Application of phyllostachys edulis PeNRAMP5 gene in reduction of Cd accumulation in rice and increase of Mn absorption

Abstract

The invention discloses application of a phyllostachys edulis PeNRAMP5 gene to reduction of Cd accumulation in rice and increase of Mn absorption. The OsNRAMP5 mutant has the advantages that the PeNRAMP5-1593 or PeNRAMP5-1602 gene of moso bamboo is supplemented in the OsNRAMP5 mutant, so that Cd absorption of a rice root system is remarkably increased, but the transfer efficiency of Cd absorbed by the rice root system to an overground part is remarkably reduced, Cd accumulation of the overground part is effectively reduced, compared with wild rice, the Cd accumulation amount of the overground part of transgenic rice can be reduced by about 29 times, and compared with the OsNRAMP5 mutant, the Cd accumulation amount of the overground part of transgenic rice can be reduced by about 29 times. And the Cd accumulation amount of the overground part of the transgenic rice can be reduced by about 3 times. Meanwhile, compared with the OsNRAMP5 mutant, the transgenic rice recovers the absorption of Mn and the accumulation of overground parts.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and specifically relates to bamboo PeNRAMP5 Application of genes in reducing Cd accumulation and increasing Mn uptake in rice. Background Art

[0002] Rice has a strong ability to absorb Cd. Cd pollution not only leads to a reduction in rice production, but also accumulates in rice, enters the human body through the food chain, and threatens human health.

[0003] Nramp5 The gene encodes a metal ion transporter that is mainly involved in the absorption and transport of Mn and Cd. It plays a key role in plant nutrient absorption and heavy metal tolerance and has important application value in agriculture and environmental remediation. Nramp5 Genes can improve the absorption characteristics of crops for metal ions and reduce the impact of heavy metal pollution on food safety.

[0004] However, using Nramp5 When genes control the absorption of Cd, the absorption of Mn is also reduced. However, Mn is an essential element for plants, and the lack of Mn will affect the normal growth of plants. Summary of the invention

[0005] In order to solve the problems and shortcomings of the prior art, the present invention aims to provide a PeNRAMP5 Application of genes in reducing Cd accumulation in rice and increasing Mn absorption. PeNRAMP5 The gene inhibits the absorption of Cd while maintaining the absorption of Mn by the aboveground part of rice.

[0006] bamboo PeNRAMP5 Application of genes in regulating reduction of Cd accumulation and increase of Mn uptake in rice.

[0007] Furthermore, bamboo PeNRAMP5 Genes include PeNRAMP5-1593 Genes and PeNRAMP5-1602 Gene.

[0008] Further, PeNRAMP5-1593 The amino acid sequence encoded by the gene is shown in SEQ ID NO.1.

[0009] Further, PeNRAMP5-1593 The nucleotide sequence of the CDS sequence of the gene is shown in SEQ ID NO.2.

[0010] Further, PeNRAMP5-1602 The amino acid sequence encoded by the gene is shown in SEQ ID NO.3.

[0011] Further, PeNRAMP5-1602The nucleotide sequence of the CDS sequence of the gene is shown in SEQ ID NO.4.

[0012] Furthermore, construct pUbi- PeNRAMP5-1593 and pUbi- PeNRAMP5-1602 The recombinant vector was transformed into rice mutants deficient in Cd and Mn absorption OsNRAMP5 middle.

[0013] Further, in OsNRAMP5 Mutant complementation of bamboo PeNRAMP5-1593 or PeNRAMP5-1602 The gene increased the absorption of Cd by rice roots and reduced the efficiency of Cd transport from roots to aboveground parts, effectively reducing Cd accumulation while restoring the absorption of Mn and its accumulation in aboveground parts.

[0014] The present invention is beneficial in that: exist OsNRAMP5 Mutant complementation of bamboo PeNRAMP5-1593 or PeNRAMP5-1602 The gene significantly increased the absorption of Cd by rice roots, but the efficiency of Cd transport from rice roots to aboveground parts was significantly reduced. Compared with wild-type rice, the accumulation of Cd in aboveground parts of transgenic rice was reduced by about 29 times. OsNRAMP5 Compared with the mutant, the Cd accumulation in the aboveground part of the transgenic rice was reduced by about 3 times. OsNRAMP5 Compared with the mutant, the transgenic rice restored Mn uptake and accumulation in the aboveground parts. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 for PeNRAMP5 Gene expression pattern.

[0016] Figure 2 for PeNRAMP5 Subcellular localization of the gene in bamboo protoplasts. The green fluorescent signal represents the GFP signal.

[0017] Figure 3 for PeNRAMP5 Subcellular localization of the gene in onion epidermal cells. The green fluorescent signal represents the GFP signal.

[0018] Figure 4 for PeNRAMP5 Genes for Cd transport activity in yeast.

[0019] Figure 5 for PeNRAMP5 Tissue localization of genes in transgenic rice. Red signals represent PeNRAMP5 Localization of proteins.

[0020] Figure 6 Rice mutants defective in Cd and Mn uptake OsNRAMP5 Moso bamboo PeNRAMP5 Effects of genes on rice growth, where * indicates p < 0.05 and ** indicates p < 0.01.

[0021] Figure 7 Rice mutants defective in Cd and Mn uptake OsNRAMP5 Bamboo PeNRAMP5 Effects of genes on Cd absorption.

[0022] Figure 8 Rice mutants defective in Cd and Mn uptake OsNRAMP5 Moso bamboo PeNRAMP5 Genetic influence on Mn absorption.

[0023] Figure 9 Rice mutants defective in Cd and Mn uptake OsNRAMP5 Moso bamboo PeNRAMP5 Genetic influence on other elements (Zn, Cu, Fe).

[0024] The results of the attached figures were analyzed by one-way ANOVA using SPSS statistical software. The Duncan method was used to test the significance of differences between groups, and the significance level was set at p < 0.05. The experimental results are expressed as mean ± standard deviation, and different lowercase superscripts represent statistically significant differences between groups. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0026] Experimental methods: 1. Nutrient solution formula: In this experiment, rice or bamboo seedlings were cultured in 1 / 2 Kimura nutrient solution. The contents of each element in the nutrient solution were as follows: MgSO4 (0.28 mM), (NH4)2SO4 (0.18 mM), Ca(NO3)2 (0.18 mM), KNO3 (0.09 mM), KH2PO4 (0.09 mM), Fe-EDTA (20 µM), H3BO3 (3 µM), MnCl2 (0.5 µM), CuSO4 (0.2 µM), ZnSO4 (0.4 µM) and (NH4)6Mo7O 24 (1 µM).

[0027] 2. Sequence information PeNRAMP5-1593 The amino acid sequence encoded by the gene is shown in SEQ ID NO.1. SEQ ID NO.1: MEIERETSTGSERGRSWRANLAQEDAKKLEDNDQLIKKPSWRRFLAHVGPGFMVSLAYLDPGNLETDLQAGANHRYELLWVILIGLIFALIIQSLAANLGVVTGRHLAEICKSEYPKSVRICLWLLAELAVIAADIPEVIGTAFAFNLLFHIPVWVGVLITGSSTLLLIGLQRYGVRKLEFVISMLVFVMAACFFAELGTVKPPAVEVMKGLFIPRLNGAGATGDAIALLGALVMPHNLFLHSALVLSRKTPASVRGIKDACRFFLYESSFALFVALLINIAVVSVSGTVCFSDNLSPEDTEKCSDLTLDSSSFLLKNVLGRSSAIVYGVALLASGQSSTITGTYSGQYIMQGFLDIKMRKWLRNLLTRSIAIAPSLVVSIIGGSSGAGRLIIIASMILSFELPFALIPLLKFSSSSSKMGPHKNSIYIIVFSWLLSLLIIGINIYFLSTSFVGWLIHNSLPKFANVLIGVAIFPLMFIYLLSVVYLTFRKDTVVTFVADSCQVDAEKAKEDDDETVPYREDLADIPLPQ。

[0028] PeNRAMP5-1593

[0029] PeNRAMP5-1602 The amino acid sequence encoded by the gene is shown in SEQ ID NO.3. SEQ ID NO.3: MEIERETSAGSERGRSWTANVAQEDAKKLEDSDQLIKEPAWKRFLAHVGPGFMVSLAYLDPGNLETDLQAGANHRYELLWVILIGLIFALIIQSLSANLGVVTGRHLAEICKSEYPKFVRICLWLLAELAVIAADIPEVIGTAFAFNLLFHIPVWVGVLITGSSTLLLLGLQKYGVRKLEFLISMLVFVMAACFFGELSIVKPPAVEVMKGLFIPRLNGAGATGDAIALLGALIMPHNLFLHSALVLSRKTPASVRGIKDACRFFLYESGFALFIALLINIAVVSVSGTVCFSDSLSPEDAEKCGDLTLDTSSFLLKNVLGRSSAIVYGVALLASGQSSTITGTYSGQYIMQGFLDIKMKKWLRNLMTRCIAIAPSLVVSIIGGSSGAGRLIIIASMILSFELPFALIPLLKFSSSSSKMGPHKNSIYIIVLSWLLGLLIIGINMYFLSTSFVGWLIHNSLPKFANVLIGVAIFPFMFIYLFAVVYLTFRKDTVVTFVADSCQLDAEKAKEAGEDDDEPVPYREDLADIPLPQ。

[0030] PeNRAMP5-1602

[0031] 3. PeNRAMP5-1593 and PeNRAMP5-1602 Gene expression pattern.

[0032] Real-time fluorescence quantitative PCR analysis of PeNRAMP5 Whether the expression of genes at the transcriptional level is regulated by Cd, the specific experimental steps are as follows: (1) Sample processing; The 40-day-old bamboo seedlings cultured in 1 / 2 Kimura nutrient solution were transferred to 1 / 2 nutrient solution containing 5µM CdSO4 or different concentrations of CdSO4 (0, 1, 3, 5 µM) for 3 days. Then, the roots and aboveground parts of the bamboo seedlings were placed in 1.5mL centrifuge tubes and stored in liquid nitrogen.

[0033] (2) Extraction of total RNA: The samples prepared above were extracted using the NGzol total RNA rapid extraction kit from Shanghai Huiling Biotechnology Co., Ltd.

[0034] (3) Synthesis of cDAN: The RNA samples were reverse transcribed using the TOROBlue® qRT Premix with gDNA Eraser 2.0 reverse transcription kit from Toyobo Biotechnology Co., Ltd., Japan, to synthesize total cDNA.

[0035] (4) Real-time fluorescence quantitative PCR; Using cDNA as template, SYBR GREEN enzyme from Toyobo Biotech Co., Ltd. was used for fluorescence quantitative PCR amplification. The UBQ gene of bamboo was used as internal reference for analysis and normalization. PeNRAMP5-1593 or PeNRAMP5-1602 The relative expression of genes at the transcriptional level was calculated using 2-ΔΔCt. The primer sequences used in the quantification process are shown in Table 1: Table 1: Quantification primer sequences

[0036] In this experiment, real-time fluorescence quantitative RCR analysis was performed PeNRAMP5-1593 or PeNRAMP5-1602 The expression pattern of the gene in different parts of bamboo and whether the expression of the gene is regulated by Cd treatment. PeNRAMP5-1602 The expression in roots and aerial parts was higher than PeNRAMP5-1593 , PeNRAMP5-1593 and PeNRMP5-1602 The expression levels of both genes in roots were higher than those in aerial parts, and the expression of these two genes in roots was downregulated by high-concentration Cd treatment, while their expression in aerial parts was not affected by Cd treatment ( Figure 1 ).

[0037] 4. PeNRAMP5 Subcellular localization of proteins; For research PeNRAMP5-1593 and PeNRAMP5-1602 The specific location of proteins in cells, construction PeNRAMP5-1593 -GFP, PeNRAMP5-1602 -GFP, GFP- PeNRAMP5-1593 and GFP- PeNRAMP5-1602 The expression vector was constructed and the recombinant plasmid was transiently transferred into bamboo protoplasts and onion epidermal cells for subcellular localization. The specific experimental process is as follows: (1) Gene cloning and vector construction; because PeNRAMP5-1593 and PeNRAMP5-1602 The CDS sequence bases of PeNRAMP5-1593 and PeNRAMP5-1602 CDS sequence and contains part of the 5'UTR and 3'UTR regions, which are named PeNRAMP5-1593 -UTR and PeNRAMP5-1602 -UTR. The product recovered from the gel was used as a template for secondary amplification PeNRAMP5-1593 and PeNRAMP5-1062 The full-length coding sequence (without the stop codon) was inserted between the CaMV35S promoter and the GFP-NOS terminator of the GFP vector to construct PeNRAMP5- 1593 -GFP and PeNRAMP5-1602 -GFP fusion expression vector. In addition, PCR amplified PeNRAMP5-1593 and PeNRAMP5-1062 The full-length coding sequence (including the stop codon) was inserted into the GFP fragment in the GFP vector (the GFP fragment has no stop codon) to construct the GFP- PeNRAMP5-1593 and GFP- PeNRAMP5-1602 Fusion expression vector. At the same time, the GFP empty expression vector was used as a control. The target gene amplification primer sequences are shown in Table 2: Table 2: Primer sequences:

[0038] (2) Isolation of bamboo protoplasts and transformation of recombinant plasmids Protoplast extraction and transformation were performed according to the method of Zhang et al. (2021). PeNRAMP5-1593 -GFP, PeNRAMP5-1602 -GFP, GFP- PeNRAMP5-1593 , GFP- PeNRAMP5-1602 and GFP empty vector were respectively transferred into bamboo protoplasts. PeNRAMP5-1593 -GFP,PeNRAMP5-1602 -GFP, GFP-PeNRAMP5 -1593,GFP- PeNRAMP5-1602 The transformation of GFP empty vector into onion cells was carried out by biolistic bombardment.

[0039] (3) Observation of GFP signal The transformed onion epidermal cells or protoplasts were cultured at 28°C in the dark for 20–24 h, and then the GFP fluorescence signal was detected using a laser confocal microscope (LSM880, Zeiss, Oberkochen, Germany). The excitation / emission wavelengths of GFP were 488 nm / 540 nm.

[0040] This study constructed PeNRAMP5-1593 -GFP, PeNRAMP5-1602 -GFP, GFP- PeNRAMP5-1593 and GFP- PeNRAMP5-1602 The fusion expression vector was used to successfully identify the PeNRAMP5 Subcellular localization of the protein: PeNRAMP5-1593 and PeNRAMP5-1602 The protein is localized on the plasma membrane ( Figure 2 , Figure 3 ).

[0041] 5. PeNRAMP5 Protein transport activity for Cd in yeast To verify PeNRAMP5-1593 and PeNRAMP-1602 We constructed PeNRAMP5-1593 -pYES2 and PeNRAMP5-1602 -pYES2 expression vector, and transform the recombinant vector into yeast Cd absorption defective mutant for functional verification. The specific experimental process is as follows: (1) Gene cloning and vector construction The cDNA of rice variety Zh11 and the UTR region amplified in experiment 4 were used to PeNRAMP-1593 and PeNRAMP- 1602 The PCR products were templates and amplified by PCR. OsNRAMP5 , PeNRAMP5-1593 and PeNRAMP5-1602 The full-length coding sequence of OsNRAMP5 -pYES2, PeNRAMP5-1593 -pYES2 and PeNRAMP5-1602 -pYES expression vector. After sequencing verification, the constructed plasmid and empty vector (pYES2) were respectively transferred into the Cd absorption defective yeast mutant strain Δycf Functional verification is performed in OsNRAMP5 The gene was used as a positive control (it has been confirmed to be involved in Cd absorption in rice), and the pYES2 empty vector was used as a negative control. The target gene amplification primer sequences are shown in Table 3: Table 3: Primer sequences:

[0042] (2) Culture medium and culture conditions The transformed yeast cells were cultured on SD-U solid medium containing 2% glucose, 0.67% yeast nitrogen base (YNB) without amino acids, and 2% uracil-free amino acid supplement. PeNRAMP5 and OsNRAMP5 To test the expression of genes, 2% glucose in the culture medium was replaced with 2% galactose. PeNRAMP5 For Cd transport activity, different concentrations of CdSO4 were added to SD-U medium containing 2% glucose or 2% galactose.

[0043] (3) Transgenic yeast Cd tolerance experiment The transformed cells were cultured in SD-U solid medium (containing 2% glucose or 2% galactose) with different Cd concentrations. OsNRAMP5 , PeNRAMP5-1593 , PeNRAMP5-1602 Yeast cells expressing pYES2 or pYES2 empty vector were used to evaluate their tolerance to Cd stress.

[0044] (4) Cd absorption measurement Yeast cells were incubated in SD-U (containing 2% glucose) liquid medium overnight with constant temperature shaking. After centrifugation, the cells were transferred to SD-U (containing 2% galactose) liquid medium for 2 hours to induce exogenous gene expression. Subsequently, the cells were transferred to SD-U (containing 2% galactose and different concentrations of CdSO4) liquid medium for 2 hours. After the culture, the cells were collected by centrifugation, washed three times with 4°C precooled deionized water, dried and digested by adding nitric acid, and the concentration of Cd in yeast cells was determined using inductively coupled plasma mass spectrometry (ICP-MS).

[0045] This study successfully constructed a yeast system NRAMP5 The expression vector of metal transporter was constructed and the Cd uptake-deficient yeast mutant was used to verify the PeNRAMP5-1593 and PeNRAMP5-1602 Protein transport activity for Cd in yeast ( Figure 4 ).

[0046] 6. PeNRAMP5-1602 and PeNRAMP5-1593 Vector construction For research PeNRAMP5-1593 and PeNRAMP-1602Effects of Cd and Mn absorption and accumulation in rice, construction of pUbi- PeNRAMP5-1593 and pUbi- PeNRAMP5-1602 The recombinant vector was then transformed into rice mutants deficient in Cd and Mn absorption OsNRAMP5 and successfully obtained transgenic PeNRAMP5-1593 and PeNRAMP-16025 The specific experimental process is as follows: (1) Gene cloning and vector construction The UTR region amplified in Experiment 4 PeNRAMP-1593 and PeNRAMP-1602 The PCR products were templates and amplified by PCR PeNRAMP5-1593 and PeNRAMP5-1602 The full-length coding sequence was cloned into the pMBb7Fm21GW-UBIL vector containing the maize Ubiquitin promoter to construct the vector pUbi- PeNRAMP5-1593 and pUbi- PeNRAMP5-1602 The target gene amplification primer sequences are shown in Table 4: Table 4: Primer sequences:

[0047] (2) Recombinant plasmid transformation of rice: Agrobacterium-mediated genetic transformation was used to introduce the constructed recombinant vector into rice mutants deficient in Cd and Mn absorption. OsNRAMP5 After transformation, the genomic DNA of the transgenic plants was amplified and detected by PCR technology, and the transgenic plants were successfully screened. PeNRAMP5-1593 and PeNRAMP5-1602 Two strains.

[0048] 7. Construction of tissue-specific expression vector of PeNRAMP5 For research PeNRAMP5-1593 and PeNRAMP5-1602 To enhance tissue-specific expression, we constructed PeNRAMP5-1593 or PeNRAMP5-1602 The expression vector of the promoter fused with GFP was transferred into rice for tissue localization. The specific experimental process is as follows: (1) Gene cloning and vector construction PCR amplification from genomic DNA of bamboo PeNRAMP5-1593 and PeNRAMP5-1602 The promoter region of the promoter was amplified and the promoter amplification primer sequences are shown in Table 5. The amplified promoter fragment was fused with the GFP fragment isolated from the pBluescript vector, and the fusion DNA was inserted into the pPZP2H-lac vector to construct the vector p PeNRAMP5-1593 -GFP and p PeNRAMP5-1602 -GFP.

[0049] Table 5: Primer sequences:

[0050] (2) Rice transformation and transgenic plant screening: The constructed vector was transformed into callus tissue of rice variety Nipponbare by Agrobacterium-mediated transformation to obtain T1 transgenic rice plants, and non-transgenic rice was used as negative control.

[0051] (3) Immunostaining and tissue sampling: Different locations of roots of transgenic and non-transgenic plants (including root tip, root base, root maturation zone, and large lateral roots) were sampled and cross-sectioned into 100-μm slices using a vibratome (VT1200S, Leica, Weztlar, Germany), followed by immunostaining of the samples using anti-GFP antibodies.

[0052] 8. Fluorescence signal observation The GFP protein fluorescence signal was detected using a laser confocal microscope (LSM880, Zeiss, Oberkochen, Germany).

[0053] This study constructed PeNRAMP5-1593 or PeNRAMP5-1602 The transgenic rice lines with promoter fused to GFP were successfully observed by immunostaining technique. PeNRAMP5-1602 Tissue-specific expression patterns in rice roots, but unfortunately, no PeNRAMP5-1593 The specific location of the rice root system. The experimental results show PeNRAMP5-1602 Expressed in all cells of rice roots ( Figure 5 ).

[0054] 9. Replanting bamboo PeNRAMP5-1593 or PeNRAMP5-1602 Effects of genes on the absorption of Cd, Mn and other elements in rice Wild-type rice (Zh11) was selected. OsNRAMP5 Mutants (Cd and Mn absorption deficient) and rice bamboo gene complementation lines PeNRAMP5-1593#1 , PeNRAMP5-1593#2 , PeNRAMP5-1602#1 , PeNRAMP5-1602#2 The wild-type rice of 35 days old was used for hydroponic experiment. OsNRAMP5The mutant and four independent transgenic lines were treated in 1 / 2 Kimura B nutrient solution containing 0.2 or 1 μM CdSO4 for 7 days. After the Cd treatment, the roots of the seedlings were washed three times with 0.5 mM CaCl2 precooled at 4°C, and the surface water was then dried. The roots of the aboveground parts were then harvested, dried, weighed and digested, and the contents of Cd, Mn, Cu, Zn and Fe in the rice roots and aboveground parts were determined using inductively coupled plasma mass spectrometry (ICP-MS).

[0055] This study compared the PeNRAMP5-1593 or PeNRAMP5-1602 of genetically modified rice, OsNRAMP5 The growth of mutants and wild-type rice under normal culture conditions and the effects of Cd stress on the accumulation of Cd, Mn, Cu, Fe and Zn in rice. The experimental results showed that the accumulation of biomass in the root and aboveground parts of transgenic rice was significantly higher than that of wild-type rice. OsNRAMP5 mutant, but slightly lower than the wild type ( Figure 6 ).

[0056] Previous studies have found that knocking out OsNRAMP5 Although the gene significantly reduced the absorption and accumulation of Cd in rice roots and aboveground parts, unfortunately, OsNRAMP5 The mutant also reduced its absorption of the beneficial element Mn. However, in this study, we found that OsNRAMP5 Phyllostachys pubescens PeNRAMP5-1593 or PeNRAMP5-1602 The gene significantly increased the absorption of Cd by rice roots, but surprisingly, the efficiency of Cd transport from rice roots to aboveground parts was significantly reduced. We also found that the accumulation of Cd in aboveground parts of transgenic rice decreased by about 29 times compared with wild-type rice. OsNRAMP5 Compared with the mutant, the Cd accumulation in the aboveground part of the transgenic rice was reduced by about 3 times ( Figure 7 ). At the same time, we found that and OsNRAMP5 Compared with the mutant, transgenic rice restored Mn uptake and accumulation in aboveground parts ( Figure 8 In addition, we further examined the effects of transgenic rice on the absorption of other beneficial metal elements Fe, Zn, and Cu, and found that OsNRAMP5 Compared with PeNRAMP5-1593 and PeNRAMP5-1602 The gene had no significant effect on the accumulation of Fe, Cu and Zn in rice roots and aboveground parts, but significantly increased the accumulation of Zn and Cu in rice roots compared with the wild type ( Figure 9 ).

[0057] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. Bamboo PeNRAMP5 Application of genes in reducing Cd accumulation and increasing Mn uptake in rice.

2. The bamboo according to claim 1 PeNRAMP5 The application of the gene in reducing Cd accumulation and increasing Mn absorption in rice is characterized by: bamboo PeNRAMP5 Genes include PeNRAMP5-1593 Genes and PeNRAMP5-1602 Gene.

3. The bamboo according to claim 2 PeNRAMP5 The application of the gene in reducing Cd accumulation and increasing Mn absorption in rice is characterized by: PeNRAMP5-1593 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

1.

4. The bamboo according to claim 2 PeNRAMP5 The application of the gene in reducing Cd accumulation and increasing Mn absorption in rice is characterized by: PeNRAMP5-1593 The nucleotide sequence of the CDS sequence of the gene is shown in SEQ ID NO.

2.

5. The bamboo according to claim 2 PeNRAMP5 The application of the gene in reducing Cd accumulation and increasing Mn absorption in rice is characterized by: PeNRAMP5-1602 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

3.

6. The bamboo according to claim 2 PeNRAMP5 The application of the gene in reducing Cd accumulation and increasing Mn absorption in rice is characterized by: PeNRAMP5-1602 The nucleotide sequence of the CDS sequence of the gene is shown in SEQ ID NO.

4.

7. The bamboo according to claim 2 PeNRAMP5 The application of the gene in reducing Cd accumulation and increasing Mn absorption in rice is characterized in that: Build pUbi- PeNRAMP5-1593 and pUbi- PeNRAMP5-1602 The recombinant vector was transformed into rice mutants deficient in Cd and Mn absorption OsNRAMP5 middle.

8. The bamboo according to claim 2 PeNRAMP5 The application of the gene in reducing Cd accumulation and increasing Mn absorption in rice is characterized in that: exist OsNRAMP5 Mutant complementation of bamboo PeNRAMP5-1593 or PeNRAMP5-1602 The gene increased the accumulation of Cd in rice roots, but reduced the accumulation of Cd in the aboveground parts. The efficiency of Cd transport from roots to aboveground parts was reduced, while the absorption of Mn and its accumulation in aboveground parts were restored.

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