Application of tae-miR9670 in enhancing cadmium stress tolerance of wheat

Abstract

The invention discloses an application of tae-miR9670 in enhancing cadmium stress tolerance of wheat. The invention claims to protect any one of the following applications of tae-miR9670 or related biological materials of tae-miR9670: (A1) enhancing the cadmium stress resistance of wheat; (A2) cultivating a wheat variety with enhanced cadmium stress resistance; and (A3) cultivating a wheat variety with reduced cadmium content. Under cadmium stress, compared with wild type wheat, tae-miR9670 overexpressed wheat strains are higher in plant height, longer in root length, heavier in fresh weight / dry weight, lower in in-vivo malondialdehyde content and lower in in-vivo cadmium content, and the tae-miR9670 overexpressed wheat strains are higher in plant height, longer in root length, higher in fresh weight / dry weight and lower in-vivo cadmium content. The method is of great significance to cultivation of wheat varieties with enhanced cadmium stress resistance and wheat varieties with reduced cadmium content.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of tae-miR9670 in enhancing wheat resistance to cadmium stress. Background Art

[0002] Although cadmium (Cd) is not an essential element for organisms, it is generally considered to be one of the most harmful 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 hot topic in current research, aiming to gain a deeper understanding of its impact and develop effective strategies to mitigate its negative impacts on agricultural production and human health.

[0003] MicroRNA (miRNA) is a class of endogenous small RNA molecules, which are usually between 20-24 nucleotides in length and widely present in plant genomes. miRNA regulates gene expression by inhibiting the translation of target genes or cleaving mRNA, and plays a vital role in regulating plant growth and development, signal transduction, and responding to various environmental stresses (Voinnet O., 2009. Origin, biogenesis, and activity of plant microRNAs. Cell 136, 669-687.). In particular, miRNA plays an important role in plant response 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.). Some 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, but most studies have focused 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. Plant Physiol 177, 1691-1703.). As one of the world's major food crops, wheat is increasingly concerned about the enrichment of cadmium in farmland and the subsequent accumulation of cadmium in wheat grains. Therefore, identifying regulatory factors that can inhibit wheat's uptake and accumulation of cadmium is crucial to address this issue. At present, little is known about the role of miRNA in regulating wheat's cadmium stress tolerance, especially whether non-conservative species-specific miRNAs in wheat are involved in the specific mechanism, which requires further in-depth research and exploration. Summary of the invention

[0004] The present invention claims to protect the use of tae-miR9670 in enhancing wheat resistance to cadmium stress.

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

[0006] The related biological material is the precursor of the tae-miR9670 or a DNA molecule that can be transcribed into the tae-miR9670 or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the DNA molecule.

[0007] In a second aspect, the present invention claims the use of tae-miR9670 or its related biological materials in any of the following: (B1) Promote the increase of wheat plant height under cadmium stress; (B2) Promotes wheat root elongation under cadmium stress; (B3) Promote the increase of wheat fresh weight under cadmium stress; (B4) Promote the increase of wheat dry weight under cadmium stress; (B5) Reducing the content of malondialdehyde in wheat under cadmium stress; (B6) Reducing the cadmium content in wheat under cadmium stress; In an embodiment of the present invention, (B5) is specifically embodied as reducing the malondialdehyde content in wheat roots and / or leaves under cadmium stress, and (B6) is specifically embodied as reducing the cadmium content in wheat roots, flag leaves, lower nodes of the ear and / or grains under cadmium stress. The same applies below.

[0008] The related biological material is the precursor of the tae-miR9670 or a DNA molecule that can be transcribed into the tae-miR9670 or an expression cassette, a recombinant vector, a recombinant bacterium or a 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] In a third aspect, the present invention claims the use of promoting the expression of tae-miR9670 in any of the following: (A1) Enhance wheat's tolerance to cadmium stress; (A2) Breed wheat varieties with enhanced tolerance to cadmium stress; (A3) Breed wheat varieties with reduced cadmium content.

[0011] Wherein, the promoting the expression of tae-miR9670 is promoting the expression of tae-miR9670 in wheat.

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

[0013] Wherein, the promoting the expression of tae-miR9670 is promoting the expression of tae-miR9670 in wheat.

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

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

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

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

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

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

[0020] Wherein, the DNA molecule can be introduced into the recipient wheat in the form of 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 above-mentioned related aspects, the sequence of tae-miR9670 is shown as SEQ ID No. 1 (mature miRNA).

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

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

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

[0026] Experiments have shown that compared with wild-type wheat, tae-miR9670 overexpression wheat lines have stronger resistance to cadmium stress. Under cadmium stress, compared with wild-type wheat, tae-miR9670 overexpression wheat lines have higher plant height, longer root length, heavier fresh weight / dry weight, lower malondialdehyde content in the body, and lower cadmium content in the body. The present invention is of great significance for breeding wheat varieties with enhanced cadmium stress resistance and breeding wheat varieties with reduced cadmium content. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the expression of tae-miR9670 in response to cadmium stress. Among them, A is the miRNA found by small RNA sequencing in response to cadmium stress. The threshold is set to a difference of more than 1.5 times. P The value was less than 0.05, and the differentially expressed miRNAs were identified under the conditions of 350 μM CdCl2 treatment for 0 h and 12 h. B shows 6 differentially expressed miRNAs according to the normalized sequencing number TPM. * P <0.05; **P <0.01 ( t -test). C is MIR9670 Sequence distribution characteristics on the precursor. MIR9670 The main product on the precursor.

[0028] Figure 2 The expression characteristics and regulated target genes of tae-miR9670. Among them, A is the spatiotemporal expression of tae-miR9670 detected by qRT-PCR. 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 ear (1YS), 3 cm long ear (3YS), 5 cm long ear (5YS), 5 days after flowering (5D), 10 days after flowering (10D), 15 days after flowering (15D). B is 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 h of treatment to detect the expression of tae-miR9670. * P <0.05; ** P <0.01 ( t -test). C is the degradation group found that tae-miR9670-5p can mediate the cleavage of the target gene TraesCS6D02G023400. D is the degradation group found that tae-miR9670-5p can mediate the cleavage of the target gene TraesCS6D02G023500.

[0029] Figure 3 Molecular identification of tae-miR9670 transgenic wheat overexpression. A is a tae-miR9670 overexpression (OE) line identified as positive by PCR, and most of the individual plants in OE1, OE2, OE3, OE4, and OE5 lines have positive amplification bands. B is the expression level of tae-miR9670 in wild-type (WT) and overexpression (OE) lines detected by qRT-PCR. **P<0.01 (t-test).

[0030] Figure 4Figure 1. Identification of cadmium tolerance in wheat overexpressing tae-miR9670 at the seedling stage. A is the phenotype of wild-type (WT) and overexpression (OE) lines under 0 and 350 μM CdCl2 treatment conditions. B and E are plant height (B), root length (C), fresh weight (D) and dry weight (E) of WT and OE lines under CdCl2 treatment conditions, respectively. F is the malondialdehyde (MDA) content in roots and leaves of WT and OE lines under CdCl2 treatment conditions. G and H are the cadmium contents in roots (G) and leaves (H) of WT and OE lines at different time points of CdCl2 treatment, respectively. * P <0.05; ** P <0.01 ( t -test).

[0031] Figure 5 Overexpression of tae-miR9670 can reduce wheat cadmium accumulation under cadmium treatment during the whole growth cycle. AD are the cadmium contents in grains (A), lower nodes of ears (B), flag leaves (C) and roots (D) of wild-type (WT) and overexpression (OE) lines under 0, 5, and 10 mg / kg cadmium treatment during the whole growth cycle. *P<0.05; **P<0.01 (t-test).

[0032] Figure 6 Overexpression of tae-miR9670 does not affect wheat yield-related traits under normal growth conditions. A is the phenotype of wild-type (WT) and overexpression (OE) lines under normal field conditions. BF are plant height (B), effective tillering (C), number of spikelets per ear (D), number of grains per ear (E), and 1000-grain weight (F) of WT and OE lines under normal field conditions, respectively. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0034] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0035] Example 1. miRNA group analysis found that tae-miR9670 responds to cadmium stress in wheat young roots The wheat variety Fielder used in this example was preserved in this laboratory. The germinated seeds were hydroponically cultured in an incubator (16 hours light / 8 hours dark) for two weeks, and the roots of the two-week-old seedlings were immersed in a 350 μM CdCl2 aqueous solution. The wheat roots that were immersed for 0 hours and 12 hours were taken, and the total RNA of the wheat roots was extracted using RNAiso Plus reagent (Takara, Beijing, China).

[0036] 1. Extract RNA (refer to reagent instructions) (1) Place about 1.5 g of wheat tissue into a 2.0 mL optimized centrifuge tube filled with steel balls (the steel balls were pre-heated with anhydrous ethanol for 1 min and then cooled), quickly freeze with liquid nitrogen, and break the sample; add 1.0 mL of RNAiso Plus, immediately vortex for 30 seconds to completely dissolve the sample, and place on ice for 10 minutes.

[0037] (2) Centrifuge the sample at 12,000 rpm in a 4°C low-temperature centrifuge for 15 min, extract all the supernatant into a new 1.5 mL optimized 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 12,000 rpm for 15 min in a 4°C low-temperature centrifuge; transfer about 700 μL of supernatant to a new 1.5 mL optimized tube; add 200 μL of RNA extraction solution (same as before), mix by inverting, and centrifuge at 12,000 rpm for 15 min; transfer the supernatant to a new 1.5 mL optimized tube, add an equal amount of isopropanol, mix, and freeze for 25 min.

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

[0040] 2. Small RNA Sequencing and miRNA Group Analysis Small RNA libraries were constructed from the extracted total RNA using a small RNA sample library construction kit (KAITAI-BIO, Hangzhou, China), and high-throughput sequencing of the small RNA libraries was performed using the Illumina Novaseq 6000 platform (the above work was completed by Hangzhou Kaitai Biotechnology Co., Ltd.). 93 known expressed 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 conditions for differentially expressed miRNAs were: the relative expression level between the treatment group and the control group changed by more than 50%, and PThe value was less than 0.05. Based on this screening condition, a total of 6 miRNAs were found to be differentially expressed under cadmium treatment ( Figure 1 Among them, the expression of 4 miRNAs (tae-miR171a, tae-miR9670-5p, tae-miR9675-3p, and tae-miR9676-5p) was significantly upregulated, while the expression of 2 miRNAs (tae-miR1436 and tae-miR6300) was significantly downregulated ( Figure 1 Middle B).

[0041] Since tae-miR9670-5p has the highest abundance and its expression difference is more significant 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 found is predicted to be a miRNA* sequence. However, our sequencing data showed that tae-miR9760-5p is significantly more abundant than tae-miR9760-3p, and the sequencing number is one order of magnitude higher ( Figure 1 Therefore, this suggests that tae-miR9760-5p is MIR9670 The real functional mature miRNA sequence produced from the precursor (SEQ ID No.2) is subsequently referred to as tae-miR9760 to refer to tae-miR9760-5p (SEQ ID No.1). In addition, we searched the genome sequences of various plant species through bioinformatics and found that tae-miR9760 and its precursor sequences only exist in closely related species such as wheat and rye, and do not exist in other monocotyledons such as rice and corn, as well as dicotyledons, indicating that miR9670 may be a miRNA unique to the wheat family.

[0042] Example 2: Expression analysis and target gene identification of tae-miR9670 1. Analysis of tae-miR9670 expression Total RNA was extracted from different developmental tissues of wheat using RNAiso Plus reagent (Takara, Beijing, China). The expression level of tae-miR9670 was quantified by qRT-PCR using miRcute miRNA cDNA Synthesis Kit and miRcute Plus miRNA qPCR Kit (Tiangen, Beijing, China) according to the instructions. Wheat U6 gene was used as an internal reference. The relative expression level was determined by calculating the fold change using the comparative CT method, and each assay was performed independently three times. The experimental method was referred to the kit instructions, and the quantitative primers were as follows (the universal downstream primer was provided by the kit): Upstream primer for detecting miR9670: 5′-TTCTTCAAGTACTCCACTTTT-3′; Upstream primer for detection of TaU6: 5'-CGGCGGCATGTAGTTACTCG-3'.

[0043] The results of qRT-PCR showed that tae-miR9670 was expressed in various tissues of wheat, including roots, stems, leaves, ears and grains at different developmental stages (under normal growth conditions), so its expression characteristic was constitutive expression ( Figure 2 In addition, we further detected the expression of tae-miR9670 in the roots of seedlings treated with 350 μM CdCl2 for different time periods. The results showed that its expression was significantly induced by CdCl2, increasing by 1.5 and 2 times at 6 and 12 hours compared with 0 hours, respectively ( Figure 2 This result further corroborated the results of the miRNA group.

[0044] 2. Identification of target genes of tae-miR9670 The function of miRNA is closely related to the target genes it regulates, and the target genes of plant miRNAs are usually almost completely complementary to 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 multiple target genes that tae-miR9670 may regulate (the number of mismatches is less than 3), all of which encode mitochondrial transcription termination factors (mTERFs). In addition, according to our existing wheat degradation group 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.), it was confirmed that tae-miR9670 mediated the cleavage of at least two of its representative target gene mRNAs, including TraesCS6D02G023400 and TraesCS6D02G023500 ( Figure 2 These results support that tae-miR9670 can target multiple mTERF genes and mediate their mRNA cleavage.

[0045] Example 3: Construction of tae-miR9670 overexpression vector and acquisition of tae-miR9670 overexpression transgenic wheat 1. 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 of tae-miR9670. First, a 543 bp sequence containing the complete precursor stem-loop sequence of tae-miR9670 was amplified from wheat genomic DNA, and then the sequence was cloned into the modified pCAMBIA3301 vector, so that it was located 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 yieldin common wheat (Triticum aestivum). New Phytol 235, 1515-1530.).

[0046] The “modified pCAMBIA3301 vector” mentioned above is obtained by double digesting the pWMB006 vector (recorded in the article “Liu H, LiH, Hao C, Wang K, Wang Y, Qin L, An D, Li T, Zhang X. TaDA1, a conservednegative regulator of kernel size, has an additive effect with TaGW2 incommon 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, which is then ligated to the large fragment of the pCAMBIA3301 vector that was also digested with HindIII and EcoRI to obtain 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 BamH I recognition sequence); miR9670-OE-R: 5'-GCTCTCTAGA ACTAGT GAATGAACTGGCAGCAAAG-3' (the underlined part is the SpeI recognition sequence).

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

[0049] 2. Obtaining transgenic wheat with tae-miR9670 overexpression The constructed tae-miR9670 overexpression vector pCAMBIA3301-miR9670 was introduced into the Agrobacterium EHA105 strain and transformed into Fielder wheat embryos by Agrobacterium transformation. By extracting the genomic DNA of the transgenic material and performing molecular detection using specific primers, five tae-miR9670 transgenic positive strains were obtained, named OE1-OE5 ( Figure 3 Middle A).

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

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

[0052] Further qRT-PCR experiments (for specific methods and primers, see 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) strains, with an increase of 2-4 times ( Figure 3 Subsequently, three overexpression strains with higher expression levels, OE1-OE3, were selected for phenotypic identification experiments.

[0053] Example 4: Overexpression of tae-miR9670 enhances the tolerance of wheat to cadmium stress at the seedling and adult stages 1. Overexpression of tae-miR9670 enhances wheat seedling 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 a nutrient solution containing 0 or 350μM CdCl2 for further culture. After 15 days of treatment, there was no significant difference between WT and OE under control conditions (0μM CdCl2); however, under 350μM CdCl2 treatment conditions, the growth of WT was significantly slowed down, and the growth state of OE strains was significantly better than that of WT ( Figure 4 Corresponding to this phenotypic result, under CdCl2 treatment, the plant height, root length, fresh weight and dry weight of the OE strain were significantly higher than those of the WT ( Figure 4 Therefore, tae-miR9670 overexpression improved the ability of wheat seedlings to tolerate cadmium stress.

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

[0055] 2. Overexpression of tae-miR9670 reduces cadmium content in mature wheat grains and other organs After germination, the seeds of tae-miR9670-overexpressing wheat (OE1-OE3) and wild type (WT) obtained in Example 3 were planted in plastic pots containing soil with different concentrations (0 mg / kg, 5 mg / kg, 10 mg / kg) of CdCl2 and grown until maturity. After harvest, we measured the cadmium content in roots, flag leaves, sub-ear nodes and grains of wild type and OE lines. The results showed that compared with WT, the overexpression lines generally showed lower cadmium content in all these tissues ( Figure 5 It is worth noting that under the treatment of 10 mg / kg CdCl2, the cadmium content in the grains of the OE strain was significantly lower than that of the WT, decreasing by 61% to 70% ( Figure 5 Similarly, the cadmium content in the sub-ear nodes of the OE lines was also reduced by 55% to 68% compared with that of the WT ( Figure 5 In contrast, the Cd content in the roots and flag leaves of the OE line was slightly lower than that of the WT ( Figure 5 These differences in Cd accumulation in different tissues indicate that overexpression of tae-miR9670 can significantly reduce Cd accumulation in upper organs such as grains and lower nodes of panicle under Cd treatment throughout the growth cycle. This reduction may be due to the reduced transport of Cd caused by overexpression of tae-miR9670, especially the slowing down of Cd transport from roots to upper organs.

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

[0057] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of tae-miR9670 or its related biomaterials in any of the following: (A1) Enhance wheat's tolerance to cadmium stress; (A2) Breed wheat varieties with enhanced tolerance to cadmium stress; (A3) Breed wheat varieties with reduced cadmium content; The related biological material is the precursor of the tae-miR9670 or a DNA molecule that can be transcribed into the tae-miR9670 or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the DNA molecule.

2. Use of tae-miR9670 or its related biomaterials in any of the following: (B1) Promote the increase of wheat plant height under cadmium stress; (B2) Promotes wheat root elongation under cadmium stress; (B3) Promote the increase of wheat fresh weight under cadmium stress; (B4) Promote the increase of wheat dry weight under cadmium stress; (B5) Reducing the content of malondialdehyde in wheat under cadmium stress; (B6) Reducing the cadmium content in wheat under cadmium stress; The related biological material is the precursor of the tae-miR9670 or a DNA molecule that can be transcribed into the tae-miR9670 or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the DNA molecule.

3. Application of promoting tae-miR9670 expression in any of the following: (A1) Enhance wheat's tolerance to cadmium stress; (A2) Breed wheat varieties with enhanced tolerance to cadmium stress; (A3) Breed wheat varieties with reduced cadmium content.

4. Application of promoting tae-miR9670 expression in any of the following: (B1) Promote the increase of wheat plant height under cadmium stress; (B2) Promotes wheat root elongation under cadmium stress; (B3) Promote the increase of wheat fresh weight under cadmium stress; (B4) Promote the increase of wheat dry weight under cadmium stress; (B5) Reducing the content of malondialdehyde in wheat under cadmium stress; (B6) Reducing cadmium content in wheat under cadmium stress.

5. Any of the following methods: Method I: A method for enhancing the cadmium stress resistance of wheat, comprising the following steps: increasing the expression level of tae-miR9670 in the recipient wheat, thereby enhancing the cadmium stress resistance of wheat; Method II: A method for breeding a wheat variety with enhanced cadmium stress tolerance, comprising the following steps: increasing the expression level of tae-miR9670 in a recipient wheat, thereby obtaining a wheat variety with enhanced cadmium stress tolerance; Method III: A method for breeding a wheat variety with reduced cadmium content, comprising the following steps: increasing the expression level of tae-miR9670 in the recipient wheat, thereby obtaining a wheat variety with reduced cadmium content.

6. Either of the following methods: Method IV: A method for promoting the increase of plant height and / or root length elongation and / or fresh weight and / or dry weight of wheat under cadmium stress, comprising the following steps: increasing the expression level of tae-miR9670 in the recipient wheat, thereby promoting the increase of plant height and / or root length elongation and / or fresh weight and / or dry weight of wheat under cadmium stress; Method V: A method for reducing the malondialdehyde content and / or reducing the cadmium content in wheat under cadmium stress, comprising the following steps: increasing the expression level of tae-miR9670 in the recipient wheat, thereby reducing the malondialdehyde content and / or reducing the cadmium content in wheat under cadmium stress.

7. The method according to claim 5 or 6, characterized in that: The expression level of the tae-miR9670 in the recipient wheat is increased by introducing a DNA molecule that can be transcribed into the tae-miR9670 into the recipient wheat.

8. The method according to claim 7, characterized in that: The DNA molecule is introduced into the recipient wheat in the form of a recombinant vector.

9. The use or method according to any one of claims 1 to 8, characterized in that: The sequence of tae-miR9670 is shown in SEQ ID No.

1.

10. The use or method according to any one of claims 1 to 9, characterized in that: The precursor of tae-miR9670 is shown in SEQ ID No. 2; or The DNA molecule is shown as SEQ ID No.3.

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