Application of Phyllostachys edulis PeNRAMP5 gene in reducing Cd accumulation and increasing Mn uptake in rice

Abstract

The present invention discloses the application of the Phyllostachys edulis gene in reducing Cd accumulation and increasing Mn absorption in rice. The advantages are as follows. PeNRAMP5 In the OsNRAMP5 mutant, complementing the PeNRAMP5‑1593 or PeNRAMP5‑1602 gene of Phyllostachys edulis significantly increases the absorption of Cd by rice roots, but the transport efficiency of Cd absorbed by rice roots to the above-ground parts is significantly reduced, effectively reducing the accumulation of Cd in the above-ground parts. Compared with wild-type rice, the Cd accumulation in the above-ground parts of transgenic rice can be reduced by about 29 times. Compared with the OsNRAMP5 mutant, the Cd accumulation in the above-ground parts of transgenic rice can be reduced by about 3 times. At the same time, compared with the OsNRAMP5 mutant, transgenic rice restores the absorption of Mn and its accumulation in the above-ground parts.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to the application of PeNRAMP5 genes of Phyllostachys edulis in reducing Cd accumulation and increasing Mn absorption in rice. Background Art

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

[0003] Nramp5 The Nramp5 gene encodes a metal ion transporter, which 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 the fields of agriculture and environmental remediation. By regulating the

[0004] However, when using the Nramp5 gene to control Cd absorption, the absorption of Mn is also reduced at the same time. 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] To solve the problems and deficiencies existing in the prior art, the purpose of the present invention is to provide the application of PeNRAMP5 genes of Phyllostachys edulis in reducing Cd accumulation and increasing Mn absorption in rice. By overexpressing the PeNRAMP5 genes of Phyllostachys edulis, while maintaining the absorption of Mn in the above-ground part of rice, the absorption of Cd is inhibited.

[0006] The application of PeNRAMP5 genes of Phyllostachys edulis in regulating and reducing Cd accumulation and increasing Mn absorption in rice.

[0007] Furthermore, the PeNRAMP5 genes of Phyllostachys edulis include PeNRAMP5-1593 genes and PeNRAMP5-1602 genes.

[0008] Furthermore, PeNRAMP5-1593 the amino acid sequence encoded by the

[0009] Furthermore, PeNRAMP5-1593 the nucleotide sequence of the CDS sequence of the

[0010] Furthermore, PeNRAMP5-1602 the amino acid sequence encoded by the

[0011] Furthermore, 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 vectors, and transform the recombinant vectors into the rice Cd and Mn uptake-deficient mutant OsNRAMP5 .

[0013] Furthermore, in the OsNRAMP5 mutant, complementing the PeNRAMP5-1593 or PeNRAMP5-1602 gene of Phyllostachys edulis increased the Cd uptake of rice roots, and the translocation efficiency of the absorbed Cd from the roots to the above-ground parts decreased, effectively reducing Cd accumulation, while restoring the Mn uptake and above-ground accumulation.

[0014] The advantages of the present invention are as follows:

[0015] In the OsNRAMP5 mutant, complementing the PeNRAMP5-1593 or PeNRAMP5-1602 gene of Phyllostachys edulis significantly increased the Cd uptake of rice roots, but the translocation efficiency of the absorbed Cd from the roots to the above-ground parts decreased significantly. Compared with wild-type rice, the Cd accumulation in the above-ground parts of transgenic rice can be reduced by about 29 times. Compared with the OsNRAMP5 mutant, the Cd accumulation in the above-ground parts of transgenic rice can be reduced by about 3 times. At the same time, compared with the OsNRAMP5 mutant, transgenic rice restored the Mn uptake and above-ground accumulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the PeNRAMP5 gene expression pattern.

[0017] Figure 2 is the PeNRAMP5 gene subcellular localization in Phyllostachys edulis protoplasts, and the green fluorescence signal represents the GFP signal.

[0018] Figure 3 is the PeNRAMP5 gene subcellular localization in onion epidermal cells, and the green fluorescence signal represents the GFP signal.

[0019] Figure 4 is the PeNRAMP5 gene Cd transport activity in yeast.

[0020] Figure 5 is the PeNRAMP5 gene tissue localization in transgenic rice, and the red signal represents the PeNRAMP5 protein localization.

[0021] Figure 6 Rice mutants defective in Cd and Mn uptake OsNRAMP5 Bamboo PeNRAMP5 Effects of genes on rice growth, where * indicates p < 0.05 and ** indicates p < 0.01.

[0022] Figure 7 Rice mutants defective in Cd and Mn uptake OsNRAMP5 Bamboo PeNRAMP5 Effects of genes on Cd absorption.

[0023] Figure 8 Rice mutants defective in Cd and Mn uptake OsNRAMP5 Bamboo PeNRAMP5 Genetic influence on Mn absorption.

[0024] Figure 9 Rice mutants defective in Cd and Mn uptake OsNRAMP5 Bamboo PeNRAMP5 Genetic influence on other elements (Zn, Cu, Fe).

[0025] 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

[0026] 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.

[0027] Experimental methods:

[0028] 1. Nutrient solution formula:

[0029] 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).

[0030] 2. Sequence Information

[0031] PeNRAMP5-1593 The amino acid sequence encoded by the gene is shown in SEQ ID NO.1. SEQ ID NO.1: MEIERETSTGSERGRSWRANLAQEDAKKLEDNDQLIKKPSWRRFLAHVGPGFMVSLAYLDPGNLETDLQAGANHRYELLWVILIGLIFALIIQSLAANLGVVTGRHLAEICKSEYPKSVRICLWLLAELAVIAADIPEVIGTAFAFNLLFHIPVWVGVLITGSSTLLLIGLQRYGVRKLEFVISMLVFVMAACFFAELGTVKPPAVEVMKGLFIPRLNGAGATGDAIALLGALVMPHNLFLHSALVLSRKTPASVRGIKDACRFFLYESSFALFVALLINIAVVSVSGTVCFSDNLSPEDTEKCSDLTLDSSSFLLKNVLGRSSAIVYGVALLASGQSSTITGTYSGQYIMQGFLDIKMRKWLRNLLTRSIAIAPSLVVSIIGGSSGAGRLIIIASMILSFELPFALIPLLKFSSSSSKMGPHKNSIYIIVFSWLLSLLIIGINIYFLSTSFVGWLIHNSLPKFANVLIGVAIFPLMFIYLLSVVYLTFRKDTVVTFVADSCQVDAEKAKEDDDETVPYREDLADIPLPQ。

[0032] PeNRAMP5-1593

[0033] PeNRAMP5-1602 The amino acid sequence encoded by the gene is shown in SEQ ID NO.3. SEQ ID NO.3: MEIERETSAGSERGRSWTANVAQEDAKKLEDSDQLIKEPAWKRFLAHVGPGFMVSLAYLDPGNLETDLQAGANHRYELLWVILIGLIFALIIQSLSANLGVVTGRHLAEICKSEYPKFVRICLWLLAELAVIAADIPEVIGTAFAFNLLFHIPVWVGVLITGSSTLLLLGLQKYGVRKLEFLISMLVFVMAACFFGELSIVKPPAVEVMKGLFIPRLNGAGATGDAIALLGALIMPHNLFLHSALVLSRKTPASVRGIKDACRFFLYESGFALFIALLINIAVVSVSGTVCFSDSLSPEDAEKCGDLTLDTSSFLLKNVLGRSSAIVYGVALLASGQSSTITGTYSGQYIMQGFLDIKMKKWLRNLMTRCIAIAPSLVVSIIGGSSGAGRLIIIASMILSFELPFALIPLLKFSSSSSKMGPHKNSIYIIVLSWLLGLLIIGINMYFLSTSFVGWLIHNSLPKFANVLIGVAIFPFMFIYLFAVVYLTFRKDTVVTFVADSCQLDAEKAKEAGEDDDEPVPYREDLADIPLPQ。

[0034] PeNRAMP5-1602

[0035] 3. PeNRAMP5-1593 and PeNRAMP5-1602 the expression patterns of genes.

[0036] Analyze whether the expression of PeNRAMP5 genes at the transcriptional level in Phyllostachys edulis is regulated by Cd through real-time fluorescence quantitative PCR. The specific experimental steps are as follows:

[0037] (1)Sample treatment;

[0038] Transfer 40-day-old Phyllostachys edulis seedlings cultured in 1 / 2 Kimura nutrient solution for days to 1 / 2 nutrient solution containing 5 µM CdSO4 or different concentrations of CdSO4 (0, 1, 3, 5 µM) and treat for 3 days. Then, separately place the roots and above-ground parts of the Phyllostachys edulis seedlings in 1.5 mL centrifuge tubes and store them in liquid nitrogen.

[0039] (2)Total RNA extraction:

[0040] Extract the above-prepared samples using the N Gzol total RNA rapid extraction kit from Shanghai Huiling Biotechnology Co., Ltd.

[0041] (3)cDNA synthesis:

[0042] Use the reverse transcription kit TOROBlue® qRT Premix with gDNA Eraser 2.0 from Toyobo Biotech Co., Ltd., Japan to reverse transcribe the RNA samples to synthesize total cDNA.

[0043] (4)Real-time fluorescence quantitative PCR;

[0044] Using cDNA as a template, perform fluorescence quantitative PCR amplification with SYBR GREEN enzyme from Toyobo Biotech Co., Ltd., Japan. Use the UBQ gene of Phyllostachys edulis as an internal reference for analysis and normalization. PeNRAMP5-1593 or PeNRAMP5-1602 The relative expression of genes at the transcriptional level is calculated using 2-ΔΔCt. The primer sequences used in the quantification process are shown in Table 1:

[0045] Table 1: Quantitative primer sequences

[0046]

[0047] This experiment analyzed PeNRAMP5-1593 or PeNRAMP5-1602 the expression patterns of genes in different parts of Phyllostachys edulis and whether the expression of this gene is regulated by Cd treatment. We found that PeNRAMP5-1602 the expression in both roots and above-ground parts is higher than PeNRAMP5-1593 ,PeNRAMP5-1593 and PeNRMP5-1602 both showed higher expression levels in roots than in shoots, and the expression of these two genes in roots was downregulated by high-concentration Cd treatment, while their expression in shoots was not affected by Cd treatment ( Figure 1 ).

[0048] 4. PeNRAMP5 Subcellular localization of proteins;

[0049] To study the specific localization of PeNRAMP5-1593 and PeNRAMP5-1602 proteins in cells, PeNRAMP5-1593 -GFP, PeNRAMP5-1602 -GFP, GFP- PeNRAMP5-1593 and GFP- PeNRAMP5-1602 expression vectors were constructed and the recombinant plasmids were transiently transfected into moso bamboo protoplasts and onion epidermal cells for subcellular localization. The specific experimental procedures are as follows:

[0050] (1) Gene cloning and vector construction;

[0051] Since PeNRAMP5-1593 and PeNRAMP5-1602 have highly similar CDS sequence bases, we first amplified the CDS sequences of PeNRAMP5-1593 and PeNRAMP5-1602 by PCR, which contained partial 5’UTR and 3’UTR regions. These regions were named PeNRAMP5-1593 -UTR and PeNRAMP5-1602 -UTR respectively. Using the products recovered by gel extraction as templates, the full-length coding sequences of PeNRAMP5-1593 and PeNRAMP5-1062 (excluding the stop codon) were amplified and 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 vectors. In addition, the full-length coding sequences of PeNRAMP5-1593 and PeNRAMP5-1062 (including the stop codon) were amplified by PCR and inserted after the GFP fragment in the GFP vector (the GFP fragment has no stop codon) to construct GFP- PeNRAMP5-1593 and GFP- PeNRAMP5-1602 fusion expression vectors. At the same time, the GFP empty expression vector was used as a control. The primer sequences for amplifying the target genes are shown in Table 2:

[0052] Table 2: Primer sequences:

[0053]

[0054] (2) Isolation of moso bamboo protoplasts and transformation of recombinant plasmids

[0055] Protoplast extraction and transformation were carried out 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 moso bamboo protoplasts. For PeNRAMP5-1593 -GFP, PeNRAMP5-1602 -GFP, GFP-PeNRAMP5 -1593, GFP- PeNRAMP5-1602 and GFP empty vector were transformed into onion cells by particle bombardment.

[0056] (3) Observation of GFP signal

[0057] The transformed onion epidermal cells or protoplasts were cultured for 20 - 24 hours at 28 °C in the dark, and then the GFP fluorescence signal was detected using a laser confocal microscope (LSM880, Zeiss, Oberkochen, Germany). The excitation / emission wavelength of GFP was 488 nm / 540 nm.

[0058] In this study, by constructing PeNRAMP5-1593 -GFP, PeNRAMP5-1602 -GFP, GFP- PeNRAMP5-1593 and GFP- PeNRAMP5-1602 fusion expression vectors, and using the moso bamboo protoplast and onion epidermal cell systems, combined with confocal microscopy technology, the subcellular localization of PeNRAMP5 protein was successfully determined: PeNRAMP5-1593 and PeNRAMP5-1602 protein was localized on the plasma membrane ( Figure 2 , Figure 3 ).

[0059] 5. PeNRAMP5 Transport activity of

[0060] To verify whether PeNRAMP5-1593 and PeNRAMP-1602 genes have transport activity for Cd. We constructed PeNRAMP5-1593 -pYES2 and PeNRAMP5-1602 -pYES2 expression vectors, and transformed the recombinant vectors into yeast Cd uptake-deficient mutants for functional verification. The specific experimental procedures are as follows:

[0061] (1) Gene cloning and vector construction

[0062] Using the cDNA of rice variety Zh11 and the UTR regions amplified in Experiment 4 PeNRAMP-1593 and PeNRAMP-1602 The PCR products were used as templates respectively, and OsNRAMP5 , PeNRAMP5-1593 and PeNRAMP5-1602 full-length coding sequences were amplified by PCR, cloned into the pYES2 vector, and OsNRAMP5 -pYES2, PeNRAMP5-1593 -pYES2 and PeNRAMP5-1602 -pYES expression vectors were constructed. After sequencing verification, the constructed plasmids and the empty vector (pYES2) were separately transformed into the Cd absorption-deficient yeast mutant strain Δycf for functional verification. OsNRAMP5 The

[0063] gene was used as a positive control (it has been confirmed that it is involved in Cd absorption in rice), and the pYES2 empty vector was used as a negative control. The primer sequences for amplifying the target gene are shown in Table 3:

[0064]

[0065] (2) Media and culture conditions

[0066] The transformed yeast cells were cultured on SD-U solid medium containing 2% glucose, 0.67% yeast nitrogen base without amino acids (YNB), and 2% uracil-free amino acid supplement. To induce the expression of PeNRAMP5 and OsNRAMP5 genes, 2% glucose in the medium was replaced with 2% galactose. To test the PeNRAMP5 transport activity of Cd, different concentrations of CdSO4 were added to the SD-U medium containing 2% glucose or 2% galactose respectively.

[0067] (3) Cd tolerance experiment of transgenic yeast

[0068] Yeast cells transformed with OsNRAMP5 , PeNRAMP5-1593 , PeNRAMP5-1602 or the pYES2 empty vector were cultured on SD-U solid medium with different concentrations of Cd (containing 2% glucose or 2% galactose) to evaluate their tolerance to Cd stress.

[0069] (4) Cd absorption determination

[0070] Yeast cells were incubated overnight at a constant temperature with shaking in a liquid medium of SD-U (containing 2% glucose). After centrifuging to collect the cells, they were transferred to a liquid medium of SD-U (containing 2% galactose) and cultured for 2 hours to induce the expression of foreign genes. Subsequently, the cells were transferred to a liquid medium of SD-U (containing 2% galactose and different concentrations of CdSO4) and cultured for 2 hours. After the culture, the cells were centrifuged to collect, washed 3 times with deionized water pre-cooled at 4 °C, dried, digested by adding nitric acid, and the concentration of Cd in yeast cells was determined using inductively coupled plasma mass spectrometry (ICP-MS).

[0071] In this study, an expression vector of NRAMP5 metal transporter was successfully constructed through a yeast system, and the PeNRAMP5-1593 and PeNRAMP5-1602 proteins in moso bamboo were verified for their Cd transport activity in yeast ( Figure 4 ).

[0072] 6. PeNRAMP5-1602 and PeNRAMP5-1593 Vector construction

[0073] To study the effects of PeNRAMP5-1593 and PeNRAMP-1602 on the absorption and accumulation of Cd and Mn in rice, pUbi- PeNRAMP5-1593 and pUbi- PeNRAMP5-1602 vectors were constructed and the recombinant vectors were transformed into the rice Cd and Mn absorption-deficient mutant OsNRAMP5 , and genetically modified PeNRAMP5-1593 and PeNRAMP-16025 genetic materials were successfully obtained. The specific experimental procedures are as follows:

[0074] (1) Gene cloning and vector construction

[0075] Using the PCR products amplified in Experiment 4 containing the UTR regions PeNRAMP-1593 and PeNRAMP-1602 as templates respectively, the full-length coding sequences of PeNRAMP5-1593 and PeNRAMP5-1602 were amplified by PCR and cloned into the pMBb7Fm21GW-UBIL vector containing the maize Ubiquitin promoter to construct the vectors pUbi- PeNRAMP5-1593 and pUbi- PeNRAMP5-1602 . The primer sequences for amplifying the target genes are shown in Table 4:

[0076] Table 4: Primer sequences:

[0077]

[0078] (2) Transformation of recombinant plasmids into rice:

[0079] Using the Agrobacterium-mediated genetic transformation method, the constructed recombinant vector was introduced into a rice mutant defective in Cd and Mn uptake OsNRAMP5 . After transformation, the genomic DNA of the transgenic plants was amplified and detected by PCR technology, and two strains were successfully screened PeNRAMP5-1593 and PeNRAMP5-1602 .

[0080] 7. Construction of the tissue-specific expression vector of PeNRAMP5

[0081] To study the tissue-specific expression of PeNRAMP5-1593 and PeNRAMP5-1602 , we constructed expression vectors carrying the fusion of the promoter of PeNRAMP5-1593 or PeNRAMP5-1602 with GFP, and transferred them into rice for tissue localization. The specific experimental procedures are as follows:

[0082] (1) Gene cloning and vector construction

[0083] The promoter regions of PeNRAMP5-1593 and PeNRAMP5-1602 were amplified by PCR from the moso bamboo genomic DNA. The primer sequences for promoter amplification are shown in Table 5. The amplified promoter fragments were fused with the GFP fragment isolated from the pBluescript vector, and the fused DNA was inserted into the pPZP2H-lac vector to construct the vectors p PeNRAMP5-1593 -GFP and p PeNRAMP5-1602 -GFP.

[0084] Table 5: Primer sequences:

[0085]

[0086] (2) Rice transformation and screening of transgenic plants:

[0087] By the Agrobacterium-mediated transformation method, the constructed vectors were transferred into the calli of the rice variety Nipponbare. T1 generation transgenic rice plants were obtained, and non-transgenic rice was used as a negative control

[0088] (3) Immunostaining and tissue sampling:

[0089] Different positions of the roots of transgenic and non-transgenic plants (including root tips, root bases, root maturation zones, and large lateral roots) were taken, and they were cross-sectioned into 100-μm thin slices using a vibratome (VT1200S, Leica, Weztlar, Germany), and then the samples were immunostained with anti-GFP antibody

[0090] 8. Observation of fluorescence signals

[0091] The fluorescence signal of GFP protein was detected using a laser confocal microscope (LSM880, Zeiss, Oberkochen, Germany).

[0092] In this study, by constructing PeNRAMP5-1593 or PeNRAMP5-1602 transgenic rice lines with the promoter fused to GFP and combining immunostaining techniques, the tissue-specific expression pattern in rice roots was successfully observed. Unfortunately, however, the specific localization in rice roots was not detected. PeNRAMP5-1602 The experimental results showed that PeNRAMP5-1593 it was expressed in all cells in rice roots ( PeNRAMP5-1602 ). Figure 5 )

[0093] 9. Effect of complementing moso bamboo PeNRAMP5-1593 or PeNRAMP5-1602 gene on the uptake of Cd, Mn and other elements by rice

[0094] Wild-type rice (Zh11), OsNRAMP5 mutants (deficient in Cd and Mn uptake) and rice moso bamboo gene complementation lines PeNRAMP5-1593#1 , PeNRAMP5-1593#2 , PeNRAMP5-1602#1 , PeNRAMP5-1602#2 were used for hydroponic experiments. 35-day-old wild-type rice, OsNRAMP5 mutants and 4 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 3 times with 0.5 mM CaCl2 pre-cooled at 4 °C, and then the surface moisture was blotted dry. Subsequently, the above-ground parts and roots were harvested separately, dried, weighed, digested, and the contents of Cd, Mn, Cu, Zn and Fe in the rice roots and above-ground parts were determined using inductively coupled plasma mass spectrometry (ICP-MS).

[0095] In this study, by comparing the growth of transgenic rice carrying moso bamboo PeNRAMP5-1593 or PeNRAMP5-1602 , OsNRAMP5 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 elements in rice. The experimental results showed that the accumulation of biomass in the roots and above-ground parts of transgenic rice was significantly higher than that of OsNRAMP5 mutants, but slightly lower than that of wild-type ( Figure 6 ).

[0096] Previous studies found that knocking out the OsNRAMP5 gene in rice significantly reduced the uptake and accumulation of Cd in the roots and above-ground parts of rice. Unfortunately, however, OsNRAMP5The absorption of the beneficial element Mn by the mutant also decreased. However, in this study, we found that OsNRAMP5 Complementation of Phyllostachys edulis in the mutant PeNRAMP5-1593 or PeNRAMP5-1602 genes significantly increased the Cd uptake by rice roots. However, surprisingly, the translocation efficiency of Cd absorbed by rice roots to the above-ground parts was significantly reduced. And we detected that, compared with wild-type rice, the Cd accumulation in the above-ground parts of transgenic rice could be reduced by about 29 times. However, compared with OsNRAMP5 mutants, the Cd accumulation in the above-ground parts of transgenic rice could be reduced by about 3 times ( Figure 7 ). At the same time, we found that, compared with OsNRAMP5 mutants, transgenic rice restored the absorption and above-ground accumulation of Mn ( Figure 8 ). In addition, we further detected the effects of transgenic rice on the absorption of other beneficial metal elements Fe, Zn, and Cu. We found that, compared with the mutant OsNRAMP5 , complementation of Phyllostachys edulis PeNRAMP5-1593 and PeNRAMP5-1602 genes had no particularly significant effect on the accumulation of Fe, Cu, and Zn in the roots and above-ground parts of rice. However, compared with the wild type, it significantly increased the accumulation of Zn and Cu in the roots of rice ( Figure 9 ).

[0097] The above-disclosed are only the preferred embodiments of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope covered by the present invention.

Claims

1. Bamboo PeNRAMP5 Application of genes in reducing Cd accumulation in rice and increasing Mn absorption, bamboo PeNRAMP5 Genes include PeNRAMP5-1593 Genes and PeNRAMP5-1602 Gene, PeNRAMP5-1593 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

1. PeNRAMP5-1602 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

3. PeNRAMP5-1593 and pUbi- PeNRAMP5-1602 The recombinant vector was transformed into rice mutants deficient in Cd and Mn absorption OsNRAMP5 middle.

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: PeNRAMP5-1593 The nucleotide sequence of the CDS sequence of the gene is shown in SEQ ID NO.2, PeNRAMP5- 1602 The nucleotide sequence of the CDS sequence of the gene is shown in SEQ ID NO.

4.

3. The bamboo according to claim 1 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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