP2 gene enhances wheat's tolerance to Cd. A: Phenotypes of TaZFP2 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 TaZFP2 gene-silenced plants. C: Cd content of TaZFP2 gene-silenced and control plants after Cd stress. ** indicates a significant difference at the P=0.01 level.

[0047] Figure 6 The results show the physiological indicators of TaZFP2 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

[0048] Example 1

[0049] 1. Cd-stressed wild-type wheat

[0050] 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.

[0051] 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.

[0052] 2. Total RNA extraction and cDNA reverse transcription from wheat roots

[0053] Cd-treated wheat roots were collected, washed thoroughly with water, flash-frozen in liquid nitrogen, and then stored at -80°C. High-quality wheat RNA was extracted using TRIzol reagent, and some of the RNA was used to synthesize cDNA according to the cDNA reverse transcription kit instructions for gene cloning and real-time quantitative PCR.

[0054] 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 cDNA library constructed in this study is of good quality.

[0055] 3. Construction and screening of two-hybrid libraries of yeast from wheat roots under Cd stress

[0056] 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.

[0057] 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. Three of the transformants encoded the transcription factor TaZFP2. Therefore, TaZFP2 was selected as the target gene for functional verification. Further, the growth curves of yeast transformed with pGADT7 and pGADT7-TaZFP2 were examined in normal and high-Cd (0.004 mol / L) media. The results showed that the growth rates of the two yeasts were comparable in normal media. Figure 4In high-Cd medium, the growth rate of the control strain transformed with pGADT7 was significantly inhibited, while the yeast strain transformed with pGADT7-TaZFP2 grew well, indicating that the expression of TaZFP2 improved the Cd tolerance of yeast. Figure 4 B).

[0058] 4. Screening of wheat genes related to Cd stress response and determination of growth curves

[0059] 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.

[0060] 5. Functional verification of Cd stress response genes

[0061] The obtained yeast plasmid was BLAST-aligned using NCBI to obtain the target gene. The CDS sequence of TaZFP2 (Accession No. XM_020309267.3) 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 homologous recombination technology and sent to a biotechnology company for sequencing. The positive recombinant vector TRV:TaZFP2 and the empty vector pYL156 were transformed into Agrobacterium GV3101, and wheat seeds were infected.

[0062] Table 1 Primer sequences

[0063]

[0064] 6. Cd content detection in wheat

[0065] Wheat plants infected with Agrobacterium tumefaciens containing the TRV:TaZFP2 recombinant plasmid and the control plasmid for 16 days were irrigated with a 40 mg / L Cd solution. Ten days later, roots and leaves of the control plants and TaZFP2 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 (Kunming) for Cd content detection using inductively coupled plasma mass spectrometry.

[0066] 7. Detection of physiological indicators of wheat

[0067] 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.

[0068] Leaves from different lines of normally growing and Cd-treated control plants and TaZFP2 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.

[0069] 8. qRT-PCR

[0070] RNA was extracted from the remaining wheat roots and leaves from step 7, reverse transcribed into cDNA, diluted 10-20 times, and subjected to qRT-PCR to analyze the relative expression level of TaZFP2. The Actin gene (Accession No. KC775780) was used as an internal reference gene, and relative quantification was performed. -ΔΔCt Perform RNA transcriptome alteration analysis.

[0071] Homologous recombination was used to construct the 482-681 bp region of the TaZFP2 gene's CDS region 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 TaZFP2-silenced plants (TRV:TaZFP2) and control plants (TRV:00) grew normally. However, TRV:00 plants subjected to 40 mg / L Cd stress showed severely inhibited growth, exhibiting stunted growth, shortened total root length, and wilted, yellowing leaves. TRV:TaZFP2 plants, after Cd stress, grew well, showing comparable growth characteristics to TaZFP2-silenced plants and the control under normal conditions. Figure 5qRT-PCR results showed that, compared with the control, the expression level of TaZFP2 in the roots and leaves of TRV:TaZFP2 plants was significantly decreased, indicating that the TaZFP2 silencing effect was good. Figure 5 (B) Inductively coupled plasma mass spectrometry (ICP-MS) was used to detect the Cd content in the roots and leaves of TRV:00 and TRV:TaZFP2 plants. The results showed that the Cd content in the roots and leaves of the TRV:00 control plants was higher than that in the TRV:TaZFP2 silenced plants, and the difference in the roots was statistically significant. Furthermore, the Cd content in the roots of both TRV:00 and TRV:TaZFP2 plants was significantly higher than that in the leaves. Figure 5 Finally, physiological indicators such as chlorophyll content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, and malondialdehyde (MDA) content in the leaves of TRV:00 control plants and TRV:TaZFP2 silenced plants were measured. The results showed that after Cd stress, the chlorophyll content, SOD and POD activities of the control plants were significantly lower than those of the TRV:TaZFP2 silenced plants. Figure 6 (A, B, and C), while the MDA content of the control plants was significantly higher than that of the TRV:TaZFP2 plants ( Figure 6 The above results indicate that silencing TaZFP2 may resist Cd toxicity to wheat by increasing chlorophyll content, SOD and POD activity, thereby reducing the Cd content in the aboveground parts and roots of wheat plants.

[0072] 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 TaZFP2 The application of genes to enhance wheat plants' tolerance to Cd is characterized by, The TaZFP2 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 TaZFP2 The CDS region of the gene is 482bp-681bp.

3. The application according to claim 1, characterized in that, The TaZFP2 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. TaZFP2 The steps of gene generation, wherein, TaZFP2 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 TaZFP2 The 482bp-681bp region of the gene's CDS region was constructed into a viral vector, and then the virus was inoculated into a seedling to silence the virus. TaZFP2 Gene.

6. The method for improving wheat's tolerance to Cd according to claim 4, characterized in that, The TaZFP2 The nucleotide sequence of the gene is shown in SEQ ID No: 2.

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