Application of tae-mir9670 in enhancing cadmium stress tolerance in wheat

Abstract

The application discloses application of tae-miR9670 in enhancing the cadmium stress resistance of wheat. The application claims the application of tae-miR9670 or a related biological material in any of the following aspects: (A1) enhancing the cadmium stress resistance of wheat; (A2) cultivating a wheat variety with enhanced cadmium stress resistance; (A3) cultivating a wheat variety with reduced cadmium content. Under cadmium stress, compared with wild-type wheat, a tae-miR9670 overexpression wheat line has a higher plant height, a longer root length, a higher fresh weight / dry weight, a lower content of malondialdehyde in the body and a lower content of cadmium in the body. The application has important significance for cultivating a wheat variety with enhanced cadmium stress resistance and cultivating a wheat variety with reduced cadmium content.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of tae-miR9670 in enhancing wheat's tolerance to cadmium stress. Background Technology

[0002] Although cadmium (Cd) is not an essential element for life, it is widely considered one of the most hazardous elements due to its high toxicity (Nordberg, GF, 2009. Historical perspectives on cadmium toxicology. Toxicol Appl Pharmacol 238, 192-200.). In recent years, cadmium pollution in agricultural environments has become increasingly serious due to factors such as industrial activities, the use of cadmium-containing phosphate fertilizers, and improper water and fertilizer management (Wang, P., Chen, H., Kopittke, PM, Zhao, FJ, 2019. Cadmium contamination in agricultural soils of China and the impact on food safety. Environ Pollut 249, 1038-1048.). Cadmium pollution has become a current research hotspot, aiming to gain a deeper understanding of its impact and develop effective strategies to mitigate its negative effects on agricultural production and human health.

[0003] MicroRNAs (miRNAs) are a class of endogenous small RNA molecules, typically 20-24 nucleotides in length, widely distributed throughout plant genomes. miRNAs regulate gene expression by inhibiting the translation of target genes or cleaving mRNA, playing a crucial role in regulating plant growth and development, signal transduction, and responses to various environmental stresses (Voinnet O., 2009. Origin, biogenesis, and activity of plant microRNAs. Cell 136, 669-687.). In particular, miRNAs play a significant role in plant responses to heavy metal stress (Srivastava, S., Suprasanna, P., 2021. MicroRNAs: Tiny, powerful players of metal stress responses in plants. Plant Physiol Biochem 166, 928-938.). Several conserved miRNAs, such as miR166, miR390, miR395, miR397, and miR398, have been found to respond to cadmium stress and regulate cadmium tolerance in different plant species. However, most current research focuses on these highly conserved miRNAs (Ding, Y., Gong, S., Wang, Y., Wang, F., Bao, H., Sun, J., Cai, C., Yi, K., Chen, Z., Zhu, C., 2018. MicroRNA166 modulates cadmium tolerance and accumulation in rice. PlantPhysiol 177, 1691-1703.). Wheat, as one of the world's major food crops, is increasingly facing concerns regarding cadmium enrichment in farmland and the resulting accumulation of cadmium in wheat grains. Therefore, identifying regulatory factors that can inhibit cadmium uptake and accumulation in wheat is crucial for addressing this issue. Currently, little is known about the role of miRNAs in regulating wheat's tolerance to cadmium stress, especially the specific mechanisms by which non-conserved species-specific miRNAs in wheat are involved, and further in-depth research and exploration are needed. Summary of the Invention

[0004] This invention claims protection for the use of tae-miR9670 in enhancing wheat tolerance to cadmium stress.

[0005] In a first aspect, the present invention claims protection for the use of tae-miR9670 or related biomaterials in any of the following: (A1) Enhances wheat's tolerance to cadmium stress; (A2) Develop wheat varieties with enhanced tolerance to cadmium stress; (A3) Develop wheat varieties with reduced cadmium content.

[0006] The relevant biological material is the precursor of tae-miR9670 or a DNA molecule capable of being transcribed into tae-miR9670, or an expression cassette, recombinant vector, recombinant bacteria, or transgenic cell line containing the DNA molecule.

[0007] Secondly, the present invention claims protection for the use of tae-miR9670 or related biomaterials in any of the following: (B1) Promotes wheat plant height increase under cadmium stress; (B2) Promotes root elongation in wheat under cadmium stress; (B3) Promotes increased fresh weight of wheat under cadmium stress; (B4) Promotes increased dry weight of wheat under cadmium stress; (B5) Reduces malondialdehyde content in wheat under cadmium stress; (B6) Reduces cadmium content in wheat under cadmium stress; In one embodiment of the present invention, (B5) specifically refers to reducing the malondialdehyde content in wheat roots and / or leaves under cadmium stress, and (B6) specifically refers to reducing the cadmium content in wheat roots, flag leaves, lower nodes, and / or grains under cadmium stress. The same applies below.

[0008] The relevant biological material is the precursor of tae-miR9670 or a DNA molecule capable of being transcribed into tae-miR9670, or an expression cassette, recombinant vector, recombinant bacteria, or transgenic cell line containing the DNA molecule.

[0009] The promotion or reduction mentioned in (B1)-(B6) are all relative to wild-type wheat. The same applies below.

[0010] Thirdly, the present invention claims protection for the use of promoting tae-miR9670 expression in any of the following: (A1) Enhances wheat's tolerance to cadmium stress; (A2) Develop wheat varieties with enhanced tolerance to cadmium stress; (A3) Develop wheat varieties with reduced cadmium content.

[0011] The promotion of tae-miR9670 expression refers to promoting the expression of tae-miR9670 in wheat.

[0012] Fourthly, the present invention claims protection for the use of promoting tae-miR9670 expression in any of the following: (B1) Promotes wheat plant height increase under cadmium stress; (B2) Promotes root elongation in wheat under cadmium stress; (B3) Promotes increased fresh weight of wheat under cadmium stress; (B4) Promotes increased dry weight of wheat under cadmium stress; (B5) Reduces malondialdehyde content in wheat under cadmium stress; (B6) Reduces cadmium content in wheat under cadmium stress.

[0013] The promotion of tae-miR9670 expression refers to promoting the expression of tae-miR9670 in wheat.

[0014] Fifthly, the present invention claims protection for any of the following methods: Method I: A method for enhancing the cadmium stress resistance of wheat, which may include the following steps: increasing the expression level of tae-miR9670 in recipient wheat, thereby enhancing the cadmium stress resistance of wheat (compared to recipient wheat).

[0015] Method II: A method for breeding wheat varieties with enhanced tolerance to cadmium stress may include the following steps: increasing the expression level of tae-miR9670 in recipient wheat to obtain wheat varieties with enhanced tolerance to cadmium stress (compared to recipient wheat).

[0016] Method III: A method for breeding wheat varieties with reduced cadmium content, comprising the following steps: increasing the expression level of tae-miR9670 in recipient wheat to obtain wheat varieties with reduced cadmium content.

[0017] Sixthly, the present invention claims protection for any of the following methods: Method IV: A method for promoting wheat plant height and / or root length elongation and / or fresh weight and / or dry weight under cadmium stress, comprising the following steps: increasing the expression level of tae-miR9670 in recipient wheat, thereby achieving the promotion of wheat plant height and / or root length elongation and / or fresh weight and / or dry weight under cadmium stress (compared to recipient wheat).

[0018] Method V: A method for reducing malondialdehyde (MDA) content and / or reducing cadmium content in wheat under cadmium stress, comprising the following steps: increasing the expression level of tae-miR9670 in recipient wheat, thereby reducing MDA content and / or reducing cadmium content in wheat (compared to recipient wheat) under cadmium stress.

[0019] In the fifth and sixth aspects above, increasing the expression level of tae-miR9670 in the recipient wheat can be achieved by introducing a DNA molecule capable of being transcribed into tae-miR9670 into the recipient wheat.

[0020] The DNA molecule can be introduced into the recipient wheat via a recombinant vector.

[0021] In one embodiment of the present invention, the promoter for initiating transcription of the DNA molecule in the recombinant vector is the Ubi promoter.

[0022] In the aforementioned relevant aspects, the sequence of tae-miR9670 is shown in SEQ ID No. 1 (mature miRNA).

[0023] In the foregoing related aspects, the precursor of tae-miR9670 is shown in SEQ ID No. 2; the DNA molecule is shown in SEQ ID No. 3.

[0024] In the aforementioned aspects, the cadmium stress is CdCl2 stress. In one embodiment of the invention, the cadmium stress is a CdCl2 concentration of 350 μM (the final concentration of CdCl2 in the nutrient solution). In another embodiment of the invention, the cadmium stress is 10 mg of CdCl2 per kg of soil.

[0025] In one embodiment of the present invention, the wheat is Fielder wheat.

[0026] Experiments have demonstrated that, compared to wild-type wheat, wheat lines overexpressing tae-miR9670 exhibit stronger tolerance to cadmium stress. Under cadmium stress, compared to wild-type wheat, tae-miR9670-overexpressing wheat lines showed greater plant height, longer root length, higher fresh / dry weight, lower malondialdehyde (MDA) content, and lower cadmium content. This invention is of significant importance for breeding wheat varieties with enhanced cadmium stress tolerance and those with reduced cadmium content. Attached Figure Description

[0027] Figure 1 The expression of tae-miR9670 was in response to cadmium stress treatment. Here, A represents the miRNA identified by small RNA sequencing as responding to cadmium stress. The threshold was set to a fold change greater than 1.5. P Values ​​less than 0.05 were used to identify differentially expressed miRNAs under two conditions: 0 hours and 12 hours of treatment with 350 µM CdCl2. B represents six differentially expressed miRNAs based on the normalized sequencing count (TPM). * P <0.05; **P <0.01 ( t -test). C is MIR9670 Sequence distribution characteristics of the precursor. tae-miR9670-5p is... MIR9670 The main product on the precursor.

[0028] Figure 2 This section describes the expression characteristics and target genes of tae-miR9670. A shows the spatiotemporal expression of tae-miR9670 detected by qRT-PCR. The data include: seedling root (SR), seedling stem (SS), seedling leaf (SL), jointing root (ER), jointing stem (ES), jointing leaf (EL), heading root (HR), heading stem (HS), heading leaf (HL), 1 cm long young spike (1YS), 3 cm long young spike (3YS), 5 cm long young spike (5YS), grains 5 days after flowering (5D), grains 10 days after flowering (10D), and grains 15 days after flowering (15D). B shows the cadmium stress response of tae-miR9670 detected by qRT-PCR. Wheat seedlings were treated with 350 μM CdCl2, and wheat roots were sampled at 0, 1, 3, 6, 12, 24, and 48 hours after treatment to detect the expression of tae-miR9670. * P <0.05; ** P <0.01 ( t -test). C represents the degradome finding that tae-miR9670-5p can mediate the cleavage of the target gene TraesCS6D02G023400. D represents the degradome finding that tae-miR9670-5p can mediate the cleavage of the target gene TraesCS6D02G023500.

[0029] Figure 3 Molecular identification of tae-miR9670 transgenic wheat overexpression. In A, PCR-positive tae-miR9670 overexpression (OE) lines were identified; most individual plants in OE1, OE2, OE3, OE4, and OE5 lines showed positive amplification bands. In B, qRT-PCR was used to detect the expression level of tae-miR9670 in wild-type (WT) and overexpression (OE) lines. **P<0.01 (t-test).

[0030] Figure 4This study aimed to identify cadmium tolerance in seedlings of tae-miR9670 transgenic wheat overexpressing the gene. In the data, A represents the phenotypes of wild-type (WT) and overexpression (OE) lines under 0 and 350 μM CdCl2 treatments. B and E represent plant height (B), root length (C), fresh weight (D), and dry weight (E) of WT and OE lines under CdCl2 treatment, respectively. F represents the malondialdehyde (MDA) content in roots and leaves of WT and OE lines under CdCl2 treatment. G and H represent the cadmium content in roots (G) and leaves (H) of WT and OE lines at different time points after CdCl2 treatment, respectively. * P <0.05; ** P <0.01 ( t -test).

[0031] Figure 5 Overexpression of tae-miR9670 reduced cadmium accumulation in wheat under cadmium treatment throughout the entire growth cycle. AD represents the cadmium content in grain (A), lower spike node (B), flag leaf (C), and root (D) of wild-type (WT) and overexpressed (OE) lines under 0, 5, and 10 mg / kg cadmium treatments throughout the entire growth cycle, respectively. *P<0.05; **P<0.01 (t-test).

[0032] Figure 6 The data represent wheat yield-related traits under normal growth conditions without the effect of tae-miR9670 overexpression. A represents the phenotypes of wild-type (WT) and overexpressed (OE) lines under normal field conditions. B and F represent plant height (B), effective tillers (C), number of spikelets per spike (D), number of grains per spike (E), and thousand-grain weight (F) of WT and OE lines under normal field conditions, respectively. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0035] Example 1: miRNA genome analysis revealed that tae-miR9670 responds to cadmium stress in wheat seedling roots. The wheat variety Fielder used in this example was preserved in our laboratory. Germinated seeds were hydroponically cultured in an incubator (16 hours light / 8 hours darkness) for two weeks. The roots of the two-week-old seedlings were then soaked in a 350 μM CdCl2 aqueous solution. Wheat roots soaked for 0 hours and 12 hours were collected, and total RNA was extracted from the wheat roots using RNAiso Plus reagent (Takara, Beijing, China).

[0036] I. RNA extraction (refer to reagent instructions) (1) Place about 1.5g of wheat tissue into a 2.0mL optimized centrifuge tube containing steel balls (the steel balls were ignited with anhydrous ethanol for 1min and then cooled beforehand), freeze quickly with liquid nitrogen, and break up the sample; add 1.0mL RNAiso Plus, immediately vortex for 30s to dissolve the sample completely, and let stand on ice for 10min.

[0037] (2) Centrifuge the sample at 12000 rpm for 15 min in a 4℃ low-temperature centrifuge, and extract all the supernatant into a new 1.5 mL optimization tube; add 200 μL of RNA extraction solution (phenol, chloroform and isopropanol in a volume ratio of 25:24:1), mix by inverting, and let stand for 5 min.

[0038] (3) Centrifuge at 4℃ for 15 min at 12000 rpm; transfer about 700 μL of supernatant to a new 1.5 mL optimization tube; add 200 μL of RNA extraction solution (as before), mix by inverting, centrifuge at 12000 rpm for 15 min; transfer the supernatant to a new 1.5 mL optimization tube, add an equal amount of isopropanol, mix, and freeze for 25 min.

[0039] (4) 12000 rpm, 15 min; discard the supernatant, add 70% alcohol (prepared with enzyme-free water), mix by inversion, 12000 rpm, 5 min; repeat this step; aspirate the supernatant and blow dry, add enzyme-free water to dissolve the RNA precipitate (40-60 μL), and freeze at -80℃ for storage.

[0040] II. Small RNA sequencing and miRNA analysis Small RNA libraries were constructed from extracted total RNA using a small RNA library construction kit (KAITAI-BIO, Hangzhou, China). High-throughput sequencing of the small RNA libraries was performed using an Illumina Novaseq 6000 platform (all performed by Hangzhou Kaitai Biotechnology Co., Ltd.). Ninety-three known wheat miRNAs were identified, and the abundance of each miRNA was normalized to transcripts per million (TPM) based on the number of sequencing reads. The criteria for differentially expressed miRNAs were: a relative expression level change greater than 50% between the treatment and control groups, and... PThe value was less than 0.05. Based on this screening criterion, a total of 6 miRNAs were found to be differentially expressed under cadmium treatment. Figure 1 (A). Among them, the expression of four miRNAs (tae-miR171a, tae-miR9670-5p, tae-miR9675-3p, and tae-miR9676-5p) was significantly upregulated, while the expression of two miRNAs (tae-miR1436 and tae-miR6300) was significantly downregulated. Figure 1 (B)

[0041] Because tae-miR9670-5p had the highest abundance and showed more significant differential expression after cadmium treatment, we chose tae-miR9670 for further research. According to the miRBase database (https: / / www.mirbase.org / ), tae-miR9760-3p is considered a mature miRNA sequence, while the tae-miR9760-5p we discovered was predicted as a miRNA* sequence. However, our sequencing data showed that tae-miR9760-5p was significantly more abundant than tae-miR9760-3p, with a sequencing count an order of magnitude higher (…). Figure 1 (C). Therefore, this indicates that tae-miR9760-5p is MIR9670 The truly functional mature miRNA sequence generated from the precursor (SEQ ID No. 2) will subsequently be referred to as tae-miR9760-5p (SEQ ID No. 1). Furthermore, our bioinformatics search of genome sequences in various plant species revealed that tae-miR9760 and its precursor sequence are found only in closely related species such as wheat and rye, and are absent in other monocotyledonous plants such as rice and maize, as well as dicotyledonous plants. This suggests that miR9670 may be a miRNA unique to the wheat tribe.

[0042] Example 2: Expression analysis of tae-miR9670 and identification of target genes I. Tae-MiR9670 Expression Analysis Total RNA was extracted from different developmental tissues of wheat using RNAiso Plus reagents (Takara, Beijing, China). The expression level of tae-miR9670 was quantified by qRT-PCR using the miRcute miRNA cDNA synthesis kit and the miRcute Plus miRNA qPCR kit (Tiangen, Beijing, China) according to the instructions. The wheat U6 gene was used as an internal control. Relative expression levels were determined by fold change using the comparative CT method, and each assay was performed independently three times. Experimental methods were performed according to the kit instructions, with the following quantitative primers (universal downstream primers were provided by the kit): The upstream primer used for detecting miR9670 is: 5'-TTCTTCAAGTACTCCACTTTT-3'; The upstream primer used for detecting TaU6 is 5'-CGGCGGCATGTAGTTACTCG-3'.

[0043] qRT-PCR results showed that tae-miR9670 was expressed in various tissues of wheat at different developmental stages (under normal growth conditions), including roots, stems, leaves, spikes, and grains; therefore, its expression characteristic was constitutive expression. Figure 2 (A). Furthermore, we further examined the expression of tae-miR9670 in the roots of seedlings treated with 350 μM CdCl2 for different durations. The results showed that its expression was significantly induced by CdCl2, increasing by 1.5-fold and 2-fold at 6 and 12 hours compared to 0 hours, respectively. Figure 2 (B), this result further corroborates the results of the miRNA group.

[0044] II. Target gene identification of tae-miR9670 The function of miRNAs is closely related to the target genes they regulate, and the target genes of plant miRNAs are usually nearly perfectly complementary to the miRNAs in sequence. Based on this principle, we predicted the target genes of tae-miR9670 in the latest wheat cDNA library (Triticum aestivum cDNA, Ensemblplant, release 43) and found several potential target genes regulated by tae-miR9670 (mismatch number less than 3), all of which encode mitochondrial transcription termination factors (mTERF). Furthermore, based on our existing wheat degradome data (Jian, C., Hao, P., Hao, C., Liu, S., Mao, H., Song, Q., Zhou, Y., Yin, S., Hou, J., Zhang, W., Zhao, H., Zhang, X., Li, T., 2022. The miR319 / TaGAMYB3module regulates plant architecture and improves grain yield in common wheat (Triticum aestivum). New Phytol 235, 1515-1530.), we confirmed that tae-miR9670 mediates the cleavage of at least two representative target gene mRNAs, including TraesCS6D02G023400 and TraesCS6D02G023500 ( Figure 2 (C and D). These results support the idea that tae-miR9670 can target multiple mTERF genes and mediate their mRNA cleavage.

[0045] Example 3: Construction of tae-miR9670 overexpression vector and obtaining tae-miR9670 overexpressing transgenic wheat I. Construction of tae-miR9670 overexpression vector To investigate the role of tae-miR9670 in wheat tolerance to cadmium stress, we constructed a wheat overexpression vector for tae-miR9670. First, a 543 bp sequence containing the complete precursor stem-loop sequence of tae-miR9670 was amplified from wheat genomic DNA. This sequence was then cloned into a modified pCAMBIA3301 vector, positioned downstream of the maize Ubi promoter (see Jian, C., Hao, P., Hao, C., Liu, S., Mao, H., Song, Q., Zhou, Y., Yin, S., Hou, J., Zhang, W., Zhao, H., Zhang, X., Li, T., 2022. ThemiR319 / TaGAMYB3 module regulates plant architecture and improves grain yield in common wheat (Triticum aestivum). New Phytol 235, 1515-1530.).

[0046] The "modified pCAMBIA3301 vector" mentioned above is obtained by double digesting the pWMB006 vector (described in the article "Liu H, LiH, Hao C, Wang K, Wang Y, Qin L, An D, Li T, Zhang X. TaDA1, a conserved negative regulator of kernel size, has an additive effect with TaGW2 in common wheat (Triticum aestivum L.). Plant Biotechnol J. 2020 May;18(5):1330-1342. doi: 10.1111 / pbi.") with HindIII and EcoRI to obtain a region containing the Ubi promoter and carrying a downstream multiple cloning site. This region is then ligated to the large fragment of the pCAMBIA3301 vector, which has been digested with the same enzymes (HindIII and EcoRI), thus obtaining the "modified pCAMBIA3301 vector".

[0047] The primer sequences for constructing the overexpression vector are as follows: miR9670-OE-F: 5'-GTCGACTCTAGA GGATCC TCCCCACCACATCCTTATC-3' (the underlined part is the BamHI recognition sequence); miR9670-OE-R: 5'-GCTCTCTAGA ACTAGT GAATGAACTGGCAGCAAAG-3' (The underlined part is the SpeI recognition sequence).

[0048] Using the wheat Fielder genome as a template, PCR amplification was performed using primers miR9670-OE-F and miR9670-OE-R, yielding a DNA fragment with the sequence “GTCGACTCTAGAGGATCC+SEQ ID No.3+ACTAGTTCTAGAGAGC”. The amplified product was double-digested with BamHI and SpeI, and the recovered product was ligated to the previously described “modified pCAMBIA3301 vector” fragment, which had undergone the same double-digestion, to obtain the recombinant vector, named pCAMBIA3301-miR9670 after sequencing verification. The structure of the recombinant expression vector pCAMBIA3301-miR9670 is described as follows: the recombinant plasmid obtained by replacing the small fragment between the BamHI and SpeI restriction sites of the previously described “modified pCAMBIA3301 vector” with the fragment shown in SEQ ID No.3.

[0049] II. Obtaining transgenic wheat overexpressing tae-miR9670 The constructed tae-miR9670 overexpression vector pCAMBIA3301-miR9670 was introduced into Agrobacterium EHA105 strain, and Fielder wheat embryos were transformed using the Agrobacterium transformation method. Genomic DNA was extracted from the transgenic materials, and molecular detection was performed using specific primers, revealing five tae-miR9670 transgenic positive lines, named OE1-OE5. Figure 3 (A)

[0050] PCR primers for positive identification of transgenic lines: PubinosF: 5'-TCGATGCTCACCCTGTTGTTTG-3'; NosR2: 5'-TGTATAATTGCGGGACTCTAATC-3'.

[0051] Positive materials can amplify a 798bp fragment (543bp as shown in SEQ ID No. 3 plus 255bp of vector sequences on both sides), while negative materials cannot amplify the fragment.

[0052] Further qRT-PCR experiments (specific methods and primers are described in Step 1 of Example 2) revealed that, compared with the wild-type (WT) Fielder control, the expression level of tae-miR9670 was significantly increased in all five overexpression (OE) lines, with an increase of 2-4 times. Figure 3 (B). Subsequently, three overexpression lines with high expression levels, OE1-OE3, were selected for phenotypic identification experiments.

[0053] Example 4: Overexpression of tae-miR9670 enhanced the wheat's tolerance to cadmium stress during seedling and mature stages. I. Overexpression of tae-miR9670 enhanced the wheat seedlings' tolerance to cadmium stress. After germination, the tae-miR9670 overexpressing wheat (OE1-OE3) and wild-type (WT) Fielder seeds obtained in Example 3 were cultured in Hoagland nutrient solution until the two-leaf stage (approximately 2 weeks), and then transferred to nutrient solutions containing 0 or 350 μM CdCl2 for further culture. After 15 days of treatment, under control conditions (0 μM CdCl2), there was no significant difference between WT and OE; however, under 350 μM CdCl2 treatment, the growth of WT was significantly slowed, while the growth of OE lines was significantly better than that of WT. Figure 4 (A). Corresponding to this phenotypic result, under CdCl2 treatment, the plant height, root length, fresh weight, and dry weight of the OE line were significantly higher than those of the WT line. Figure 4 (BE). Therefore, tae-miR9670 overexpression improved the ability of wheat seedlings to tolerate cadmium stress.

[0054] The enhanced cadmium tolerance in the tae-miR9670-OE strain was associated with changes in malondialdehyde (MDA) content. Under control conditions (0 μM CdCl2), there was no significant difference in MDA content between the WT and OE strains; however, under 350 μM CdCl2 treatment, the MDA content in the roots and leaves of the OE strain was significantly lower than that in the WT strain. Figure 4 (F). We also compared the cadmium content in WT and OE lines after treatment with 350 μM CdCl2 for different times (24-168 hours). Cadmium content was determined using atomic absorption spectrometry. With increasing treatment time, the cadmium content in the roots and leaves of both WT and OE lines increased. In contrast, the average cadmium content of the OE line was significantly lower than that of WT, with a reduction of approximately 10% in roots and more than 20% in leaves. Figure 4 (G and H). All the above results indicate that tae-miR9670 overexpression may enhance the ability of wheat seedlings to resist cadmium stress by reducing cadmium uptake and translocation.

[0055] II. Overexpression of tae-miR9670 reduces cadmium content in organs such as mature wheat grains. After germination, the tae-miR9670 overexpressing wheat (OE1-OE3) and wild-type (WT) seeds obtained in Example 3 were planted in plastic pots containing soils containing different concentrations (0 mg / kg, 5 mg / kg, 10 mg / kg) of CdCl2 and grown until maturity. After harvest, the cadmium content in the roots, flag leaves, lower nodes, and grains of the wild-type and OE lines was determined. The results showed that, compared with WT, the overexpressing lines generally exhibited lower cadmium content in all these tissues. Figure 5 (AD). Notably, under the treatment condition of 10 mg / kg CdCl2, the cadmium content in the grains of the OE strain was significantly lower than that of the WT strain, decreasing by 61% to 70%. Figure 5 (A). Similarly, the cadmium content in the lower internodes of the OE line was also reduced by 55% to 68% compared to WT. Figure 5 (Middle B). In contrast, the cadmium content in the roots and flag leaves of the OE series was slightly lower than the WT level ( Figure 5 (C and D). These differences in cadmium accumulation in different tissues suggest that, under cadmium treatment conditions throughout the entire growth cycle, overexpression of tae-miR9670 can significantly reduce cadmium accumulation in upper organs such as grains and lower nodes of the ear. This reduction may be due to the reduced cadmium translocation caused by overexpression of tae-miR9670, particularly slowing down the translocation of cadmium from the roots to the upper organs.

[0056] Example 5: Effects of tae-miR9670 overexpression on wheat yield-related traits Generally, increased stress resistance often leads to decreased crop yield. Therefore, we tested whether tae-miR9670 overexpression also affects yield-related traits in wheat. We planted the tae-miR9670-overexpressing wheat (OE1-OE3) obtained in Example 3 and wild-type (WT) wheat at the Shunyi Transgenic Experimental Base of the Chinese Academy of Agricultural Sciences. Each line was planted in 4 rows, with approximately 20 individual plants per row. The row length was 2 meters, the row spacing was 30 centimeters, and the plant spacing was 10 centimeters. The wheat was grown normally under natural field conditions, with field management consistent with that of ordinary wheat. The results showed that there was no significant difference in developmental traits between the WT and OE lines throughout the entire growth cycle. Figure 6 (A). After harvest, we measured and compared the main yield-related traits between the WT and OE lines, including plant height, number of effective tillers, number of spikelets per spike, number of grains per spike, and 1000-grain weight. Statistical analysis showed no significant differences in these yield-related traits between the WT and OE lines. Figure 6(BF). Therefore, overexpression of tae-miR9670 in wheat does not have a negative impact on yield-related traits, while enhancing tolerance to cadmium stress without affecting yield traits.

[0057] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of tae-miR9670 or its related biomaterials in any of the following: (A1) Enhances wheat's tolerance to cadmium stress; (A2) Develop wheat varieties with enhanced tolerance to cadmium stress; (A3) Develop wheat varieties with reduced cadmium content; The relevant biological material is a DNA molecule capable of being transcribed into the tae-miR9670 or an expression cassette, recombinant vector, recombinant bacteria or transgenic cell line containing the DNA molecule; The sequence of tae-miR9670 is shown in SEQ ID No. 1; The DNA molecule is shown in SEQ ID No. 3; The expression of the tae-miR9670 or the DNA molecule is enhanced in wheat.

2. Application of tae-miR9670 or its related biomaterials in any of the following: (B1) Promotes wheat plant height increase under cadmium stress; (B2) Promotes root elongation in wheat under cadmium stress; (B3) Promotes increased fresh weight of wheat under cadmium stress; (B4) Promotes increased dry weight of wheat under cadmium stress; (B5) Reduces malondialdehyde content in wheat under cadmium stress; (B6) Reduces cadmium content in wheat under cadmium stress; The relevant biological material is a DNA molecule capable of being transcribed into the tae-miR9670 or an expression cassette, recombinant vector, recombinant bacteria or transgenic cell line containing the DNA molecule; The sequence of tae-miR9670 is shown in SEQ ID No. 1; The DNA molecule is shown in SEQ ID No. 3; The expression of the tae-miR9670 or the DNA molecule is enhanced in wheat.

3. Any of the following methods: Method I: A method for enhancing wheat's tolerance to cadmium stress, comprising the following steps: increasing the expression level of tae-miR9670 in recipient wheat, thereby enhancing wheat's tolerance to cadmium stress; Method II: A method for breeding wheat varieties with enhanced tolerance to cadmium stress, comprising the following steps: increasing the expression level of tae-miR9670 in recipient wheat, thereby obtaining wheat varieties with enhanced tolerance to cadmium stress; Method III: A method for breeding wheat varieties with reduced cadmium content, comprising the following steps: increasing the expression level of tae-miR9670 in recipient wheat to obtain wheat varieties with reduced cadmium content; The sequence of tae-miR9670 is shown in SEQ ID No.

1.

4. Any of the following methods: Method IV: A method for promoting wheat plant height and / or root length elongation and / or fresh weight and / or dry weight under cadmium stress, comprising the following steps: increasing the expression level of tae-miR9670 in recipient wheat, thereby promoting wheat plant height and / or root length elongation and / or fresh weight and / or dry weight under cadmium stress. Method V: A method for reducing malondialdehyde (MDA) content and / or reducing cadmium content in wheat under cadmium stress, comprising the following steps: increasing the expression level of tae-miR9670 in recipient wheat, thereby reducing MDA content and / or reducing cadmium content in wheat under cadmium stress; The sequence of tae-miR9670 is shown in SEQ ID No.

1.

5. The method according to claim 3 or 4, characterized in that: The expression level of tae-miR9670 in the recipient wheat was increased by introducing a DNA molecule capable of being transcribed into tae-miR9670 into the recipient wheat. The DNA molecule is shown in SEQ ID No.

3.

6. The method according to claim 5, characterized in that: The DNA molecule was introduced into the recipient wheat in the form of a recombinant vector.

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