Application of OsWHY2 gene in regulation and control of cadmium tolerance of rice

Abstract

The invention relates to the technical field of gene engineering, in particular to application of an OsWHY2 gene in regulation and control of cadmium tolerance of rice. The invention provides application of an OsWHY2 gene in regulation and control of cadmium tolerance of rice. The amino acid sequence of protein coded by the OsWHY2 gene is shown as SEQ ID NO. 1. Whole genome correlation analysis is carried out based on cadmium stress response of nearly thousands of core rice varieties in the world, and a key gene OsWHY2 capable of regulating and controlling cadmium tolerance of rice is obtained through identification. The OsWHY2 gene knockout mutant rice root growth inhibition degree is obviously lower than that of a wild type, and the Cd content in the underground part, the overground part (stem and leaf) and grains is also lower than that of the wild type, so that the deletion of the OsWHY2 gene function can obviously improve the Cd tolerance of the rice, and gene resources and technical supports are provided for the breeding of low-Cd-accumulation rice varieties and the cultivation of high-Cd-tolerance plants.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to application of OsWHY2 gene in regulating cadmium tolerance of rice. Background Art

[0002] Cadmium (Cd) is one of the most toxic trace heavy metal elements to plants and animals. It is highly mobile and is more easily absorbed by crop roots and transferred to edible parts of crops. It then enters the human body through the food chain, causing harm to human health. In recent decades, domestic and foreign scholars have used physical, chemical, biological and chemical-biological combined technologies to repair cadmium-contaminated farmland soils and have achieved a series of research results. However, due to the complexity of the soil itself and the strong dependence of crops on the growth environment, most of the above research results cannot be applied outside the laboratory in real environments or on a large scale. In addition, farmland ecosystems are extremely susceptible to environmental and human factors. Therefore, cadmium bioavailability regulation technologies based on physical, chemical or biological methods cannot last. Fluctuations in environmental factors are likely to cause changes in cadmium forms, thereby affecting crop cadmium absorption and accumulation processes. Therefore, screening and breeding tolerant varieties are of great practical significance for rice production to avoid soil environmental risks. Summary of the invention

[0003] In order to solve the above problems, the present invention provides the application of OsWHY2 gene in regulating cadmium tolerance of rice. The present invention discovered the OsWHY2 gene that plays a negative role in rice Cd tolerance. Knocking out the OsWHY2 gene can not only alleviate the inhibition of Cd on rice root growth, reduce the accumulation of Cd in the root system, aboveground parts (stems and leaves) and grains, but also has little effect on agronomic traits such as plant height and yield, providing genetic resources and technical support for the selection and breeding of low Cd accumulation rice varieties and the cultivation of highly Cd-tolerant plants.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides application of OsWHY2 gene in regulating cadmium tolerance of rice. The amino acid sequence of the protein encoded by the OsWHY2 gene is shown in SEQ ID NO.1.

[0006] Preferably, the regulation is that the OsWHY2 gene plays a negative regulatory role in regulating the cadmium tolerance of rice; the negative regulation is: knocking out or silencing the OsWHY2 gene to improve the cadmium tolerance of rice, or overexpressing the OsWHY2 gene to increase the cadmium sensitivity of rice.

[0007] Preferably, the improving the cadmium tolerance of rice includes: reducing the inhibition of cadmium on rice growth and / or reducing the cadmium accumulation of rice; the increasing the cadmium sensitivity of rice includes: enhancing the inhibition of cadmium on rice growth and / or increasing the cadmium accumulation of rice.

[0008] Preferably, the inhibition of rice growth by cadmium includes: reducing one or more of rice plant height, root length, root biomass and aboveground biomass; the inhibition of rice growth by cadmium is the inhibition of rice growth at the seedling stage by cadmium; the rice cadmium accumulation includes: one or more of rice root cadmium accumulation, aboveground cadmium accumulation and grain cadmium accumulation.

[0009] The present invention provides a biological material for improving cadmium tolerance of rice, comprising one or more of 1) to 4):

[0010] 1) A target nucleic acid molecule of the OsWHY2 gene; the nucleotide sequence of the target nucleic acid molecule is shown in SEQ ID NO.3;

[0011] 2) an sgRNA containing the target nucleic acid molecule described in 1);

[0012] 3) a knockout vector containing the sgRNA described in 2);

[0013] 4) An engineered bacterium containing the knockout vector described in 2).

[0014] The present invention provides a biomaterial for increasing the cadmium sensitivity of rice, comprising: a recombinant vector and / or an engineered bacterium overexpressing an OsWHY2 gene; the engineered bacterium contains the recombinant vector; and the amino acid sequence of the protein encoded by the OsWHY2 gene is shown in SEQ ID NO.1.

[0015] The present invention provides the use of the biological material for improving the cadmium tolerance of rice described in the above technical solution in improving the cadmium tolerance of rice and / or cultivating cadmium-resistant rice varieties.

[0016] Preferably, the cadmium-resistant rice variety is a rice variety with the OsWHY2 gene knocked out or silenced. Compared with wild-type rice, the rice variety with the OsWHY2 gene knocked out or silenced has improved cadmium tolerance, reduced cadmium content in grains, and other agronomic traits at maturity are comparable.

[0017] The present invention provides the use of the biological material for increasing the cadmium sensitivity of rice according to the above technical solution in increasing the cadmium sensitivity of rice and / or cultivating cadmium-sensitive rice varieties.

[0018] Preferably, the cadmium-sensitive rice variety is a rice variety in which the OsWHY2 gene is overexpressed. Compared with wild-type rice, the rice variety in which the OsWHY2 gene is overexpressed has increased cadmium sensitivity, and other agronomic traits are comparable in a cadmium-free environment.

[0019] Beneficial effects:

[0020] The present invention provides an application of the OsWHY2 gene in regulating rice cadmium tolerance, wherein the amino acid sequence of the protein encoded by the OsWHY2 gene is shown in SEQ ID NO. 1. Based on a genome-wide association analysis of the cadmium stress response of nearly a thousand core rice varieties in the world, the present invention identifies a key gene OsWHIRLY2 (denoted as OsWHY2; MSU: LOC_Os02g06370) that can regulate rice cadmium tolerance. A mutant oswhy2 with a loss of function of the gene is created by CRISPR / Cas9 gene editing technology. After exogenous addition of Cd, it was found that the mutant plants had significantly improved tolerance to Cd. Specifically, the root length of the OsWHY2 mutant plants was significantly less inhibited than that of the wild-type plant Zhonghua 11 (ZH11) after Cd treatment, and the Cd content in the roots and aboveground parts was significantly lower than that of the wild-type. At the same time, the mutant and the wild-type Zhonghua 11 were planted together in farmland with moderate to light Cd pollution. After the rice matured, the rice grains were collected for Cd content determination. The results also proved that the Cd content in the grains of the OsWHY2 mutant was significantly lower than that of the wild-type, but the agronomic traits related to yield were basically unaffected. In addition, the genetic material overexpressing the OsWHY2 gene showed a significant Cd-sensitive phenotype in Cd, the root system was significantly more inhibited than the wild-type ZH11, and the Cd accumulation in the roots and aboveground parts was significantly higher than that of the wild-type. This shows that OsWHY2 plays an important role in regulating the Cd tolerance of rice, providing genetic resources and technical support for the selection of low-Cd accumulation rice varieties and the cultivation of highly Cd-tolerant plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 The results of sequencing and identification of oswhy2 mutants;

[0023] Figure 2 The phenotypes of oswhy2 mutant and wild-type ZH11 rice under Cd treatment;

[0024] Figure 3 The results of root elongation, root biomass and aboveground biomass of oswhy2 mutant and wild-type ZH11 rice under Cd treatment are shown in Figure 2.

[0025] Figure 4 The results of the determination of cadmium content in the aboveground and underground roots of the oswhy2 mutant and wild-type ZH11 rice under Cd treatment;

[0026] Figure 5 This is a comparison of the phenotypes of oswhy2 mutant and wild-type ZH11 rice plants at maturity in a conventional field environment;

[0027] Figure 6 This is a comparison of the plant phenotype and yield per plant of the oswhy2 mutant and wild-type ZH11 rice at maturity in paddy fields with moderate to mild Cd pollution;

[0028] Figure 7 The phenotypes of wild-type Zhonghua 11, overexpression materials OsWHY2-OX-1 and OsWHY2-OX-2 under Cd treatment;

[0029] Figure 8 The results of root elongation, root biomass and aboveground biomass of wild-type Zhonghua11, overexpression materials OsWHY2-OX-1 and OsWHY2-OX-2 under Cd treatment are shown;

[0030] Fig. 9 The results are as follows: the results of the determination of cadmium content in the aboveground and underground roots of wild-type Zhonghua 11, overexpression materials OsWHY2-OX-1 and OsWHY2-OX-2 under Cd treatment;

[0031] Fig.10 This is a comparison of the phenotypes of mature plants of wild-type rice Zhonghua 11, overexpression materials OsWHY2-OX-1 and OsWHY2-OX-2 in a field environment without cadmium pollution;

[0032] Among them, * indicates P<0.05. DETAILED DESCRIPTION

[0033] The present invention provides an application of the OsWHY2 gene in regulating cadmium tolerance in rice. The amino acid sequence of the protein encoded by the OsWHY2 gene is shown in SEQ ID NO.1, which is as follows:

[0034] MQRLSRFVPSSSSRRVTDLKDALWSGSLTFQHALSTFAADENTSGRKFASYTVFKGKAALSMQPILPSFSKLESGGSRVNKNGSVMLTFFPAVGQRKYDYSKKQLFALSPTEVGS LISLGPAESCEFFHDPSMKSSHEGQVKKSLSVTPLGNDSGYFLNITVLNNLQKTTERLSLPISKAEFTVMRTALSFALPHILGWDQALTNHQPSPSPASKPRVERPHPDSEWER.

[0035] As an embodiment, the nucleotide sequence of the CDS of the OsWHY2 gene is shown in SEQ ID NO.2, which is as follows:

[0036] 5'-ATGCAGCGCCTCCCGCTTCGTCCCCTCCTCCTCCAGGAGAGTCA -3'.

[0037] As an embodiment, the regulation is that the OsWHY2 gene plays a negative regulatory role in regulating rice cadmium tolerance; the negative regulation is: knocking out or silencing the OsWHY2 gene to improve rice cadmium tolerance, or overexpressing the OsWHY2 gene to increase rice cadmium sensitivity.

[0038] As an embodiment, the improving cadmium tolerance of rice includes: reducing the inhibition of cadmium on rice growth and / or reducing the cadmium accumulation of rice; the increasing cadmium sensitivity of rice includes: enhancing the inhibition of cadmium on rice growth and / or increasing the cadmium accumulation of rice.

[0039] As an embodiment, the inhibition of rice growth by cadmium includes: reducing one or more of rice plant height, root length, root biomass and aboveground biomass; the inhibition of rice growth by cadmium is the inhibition of rice growth in the seedling stage by cadmium; the rice cadmium accumulation includes: one or more of rice root cadmium accumulation, aboveground cadmium accumulation and grain cadmium accumulation.

[0040] The present invention uses CRISPR / Cas9 gene editing technology to create a mutant oswhy2 with a loss of function of the gene OsWHY2 (LOC_Os02g06370), and conducts field experiments on exogenous Cd stress and moderate to mild Cd pollution together with the wild type Zhonghua 11, and measures the Cd content in the aboveground part, underground part and grain of rice. The results show that the degree of inhibition of root growth of the mutant is significantly lower than that of the wild type, and the Cd content in the underground part, aboveground part (stems and leaves) and grain is also lower than that of the wild type, but the yield The agronomic traits such as the amount of OsWHY2 and the growth rate were comparable to those of the wild type, indicating that the loss of OsWHY2 gene function could significantly improve the tolerance of rice to Cd. In addition, the OsWHY2 gene overexpression genetic material showed a significant Cd-sensitive phenotype under Cd stress, with the degree of root inhibition significantly stronger than that of the wild type ZH11, and the Cd accumulation in the roots and aboveground parts significantly higher than that of the wild type, further indicating that the OsWHY2 gene can not only be used to improve the regulatory network of rice Cd stress, but also provide technical support for the breeding of rice varieties with low Cd accumulation.

[0041] In order to knock out or silence the OsWHY2 gene to improve the cadmium tolerance of rice, the present invention provides a biological material for improving the cadmium tolerance of rice, comprising:

[0042] 1) A target nucleic acid molecule of the OsWHY2 gene; the nucleotide sequence of the target nucleic acid molecule is shown in SEQ ID NO.3, specifically: 5'-TGACCTAAAGGATGCTCTC-3';

[0043] 2) an sgRNA containing the target nucleic acid molecule described in 1);

[0044] 3) a knockout vector containing the sgRNA described in 2);

[0045] 4) An engineered bacterium containing the knockout vector described in 2).

[0046] As an embodiment, the sgRNA includes sgRNA-S and sgRNA-A; the nucleotide sequences of the sgRNA-S and sgRNA-A are shown in SEQ ID No.4 and SEQ ID No.5, respectively, as follows:

[0047] SEQ ID No.4: 5'-ggcaTGACCTAAAGGATGCTCTC-3';

[0048] SEQ ID No. 5: 5'-aaacGAGAGCATCCTTTAGGTCA-3'.

[0049] As an embodiment, the intermediate vector for constructing the knockout vector includes SK-gRNA; the basic vector pC1300-Cas9 vector for constructing the knockout vector. The SK-gRNA and pC1300-Cas9 vectors described in the present invention are both deposited in the China National Rice Research Institute (Hangzhou) and disclosed in the document [Upgrading the genome of an elite japonica rice variety Kongyu 131for lodging resistance improvement], and the original link is https: / / doi.org / 10.1111 / pbi.13963.

[0050] As an embodiment, the original strain of the engineered bacteria includes Agrobacterium. As another embodiment, the Agrobacterium can be Agrobacterium GV3101.

[0051] The biomaterial for improving cadmium tolerance of rice provided by the present invention is to cause the OsWHY2 gene in the rice plant to mutate (base deletion / addition / denaturation), so that the OsWHY2 gene function is lost or silenced, thereby significantly improving the tolerance of rice to Cd without affecting agronomic traits such as yield. It provides genetic resources and technical support for the selection and breeding of low Cd accumulation rice varieties and the cultivation of high Cd tolerance plants.

[0052] In order to increase the cadmium sensitivity of rice, the present invention provides a biological material for increasing the cadmium sensitivity of rice, including: a recombinant vector and / or an engineered bacterium that overexpresses the OsWHY2 gene; the engineered bacterium contains the recombinant vector; the amino acid sequence of the protein encoded by the OsWHY2 gene is shown in SEQ ID NO.1.

[0053] As an embodiment, the basic vector of the recombinant vector for overexpressing the OsWHY2 gene includes the pCAMBIAsuper1300-GFP vector.

[0054] As an embodiment, the original strain of the engineered bacteria includes Agrobacterium. As another embodiment, the Agrobacterium can be Agrobacterium GV3101.

[0055] The biological material for increasing the cadmium sensitivity of rice provided by the present invention increases the cadmium sensitivity of rice by increasing the expression amount of the rice OsWHY2 gene, and can be used to cultivate cadmium-sensitive rice varieties.

[0056] Based on the above advantages, the present invention provides the use of the biological material for improving the cadmium tolerance of rice described in the above technical solution in improving the cadmium tolerance of rice and / or cultivating cadmium-resistant rice varieties.

[0057] As an embodiment, the cadmium-tolerant rice variety is a rice variety with knockout or silenced OsWHY2 gene, and compared with wild-type rice, the rice variety with knockout or silenced OsWHY2 gene has improved cadmium tolerance, reduced grain cadmium content, and other agronomic traits at maturity are equivalent. As an embodiment, the other agronomic traits include: plant height and / or single plant yield.

[0058] As an embodiment, the biological material is transferred into rice by Agrobacterium-mediated rice genetic transformation, which can improve the cadmium resistance of rice and / or cultivate cadmium-resistant rice varieties.

[0059] Based on the above advantages, the present invention provides the use of the biological material for increasing the cadmium sensitivity of rice as described in the above technical solution in increasing the cadmium sensitivity of rice and / or cultivating cadmium-sensitive rice varieties. As an embodiment, the biological material is transferred into rice by Agrobacterium-mediated rice genetic transformation, which can increase the cadmium sensitivity of rice and / or cultivate cadmium-sensitive rice varieties. As an embodiment, the cadmium-sensitive rice variety is a rice variety with overexpression of the OsWHY2 gene. Compared with wild-type rice, the rice variety with overexpression of the OsWHY2 gene has increased cadmium sensitivity, and other agronomic traits are comparable in a cadmium-free environment.

[0060] To further illustrate the present invention, the application of the OsWHY2 gene provided by the present invention in regulating the cadmium tolerance of rice is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0061] Example 1

[0062] A recombinant vector for knocking out the OsWHY2 gene, the construction method is as follows:

[0063] 1) Designing sgRNA primers according to the OsWHY2 gene and entrusting Nanjing Qingke Biotechnology Co., Ltd. to synthesize them, wherein the sgRNA consists of sgRNA-S and sgRNA-A, and the nucleotide sequences of the sgRNA-S and sgRNA-A are shown in SEQ ID No. 4 and SEQ ID No. 5, respectively.

[0064] 2) Mix sgRNA-S: sgRNA-A: ddH2O in a volume ratio of 5:5:15 to obtain a mixture, place it in a 100°C metal bath for incubation for 5 minutes, and cool it at room temperature for 10 minutes to obtain an oligo dimer for use; the concentration of the sgRNA-S and sgRNA-A is 10 μM.

[0065] 3) The SK-gRNA vector was treated with Aar I restriction endonuclease in a 50°C water bath for 2 h, and then subjected to agarose gel electrophoresis and gel recovery to obtain the SK-gRNA digestion product.

[0066] 4) Use T4 DNA ligase to connect the vector, mix SK-gRNA digestion product: Oligo dimer: T4 DNA ligase: T4 enzyme buffer at a volume ratio of 1:7:1:1, and let it stand at room temperature for 4 hours to obtain a primary recombinant vector; transfer the constructed primary recombinant vector into DH5α competent cells, and use ampicillin-resistant LB solid culture medium for monoclonal screening, detect positive clones by colony PCR, and obtain a successfully constructed intermediate recombinant vector after successful sequencing and sequence alignment by the company.

[0067] 5) double-digesting the intermediate recombinant vector with Kpn I and Bgl II restriction endonucleases to obtain an intermediate recombinant vector digestion product; double-digesting the pC1300-Cas9 vector with Kpn I and BamH I restriction endonucleases to obtain a pC1300-Cas9 vector digestion product; mixing the intermediate recombinant vector digestion product: pC1300-Cas9 vector digestion product: T4 DNA ligase: T4 Buffer at a volume ratio of 4:4:1:1, and standing at room temperature for 4 hours to obtain the recombinant vector for knocking out the OsWHY2 gene.

[0068] Example 2

[0069] The recombinant vector for knocking out the OsWHY2 gene constructed in Example 1 was transferred into DH5α competent cells and positive single clones were screened using kanamycin-resistant LB solid medium to obtain positive clone bacterial solution. The obtained positive clone bacterial solution was used to extract the recombinant plasmid using a plasmid extraction kit.

[0070] The extracted recombinant plasmid was entrusted to Wuhan Boyuan Biotechnology Co., Ltd. for transgenic rice cultivation. The transgenic rice cultivation method was Agrobacterium-mediated rice genetic transformation, including: transferring the recombinant plasmid into Agrobacterium GV3101 and then infecting the callus of the japonica rice variety Zhonghua 11 (ZH11), and screening to obtain the positive transgenic mutant oswhy2. The screening method includes:

[0071] When the transgenic mutant seedlings grew to 10 cm, DNA was extracted from the wild-type Zhonghua 11 and the transgenic mutant plant samples to be identified to obtain DNA sample test fluid.

[0072] The DNA sample to be tested is used as a template, and genomic PCR amplification is performed using hygromycin sequence primers Hyg-F and Hyg-R to obtain PCR products; the nucleotide sequences of Hyg-F and Hyg-R are as follows:

[0073] Hyg-F: 5'-TTTCTTTGCCCTCGGACGAGT-3' (SEQ ID No. 6);

[0074] Hyg-R: 5'-ATGAAAAACCTGAACTCACC-3' (SEQ ID No. 7).

[0075] The reaction system of the PCR amplification is: 10 μL 2×Rapid Taq Master Mix, 8 μL ddH2O, 0.5 μL Hyg-F (10 μM), 0.5 μL Hyg-R (10 μM) and 1 μM DNA sample.

[0076] The reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 57°C for 15 s, extension at 72°C for 15 s, 32 cycles; final extension at 72°C for 3 min, and storage at 4°C.

[0077] The PCR amplification product is subjected to band detection by agarose gel electrophoresis, and the transgenic mutant containing the target band is initially identified as a positive transgenic mutant into which the target fragment has been successfully transferred.

[0078] The transgenic seedlings initially identified as positive were further sequenced and identified using primers OsWHY2-F and OsWHY2-R. The PCR amplification reaction system and reaction procedure were the same as above, except that Hyg-F and Hyg-R were replaced by OsWHY2-F and OsWHY2-R, respectively. The primer sequences are as follows:

[0079] OsWHY2-F: 5'-TACAATGGTATCTTCAAGTCAATCC-3' (SEQ ID No. 8);

[0080] OsWHY2-R: 5'-GTCTTCACCGTTCTGCTGCAAATGTT-3' (SEQ ID No. 9).

[0081] The obtained PCR products were sent to a testing company for first-generation sequencing, and the obtained sequencing results were compared with the PCR amplification sequencing results of wild-type (WT) Zhonghua 11 to determine whether the OsWHY2 gene was successfully mutated.

[0082] The sequencing results are shown in Figure 1The results showed that the oswhy2-1 mutant (OsWHIRLY2-1) had a single base insertion, a T base inserted between 16bp and 17bp of the target site; the oswhy2-2 mutant (OsWHIRLY2-2) had a single base insertion, an A base inserted between 16bp and 17bp of the target site.

[0083] The successful mutant positive seedlings were propagated, and after at least two generations of reproduction, two stable homozygous mutant strains were obtained, named oswhy2-1 and oswhy2-2.

[0084] Application Example 1 Tolerance of OsWHY2 gene mutants to Cd treatment

[0085] In order to determine the regulatory role of OsWHY2 gene on Cd tolerance in rice, wild type Zhonghua 11, oswhy2-1 and oswhy2-2 were used as experimental materials and treated with Cd at the same time to observe their phenotypic changes, as follows:

[0086] After the rice seeds turned white, they were germinated in 0.5 mM CaCl2 solution under dark conditions. After the roots grew to about 2 cm in length, they were transferred to Kimura B rice nutrient solution and cultured in the greenhouse for another week. Seedlings with consistent root length and aboveground growth were selected and transferred to Kimura B rice nutrient solution containing 1 μM CdSO4 (denoted as +Cd) for continued cultivation. At the same time, Kimura B rice nutrient solution without 1 μM CdSO4 (denoted as -Cd) was set up as a control group.

[0087] After 7 days of cultivation, the phenotypic data of rice plants were collected and measured, including plant height, root length, root biomass and aboveground biomass, with 10 plants measured for each. At the same time, the aboveground and underground parts of the mutants and wild types under Cd treatment conditions were harvested and repeatedly rinsed with deionized water to remove the residual Cd on the plants. After absorbing the water, the plant biomass was weighed, and after complete digestion with 2mL nitric acid, it was adjusted to 10mL with ultrapure water. The Cd content was determined by inductively coupled plasma-mass spectrometry. Four biological replicates were set for each of the three materials. The results are shown in Figure 2 to Figure 4 .

[0088] Depend on Figure 2 It can be seen that under the treatment conditions without Cd, the growth of oswhy2-1, oswhy2-2 and the wild type Zhonghua11 were basically the same; under the treatment conditions of 1μM Cd, the growth of oswhy2-1 and oswhy2-2 was significantly better than that of Zhonghua11.

[0089] Depend on Figure 3It can be seen that under normal conditions, there was no difference in root length and biomass between the mutant and Zhonghua 11. Under Cd stress, the root length of the mutant plant was significantly longer than that of Zhonghua 11, that is, the degree of Cd inhibition of the root growth of the mutant was significantly lower than that of Zhonghua 11.

[0090] Depend on Figure 4 It can be seen that the Cd content in the aboveground and underground parts of the mutant plants was significantly lower than that in Zhonghua11.

[0091] Based on the above results, it can be determined that the mutants oswhy2-1 and oswhy2-2 plants have higher tolerance to Cd, and the OsWHY2 gene has a negative regulatory effect on rice Cd tolerance.

[0092] Application Example 2

[0093] Wild-type rice ZH11 and mutant materials oswhy2-1 and oswhy2-2 were planted in a conventional soil environment and a soil with a Cd content of 0.76 mg / kg and a pH value of 4.65, respectively. After the grains matured, the yield per plant of the mutant and wild-type in the conventional soil was measured and the plant phenotype was observed. The results are shown in Figure 5 and Figure 6 The seeds on the Cd soil were collected to determine the Cd content in the seeds. They were threshed, washed and dried respectively. 40 seeds with uniform growth were weighed respectively, 5 mL of HNO3 was added, and the seeds were allowed to digest overnight. Then they were heated and digested in a 120°C digestion furnace until the liquid was clear and transparent. After cooling, deionized water was added to make the volume 10 mL. The cadmium content in the seeds was determined by inductively coupled plasma-mass spectrometry. Four biological replicates were set for the three materials. The results are shown in Table 1.

[0094] Table 1 Cadmium content in different rice grains (unit: mg / kg)

[0095] Group ZH11 Oswhy2-1 Oswhy2-2 Repeat 1 0.285 0.139 0.141 Repeat 2 0.266 0.170 0.175 Repeat 3 0.243 0.142 0.128 Repeat 4 0.211 0.156 0.190

[0096] From Table 1, Figure 5 and Figure 6 It can be seen that the Cd content in rice grains of mutant materials oswhy2-1 and oswhy2-2 was significantly lower than that of wild-type rice variety Zhonghua 11. However, agronomic traits such as yield at maturity and plant height of mutant materials were comparable to those of wild-type rice variety ZH11 under conventional and moderately to lightly Cd-polluted field conditions (i.e., there was no significant difference).

[0097] Example 3

[0098] A recombinant vector for overexpressing the OsWHY2 gene, the construction method is as follows:

[0099] 1) Using Nipponbare cDNA as a template, amplify the complete OsWHY2 CDS coding sequence (shown in SEQ ID No. 2). The primer sequences used for amplification are as follows:

[0100] OsWHY2-P-super-1300-F (SEQ ID No.10):

[0101] 5'-CTAGAAAGCTTCTGCAGATGCAGCGCCTCTCCCGCTTCGT-3';

[0102] OsWHY2-P-super-1300-R(SEQ ID No.11):

[0103] 5'-ACTAGTATTTAAATGTCGACCCTTTCCCACTCTGAATCTGGGTGT-3'.

[0104] The high success rate PCR enzyme KOD FX from TOYOBO was used for amplification.

[0105] The amplification reaction system was: 15 μL 2× PCR buffer, 6 μL dNTPs, 5.25 μL ddH2O, 0.75 μL KODFIX enzyme, 0.75 μL OsWHY2-P-super-1300-F (10 μM), 0.75 μL OsWHY2-P-super-1300-F (10 μM) and 1.5 μL cDNA sample.

[0106] The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, annealing at 57°C for 30 s, extension at 68°C for 30 s, 32 cycles; final extension at 68°C for 10 min, and storage at 4°C.

[0107] The PCR product was run on agarose gel electrophoresis and then recovered to obtain the OsWHY2-P-super-1300 product.

[0108] 2) The P-super-1300 vector was digested with Pst I and Sal I restriction endonucleases to obtain a linearized P-super-1300 vector; the OsWHY2-P-super-1300 product and the linearized P-super-1300 vector were connected with Thermo Fisher's seamless cloning enzyme, and the mixture was mixed at a volume ratio of OsWHY2-P-super-1300 product: linearized P-super-1300 vector: seamless cloning enzyme = 3:2:5, and then incubated in a water bath at 50°C for 20 minutes to obtain a recombinant vector.

[0109] 3) The recombinant vector constructed in step 2) was transferred into DH5α competent cells, and single clone screening was performed using kanamycin-resistant LB solid medium. Positive clones were detected by colony PCR. After successful sequencing and sequence alignment by the company, the recombinant plasmid was extracted from the obtained positive clone bacterial liquid using a plasmid extraction kit to obtain a recombinant vector overexpressing the OsWHY2 gene.

[0110] Example 4

[0111] The recombinant plasmid extracted in step 3) of Example 1 was entrusted to Wuhan Boyuan Biotechnology Co., Ltd. for transgenic rice cultivation. The transgenic rice cultivation method was Agrobacterium-mediated rice genetic transformation, including: transferring the recombinant plasmid into Agrobacterium GV3101 and then infecting the callus of the japonica rice variety Zhonghua 11, and screening to obtain positive transgenic OsWHY2 overexpression materials (OsWHY2-OX). The initial identification method of the positive seedlings was the same as that of Example 2.

[0112] Total RNA was extracted from the positive seedlings that were successfully identified, and real-time fluorescence quantitative q-PCR was used to detect the gene expression of the target fragment. Total RNA from the roots of wild-type Zhonghua 11 and positive plant samples was extracted, and then the RNA was reverse transcribed into cDNA, and real-time fluorescence quantitative q-PCR was used to detect the gene expression of the target fragment. -ΔΔ The raw data were analyzed by the PCR amplification method, and OsActin and OsUBQ were used as internal reference genes to normalize the expression levels to ensure accurate quantification of gene expression. The primer sequences are shown in Table 2.

[0113] Table 2 Primers used in q-PCR

[0114] Primer name Primer sequences OsWHY2-F 5'-TGAGCGACTTTCACTCCCAAT-3'(SEQ ID NO.12) OsWHY2-R 5'-TGAATCTGGGTGTGGGCGCT-3'(SEQ ID NO.13) OsActin-F 5'-CAACACCCCTGCTATGTACG-3'(SEQ ID NO.14) OsActin-R 5'-CATCACCAGAGTCCAACACAA-3'(SEQ ID NO.15) OsUBQ-F 5'-GCTCCGTGGCGGTATCAT-3'(SEQ ID NO.16) OsUBQ-R 5'-CGGCAGTTGACAGCCCTAG-3'(SEQ ID NO.17)

[0115] According to the gene expression test results, transgenic seedlings with expression levels greater than or equal to 2 times were selected and propagated individually. After at least 2 generations of breeding, two stable overexpression strains were obtained and named OsWHY2-OX-1 and OsWHY2-OX-2.

[0116] Application Example 3 Tolerance of OsWHY2 overexpressing genetic material to Cd treatment

[0117] The wild type Zhonghua 11, OsWHY2-OX-1 and OsWHY2-OX-2 were used as experimental materials and Cd treatment was performed at the same time to observe the phenotypic changes of each. The experimental process and detection indicators were similar to those of Application Example 1, except that the mutant material was replaced with the OsWHY2 overexpression strain. The results are shown in Figure 7 to Figure 9 .

[0118] Depend on Figure 7It can be seen that under the treatment conditions without Cd, the growth of OsWHY2-OX-1, OsWHY2-OX-2 and the wild type Zhonghua 11 were basically the same; under the treatment conditions of 1 μM Cd, the growth of OsWHY2-OX-1 and OsWHY2-OX-2 was significantly more inhibited by Cd than that of Zhonghua 11.

[0119] Depend on Figure 8 It can be seen that under normal conditions, there was no difference in root length and biomass between overexpression and Zhonghua 11. Under Cd stress, the root length of the overexpression plant was significantly shorter than that of Zhonghua 11, that is, the root growth of the overexpression plant was significantly more inhibited by Cd than that of Zhonghua 11.

[0120] Depend on Fig. 9 It can be seen that the Cd content in the aboveground and underground parts of the overexpression plants was significantly lower than that in Zhonghua11.

[0121] Based on the above results, it can be determined that rice plants overexpressing the OsWHY2 gene are more sensitive to Cd, which further verifies that the OsWHY2 gene has a negative regulatory effect on rice Cd tolerance.

[0122] Application Example 4

[0123] Phenotypic comparisons of overexpression materials (OsWHY2-OX-1, OsWHY2-OX-2) and Zhonghua 11 at maturity were taken under conventional field conditions. The experimental process was similar to that of Application Example 2, except that the mutant material was replaced with the OsWHY2 overexpression strain.

[0124] Results Fig.10 The results showed that the plant phenotypes of the overexpression materials and the wild-type rice variety ZH11 at maturity were comparable (i.e., there was no significant difference) under cadmium-free conditions.

[0125] In summary, the present invention mutates the OsWHY2 gene by genetic engineering technology, and obtains the mutant strains oswhy2-1 and oswhy2-2 of the OsWHY2 gene through screening, and experimentally verifies that under Cd treatment conditions, the phenotypic data of the mutant strains are better than those of the wild type Zhonghua 11, and the Cd content in the aboveground part, underground part and grain is significantly lower than that in Zhonghua 11, indicating that the mutant strains are more tolerant to Cd. At the same time, by constructing genetic materials for overexpression of the OsWHY2 gene, the present invention confirms that overexpression of the OsWHY2 gene increases the sensitivity of rice to Cd and plays a negative role in Cd tolerance. In view of the application of the OsWHY2 gene under Cd stress, it can be considered that the gene has potential application value in improving the resistance of rice to Cd stress. At the same time, the present invention also provides genetic resources and technical support for the breeding of Cd-resistant rice varieties and low Cd accumulation rice varieties using the OsWHY2 gene.

[0126] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of OsWHY2 gene in regulating cadmium tolerance of rice, the amino acid sequence of the protein encoded by the OsWHY2 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The regulation is that the OsWHY2 gene plays a negative regulatory role in regulating the cadmium tolerance of rice; the negative regulation is: knocking out or silencing the OsWHY2 gene to improve the cadmium tolerance of rice, or overexpressing the OsWHY2 gene to increase the cadmium sensitivity of rice.

3. The use according to claim 2, characterized in that: The method of improving the cadmium tolerance of rice includes: reducing the inhibition of cadmium on rice growth and / or reducing the cadmium accumulation of rice; the method of increasing the cadmium sensitivity of rice includes: enhancing the inhibition of cadmium on rice growth and / or increasing the cadmium accumulation of rice.

4. The use according to claim 3, characterized in that: The inhibition of rice growth by cadmium includes: reducing one or more of rice plant height, root length, root biomass and aboveground biomass; the inhibition of rice growth by cadmium is the inhibition of rice growth at the seedling stage by cadmium; the rice cadmium accumulation includes: one or more of rice root cadmium accumulation, aboveground cadmium accumulation and grain cadmium accumulation.

5. A biomaterial for improving cadmium tolerance in rice, characterized in that: Including one or more of 1) to 4): 1) A target nucleic acid molecule of the OsWHY2 gene; the nucleotide sequence of the target nucleic acid molecule is shown in SEQ ID NO.3; 2) an sgRNA containing the target nucleic acid molecule described in 1); 3) a knockout vector containing the sgRNA described in 2); 4) An engineered bacterium containing the knockout vector described in 2).

6. A biomaterial for increasing cadmium sensitivity in rice, characterized in that: include: A recombinant vector and / or engineered bacteria overexpressing the OsWHY2 gene; the engineered bacteria contains the recombinant vector; the amino acid sequence of the protein encoded by the OsWHY2 gene is shown in SEQ ID NO.

1.

7. Use of the biomaterial according to claim 5 in improving the cadmium tolerance of rice and / or cultivating cadmium-tolerant rice varieties.

8. The use according to claim 7, characterized in that: The cadmium-tolerant rice variety is a rice variety with the OsWHY2 gene knocked out or silenced. Compared with wild-type rice, the rice variety with the OsWHY2 gene knocked out or silenced has improved cadmium tolerance, reduced cadmium content in grains, and other agronomic traits at maturity are comparable.

9. Use of the biomaterial according to claim 6 in increasing the cadmium sensitivity of rice and / or cultivating cadmium-sensitive rice varieties.

10. The use according to claim 9, characterized in that: The cadmium-sensitive rice variety is a rice variety in which the OsWHY2 gene is overexpressed. Compared with wild-type rice, the rice variety in which the OsWHY2 gene is overexpressed has increased cadmium sensitivity, and other agronomic traits are comparable in a cadmium-free environment.

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