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Corn ZmHSMT3 gene and application thereof in reducing cadmium accumulation of rice grains

A corn and gene technology, applied in the field of genetic engineering, can solve the problems of reduced tolerance and increased absorption of cadmium by barley, and achieve the effects of enhanced tolerance and reduced cadmium content

Active Publication Date: 2021-12-24
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Another example is the patent document CN109880829A which discloses a gene HvPAA1 cloned from barley with cadmium-tolerant characteristics, which can regulate the absorption of cadmium by barley. After silencing, the absorption of cadmium by barley is increased, and the tolerance is significantly reduced.

Method used

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  • Corn ZmHSMT3 gene and application thereof in reducing cadmium accumulation of rice grains
  • Corn ZmHSMT3 gene and application thereof in reducing cadmium accumulation of rice grains
  • Corn ZmHSMT3 gene and application thereof in reducing cadmium accumulation of rice grains

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Cloning and analysis of ZmHSMT3 gene CDS region and promoter region

[0030] 1. Cloning of CDS region of ZmHSMT3 gene

[0031] Taking the genotypes with high and low accumulation of cadmium in maize grains screened in the early stage as materials, root transcriptome sequencing was carried out after cadmium stress. From the results, a HSMT family gene highly expressed in the genotype L63 with low accumulation of cadmium in maize grains was identified and cloned. The full-length CDS sequence of the gene was obtained, the corresponding nucleotide sequence is shown in SEQ ID NO.1, and it was named ZmHSMT3.

[0032] The total RNA of L63 leaves was extracted with a total RNA extraction kit, the genomic DNA contamination was removed with DNaseI, and then reverse-transcribed into single-stranded cDNA using the PrimeScripffM II 1st Strand cDNA Synthesis Kit. Primers were designed according to the sequence obtained by Blast and amplified with KOD ONE high-fidelity enzy...

Embodiment 2

[0039] Embodiment 2 transgenic rice

[0040] 1. Construction of recombinant plasmids

[0041] According to the previously cloned sequence, design specific primers, the primer sequence is as follows (the underline is the restriction site):

[0042] ZmHSMT3-PCR-F:

[0043] 5'-TCCC CCCGGG ATGGTGGGGACGGCGGAAGG-3';

[0044] ZmHSMT3-PCR-R:

[0045] 5'-CTAG TCTAGA TGCTACAGGTAACTGATGCTTGTGGCAG-3'

[0046] Use LA Taq DNA Polymerase to amplify the full-length CDS, after the rubber is tapped and recovered, connect the pMD18-T vector, transform the strain, pick a single clone for sequencing, and use XmaI and XbaI endonuclease double enzymes after the M13 forward and reverse primers are sequenced correctly cut and ligated into the 35S empty vector digested with the same endonuclease. The ligation product was heat-shocked to transform E. coli DH5a competent cells, and after shaking the bacteria, the monoclonal shaking bacteria were picked on a drawing board, and the plasmid was dig...

Embodiment 3

[0058] Effect of embodiment 3 cadmium stress on rice seedling growth and cadmium content

[0059] Seeds of wild type (WT), overexpression lines L11 and L19 were soaked in 2% H 2 o 2 Disinfect in medium for 20 minutes, rinse thoroughly with distilled water, soak seeds in greenhouse for 2 days, and accelerate germination at 30°C for 1 day. The whole and healthy germinated seeds were selected and planted in the sand bed, and pre-cultivated in an artificial climate box at a temperature of 30°C / 26°C and a relative humidity of 85%. At the second leaf stage, select healthy rice seedlings with consistent growth and transplant them into the nutrient solution. After 7 days of pre-culture, cadmium treatment was performed. Two treatments were set up in the experiment: Control (basic nutrient solution, BNS) and Cd (BNS+10μM Cd).

[0060] The result is as Figure 4 It was shown that after cadmium treatment for 5 days, the cadmium tolerance of L11 and L19 strains was significantly stron...

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Abstract

The invention discloses a corn ZmHSMT3 gene and application thereof in reducing cadmium accumulation of rice grains, and belongs to the technical field of genetic engineering. The nucleotide sequence of a CDS region of the corn ZmHSMT3 gene is shown as SEQ ID NO.1. The corn ZmHSMT3 gene is subjected to cloning analysis, and is subjected to heterologous overexpression in rice in combination with a transgenic technology to perform functional verification on the gene, so that the tolerance of a ZmHSMT3 overexpression plant to cadmium is remarkably enhanced, and the cadmium content in the grains is remarkably reduced. The invention provides a feasible technical scheme for low-accumulation breeding and production of rice grains.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the corn ZmHSMT3 gene and its application in regulating rice cadmium tolerance and reducing rice grain cadmium accumulation. Background technique [0002] Cadmium (Cadmium, Cd) is one of the most common heavy metals with strong biological toxicity and polluted farmland. Cadmium has strong mobility and is easily absorbed by plants and transferred to the aboveground. The decline in yield and quality is more likely to seriously threaten human health through the food chain. Therefore, reducing the accumulation of heavy metals in crop edible organs has become an important scientific task for the sustainable and efficient use of land resources and the quality and safety of agricultural products. [0003] For polluted soil, although various methods (techniques) including bioremediation and reducing soil heavy metal content have been proposed, they have not been effectively ...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/84A01H5/10A01H6/46
CPCC12N9/1007C12N15/8271C12N15/8205Y02A40/146
Inventor 曹方彬林凯娜
Owner ZHEJIANG UNIV
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