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Application of rice aquaporin-encoding gene osnip3;3

A technology of aquaporin and coding gene, which is applied in the field of rice aquaporin coding gene OsNIP3, and can solve the problem that aquaporin gene is rarely reported

Active Publication Date: 2021-12-10
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aquaporin genes involved in As(III) transport in rice are rarely reported except Lsi1

Method used

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  • Application of rice aquaporin-encoding gene osnip3;3
  • Application of rice aquaporin-encoding gene osnip3;3
  • Application of rice aquaporin-encoding gene osnip3;3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 The transport activity test of the aquaporin-encoding gene OsNIP3;3 in Xenopus laevis oocytes.

[0035] PCR primers were designed according to the cDNA reading frame sequences of NIP3;3, NIP2;1(Lsi1) and NIP3;1 genes, and restriction endonuclease sites were introduced into the upstream and downstream primers respectively. The primer sequences are shown in the table below.

[0036]

[0037]

[0038] The amplified PCR product was detected by agarose gel electrophoresis, and after separation, the gel was cut and recovered. After the product was recovered, the corresponding restriction endonuclease was used to digest the product. At the same time, the frog egg expression vector pT7TS was digested. The PCR fragments were ligated with T4 ligase, transformed into E. coli, and positive clones were extracted for restriction digestion and DNA sequencing identification. After the correct plasmid was linearized, the single-stranded cRNA of the NIP gene was synthesiz...

Embodiment 2

[0040] Example 2 Construction of OsNIP3;3 overexpression vector material

[0041]1) Extraction of total RNA: After sterilizing the rice seeds with 30% sodium hypochlorite solution for 30 minutes, wash them with sterile water for 4-5 times, and then cultivate them with 1 / 2 MS medium for about 2 weeks, and select seedlings with the same size and transfer them to culture In a bucket, the leaves were cultured with 1 / 2 Kimura nutrient solution for a week, and then the leaves were taken and stored in liquid nitrogen, and RNA was extracted using a plant total RNA extraction kit (Beijing Biotech Company).

[0042] 2) The total cDNA was synthesized using a reverse transcription kit (Nanjing Novozymes).

[0043] 3) Obtaining the full length of OsNIP3;3 gene and constructing the overexpression vector:

[0044] The overexpression primers were designed according to the full-length cDNA sequence of OsNIP3;3. The primer sequences are as follows:

[0045] OsNIP3;3-F:AGAGGATCCCCGGGTACCATGGAA...

Embodiment 3

[0049] Example 3 Aquaporin-encoding gene OsNIP3; 3 Acquisition of overexpressed transgenic material

[0050] The Agrobacterium transfected with the pTCK303-OsNIP3;3 plasmid obtained in Example 2 was used to infect Nipponbare rice callus, and after co-cultivation for 2 days, the T0 generation transgenic plants were obtained through selective culture, differentiation, rooting and hardening.

[0051] abbreviations for reagents and solutions

[0052] The English abbreviations used in the culture medium of the present invention are expressed as follows: 6-BA (6-benzyl adenine); Car (carbenicillin); NAA (naphthalene acetic acid); IAA (indole acetic acid); 2,4 -D (2,4-dichlorophenoxyacetic acid); AS (acetosyringone); CH (hydrolyzed casein); L-pro (L-proline); L-Glu (L-glutamine); MES (2-morpholine ethanesulfonic acid); N6 (N6 major element solution); B5 (B5 trace element solution); AA (AA major element); Agar (agar).

[0053] Solution and Media Formulations

[0054] Ⅰ Hormone prep...

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Abstract

The invention discloses the application of the rice water channel protein coding gene OsNIP3;3. An aquaporin coding gene OsNIP3;3, the accession number in Genbank is AB710141. The gene can be used in reducing the concentration of trivalent As in rice stems, reducing the loading of trivalent As to xylem, and significantly reducing the accumulation of arsenic in rice shoots and grains. The present invention discovers the biological function of the aquaporin coding gene OsNIP3;3 in rice through a large number of experiments, and after the gene is overexpressed, the As content in the shoot of rice is reduced. Overexpression of this gene significantly reduced the accumulation of As in rice leaves, seed husks and grains.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to the application of the rice aquaporin coding gene OsNIP3;3. Background technique [0002] At present, the problem of heavy metal pollution in the environment has become increasingly prominent, and the arsenic (As) pollution of cultivated soil is also becoming more and more serious. Rice is an important food crop, and rice is the main food crop for about half of the world's population. Due to the enrichment of arsenic in rice, rice accounts for 60% of the dietary arsenic intake of the Chinese population (Li et al. 2009). Therefore, it is very important to analyze the absorption and detoxification mechanism of As in rice and to develop methods to reduce As accumulation in rice grains through genetic engineering technology. In soil, arsenic exists in the form of pentavalent arsenic As(V) and trivalent arsenic As(III) under aerobic and flooded conditions, respectively (Zh...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N15/66
CPCC07K14/415C12N15/8271
Inventor 赵方杰孙晟凯唐仲黄新元马建锋
Owner NANJING AGRICULTURAL UNIVERSITY
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