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atZA-containing ti vector construction and transgenic rice preparation method

A technology of transgenic rice and atrazine, applied in the field of plant genetic engineering, can solve the problems of transgenic rice without herbicide-resistant atrazine, etc., and achieve the effect of low price

Inactive Publication Date: 2009-06-10
NANKAI UNIV +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are no literature and patent reports on transforming rice with atrazine chlorohydrolase gene and obtaining herbicide-resistant atrazine transgenic rice

Method used

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  • atZA-containing ti vector construction and transgenic rice preparation method

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Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 The construction of a Ti carrier containing atrazine chlorohydrolase gene comprises the following steps:

[0034] ①The plasmid pGEM-T-atzA containing the atrazine chlorohydrolase gene was digested with EcoR I and HindIII, and the atrazine chlorohydrolase gene was isolated;

[0035] ② digest the plasmid pBluscript SK (+ / -) with EcoR I and HindIII, separate the vector fragment, and connect it with the atrazine chlorohydrolase gene to form the intermediate vector pBSSK (+ / -)-atzA;

[0036] ③ Digest the intermediate carrier pBSSK(+ / -)-atzA with Sal I and Sac I, separate the small fragment containing atrazine chlorohydrolase gene, digest the plasmid pCAMBIA1301 with Sal I and Sac I, separate the intermediate carrier fragment, The small fragments of the terazine chlorohydrolase gene were connected to form the Ti vector p1301-atzA, and the construction results are shown in Figure 1;

[0037] ④ Transform the Ti vector p1301-atzA into Escherichia coli DH5α, extract...

Embodiment 2

[0038] Embodiment 2 The preparation of the transgenic rice resistant to the herbicide atrazine comprises the following steps:

[0039] ① Rice transformation mediated by Agrobacterium tumefaciens: Shelled mature rice seeds were sterilized on the surface, placed on mature embryo callus induction medium, cultured in the dark at 26°C for 15 days, and peeled off the scutellaria of mature embryos. The callus was transferred to mature embryo subculture medium for subculture for 4 days;

[0040] Streak the EHA105 strain containing the atrazine chlorohydrolase gene on a plate containing 50 mg / mL kanamycin, culture it in the dark at 28°C for 3 days, suspend it in a liquid medium, add acetosyringone, and make Its final concentration is 100 μ mol / L, and this is the Agrobacterium suspension used for transforming rice;

[0041] Select the rice callus that has been subcultured for a certain period of time in a better state and put it into a sterile container, add 70mL of Agrobacterium suspe...

Embodiment 3

[0047] Embodiment 3 The preparation of the transgenic rice resistant to the herbicide atrazine comprises the following steps:

[0048] ① Rice transformation mediated by Agrobacterium tumefaciens: Shelled mature rice seeds were sterilized on the surface, placed on the mature embryo callus induction medium, cultured at 20°C in the dark for 20 days, and peeled off the scutellum of the mature embryo. The callus was transferred to mature embryo subculture medium for subculture for 5 days;

[0049] Streak the EHA105 strain containing the atrazine chlorohydrolase gene on a plate containing 50 mg / mL kanamycin, culture it in the dark at 25°C for 5 days, suspend it in a liquid medium, add acetosyringone, and make Its final concentration is 100 μ mol / L, and this is the Agrobacterium suspension used for transforming rice;

[0050] Select the rice callus that has been subcultured for a certain period of time in a better state and put it into a sterile container, add 100mL of Agrobacterium...

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Abstract

The invention discloses an atZA-containing Ti vector construction method, preparation of herbicide atrazine resistant transgenic rice and use in hybrid seeding, wherein the vector construction is realized through interpolating plasmid pCAMBIA1301 into the atrazine chlorine hydrolytic enzyme genes, obtaining Ti vector p1301-atzA for transforming rice, transforming the Ti vector into agrolbacterium EHA105, transforming the rice with the agrolbacterium.

Description

technical field [0001] The invention belongs to plant genetic engineering, in particular to the construction of a carrier and the preparation of transgenic plants. Background technique [0002] Rice is one of the most important cereal crops, and about 40% of the world's population uses rice as a staple food source. With the increase of population and the improvement of people's requirements for food nutrition, increasing the yield of rice and improving the quality of rice has become an important challenge we are facing. In the process of solving this problem, modern biotechnology, especially transgenic technology, plays a very important role. Obtaining herbicide-resistant transgenic rice through transgenic technology is a rapidly developing plant biotechnology in recent years [Giri C C, Laxmi G V. Production of transgenic rice with agronomically useful genes: anassessment. Biotechnology Advances, 2000, 18: 653-683; Duan Faping, Liang Chengye, Li Yuanqing: Research progress...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84C12N15/55C12N15/70C12N1/21C12N5/10A01H5/00A01H1/00C12Q1/68
Inventor 蔡宝立王松文孙宗修施利利刘霞韩宇宁
Owner NANKAI UNIV
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