Vector with double expression cassettes and high glyphosate resistance and application thereof to rice

A glyphosate-resistant and plant expression vector technology is applied in the application field of cultivating glyphosate-resistant rice, and can solve the problems of affecting yield, affecting the normal differentiation of young ears of rice, reducing rice yield, and the like, and achieving the effect of ensuring yield

Inactive Publication Date: 2016-08-17
BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Rice panicle differentiation is closely related to rice yield, and the young panicle differentiation stage is very sensitive to the environment. Unfavorable environmental conditions can easily affect the normal differentiation of rice young panicle, thereby affecting the yield.
Before and after the rice panicle differentiation stage, if glyphosate is not applied properly, it will cause rice yield reduction

Method used

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  • Vector with double expression cassettes and high glyphosate resistance and application thereof to rice
  • Vector with double expression cassettes and high glyphosate resistance and application thereof to rice
  • Vector with double expression cassettes and high glyphosate resistance and application thereof to rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Double expression cassette glyphosate-resistant plant expression vector

[0020] 1. Rfl promoter cloning: Rice genomic DNA was extracted by CTAB method. Search rice Rfl gene sequence from NCBI database, intercept 2000 bp upstream of the start codon of Rfl gene as promoter region, design primers: F: 5'-TACAAAAATTTCGACTAAACTCG-3', R: 5'-TTTATCGTCATCATGGCCACCGC-3' clone Promoter, obtain a fragment with a length of 2001bp. The fragment was recovered and purified, and polyA tailed after sequencing.

[0021] 2. Construction of High Efficiency Specific Plant Expression Vector with Double Expression Cassettes

[0022] (1) Excise the Ubi promoter in PUEP (pCAMBIA1300-Ubi-epspsM-Tnos).

[0023] (2) Ligate the Rfl promoter fragment to the above-mentioned PUEP backbone without the Ubi promoter, and partially cut it with HindIII+EcoRV double enzymes for 5 minutes to obtain the Rfl-Osctp-epspsM-Tnos expression cassette fragment.

[0024] (3) Digest the pCDMAR-Hyg vect...

Embodiment 2

[0027] Example 2: Transformation of pCDMAR-Ubi-Osctp-OsEpspsM-Tnos-Rfl-Osctp-OsEpspsM rice material to obtain

[0028] Using the Agrobacterium-mediated method to obtain transgenic pCDMAR-Ubi-Osctp-OsEpspsM-Tnos-Rfl-Osctp-OsEpspsM rice, the specific steps are as follows:

[0029] 1. Induction and subculture of embryogenic callus

[0030] Take the immature ears of rice 15-20 days after pollination, soak them in 75% ethanol for 1 min after shelling, then transfer them to 25wt.% sodium hypochlorite solution for vibration sterilization for 25 min, and wash them in sterile water on a clean bench for 3-5 times , Pick out the immature embryos of the sterilized seeds and inoculate them in the induction medium, about 30 grains per dish, culture them in the dark at 28°C for 7 days, remove the radicle, continue to culture them for 7 days, and subculture after the callus grows up. Appropriate embryogenic calli can be selected for Agrobacterium transformation from the third generation onw...

Embodiment 3

[0043] Example 3 Molecular detection of rice materials transformed with pCDMAR-Ubi-Osctp-OsEpspsM-Tnos-Rfl-Osctp-OsEpspsM

[0044] 1. Extract the total DNA of transgenic rice and non-transgenic rice leaves, and design specific primers according to the gene sequence, F: 5'-GAAAAAGCCTGAACTCACCGC-3', R: 5'-TGCTCCATACAAGCCAACCAC- 3'

[0045] Perform PCR amplification detection, and screen the positive strains whose PCR amplified fragment size is consistent with the expected size for further molecular detection.

[0046] 2. Enzyme PCR reaction system

[0047] Template 1μg

[0048] Primer1 (5μM) 1μL

[0049] Primer2 (5μM) 1μL

[0050] Golden DNA Polymerase 0.4μL

[0051] 2×Reaction Mix 12.5μL

[0052] ddH2O 9.1μL

[0053] 3. PCR reaction program:

[0054] 94°C for 5 minutes; {94°C 45s / 90s, 60°C / 65°C / 56°C (set the annealing temperature according to the primer) 30s / 60s, 72°C 45s / 90s (determine the extension temperature according to the length of the amplified fragment)} 35 c...

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Abstract

The invention relates to the field of genetic engineering. The invention discloses a vector. The vector can achieve whole course expression at the growth and development stage of rice and specifically enhance expression in floral organs and tissues at the young panicle differentiation stage. The vector can be used for culturing glyphosate resistant crops. The vector comprises double expression cassettes, wherein one expression cassette comprises a specific expression gene Rf1 promoter at the rice young panicle differentiation stage, a rice chloroplast transit peptide, a glyphosate resistant gene and a terminator; the other expression cassette comprises a constitutive promoter Ubiquitin, a chloroplast transit peptide, a glyphosate resistant gene and a terminator. The transgenic rice obtained by utilizing the vector has higher resistance to glyphosate in all the growth and development tissues and organs and has the effect of improving the resistance of floral organs and tissues to glyphosate at the rice young panicle differentiation stage, thus relieving damages of glyphosate to the floral organs of rice and ensuring the yield of rice.

Description

technical field [0001] The present invention relates to the field of plant genetic engineering, in particular to a vector that can be expressed in all tissues during the vegetative growth period of rice, and at the same time specifically enhance expression in floral organ tissues during the young panicle differentiation period and its application in cultivating glyphosate-resistant rice Applications. Background technique [0002] Glyphosate is currently the most widely used herbicide species. Since glyphosate is a conductive broad-spectrum herbicide, it can effectively control most annual and perennial weeds and some difficult-to-control weeds that are closely related to food crops, such as: wild rice, red rice, Lisigrass, creeping Bentgrass, etc., are widely used in the weed control of important food crops such as rice; and glyphosate is cheap, does not metabolize in humans and mammalian plants, is environmentally friendly, has no residual activity, and is not easy for pla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00
CPCC12N15/8275
Inventor 苏军王锋胡太蛟刘霞林智敏李刚颜静宛
Owner BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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