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Preparation method of glyphosate resistant transgenic rice

A technology of transgenic rice and glyphosate resistance, which is applied in the fields of botanical equipment and methods, horticultural methods, horticultural tools/equipment, etc. Simple operation and low copy number effect

Inactive Publication Date: 2014-08-27
CHINA NAT SEED GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the transgenic rice cultivated by this screening method will bring in redundant screening marker genes, which will bring troubles to the application of future varieties

Method used

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  • Preparation method of glyphosate resistant transgenic rice
  • Preparation method of glyphosate resistant transgenic rice
  • Preparation method of glyphosate resistant transgenic rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The synthesis of embodiment 1Epsps-1 and Epsps-2 gene and the construction of genetic transformation vector

[0037] 1. Artificially synthesized glyphosate resistance genes Epsps-1 (US_RE39247) and Epsps-2 (CN88103827.X), the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0038] 2. Using the PU130 vector as the backbone (the construction process of the PU130 vector is: the pCambia3300 vector is digested with XhoI and HindIII, and the P35s-Bar part is replaced with Pubi to obtain PU130), and the genetic transformation vectors are constructed and named pZZ00011 and pZZ00013 respectively. The schematic diagrams of the T-DNA regions of the two genetic transformation vectors are shown in Figure 1A , Figure 1B .

[0039] (1) The artificially synthesized genes Epssps-1 and Epsps-2 were digested with BamHI and SacI, and then inserted into PU130 treated with BamHI and SacI to obtain intermediate vectors pZZ00002 (Pubi-Epsps-1-35spolyA),...

Embodiment 2

[0053] Embodiment 2 Acquisition and Identification of Transformed Agrobacterium

[0054] The pZZ00011 and pZZ00013 vectors were respectively introduced into Agrobacterium strain EHA105 by electrotransformation to obtain Agrobacterium for transformation and sequenced.

[0055] The sequencing steps include:

[0056] 1. Agrobacterium transformation

[0057] (1) Thaw the EHA105 competent state on ice, and pre-cool the electroporation cup on ice.

[0058] (2) Add 1 ul of the DNA of the pZZ00011 or pZZ00013 vector prepared in Example 1, mix gently, and add the mixture to the pre-cooled electroporation cup

[0059] (3) Select the agr conversion file, electric shock, and use 500ulLB to blow out the bacterial liquid

[0060] (4) Recovery: 28°C, 200rpm, 45min

[0061] (5) Apply the resuscitated product on a Rif+Kan resistant plate, 28°C, 40h

[0062] (6) Pick the monoclonal shaking bacteria and extract the plasmid

[0063] (7) Using the plasmid extracted from Agrobacterium, transf...

Embodiment 3

[0085] Example 3 Preparation of glyphosate-resistant transgenic Kongyu 131 rice

[0086] 1. Induction of callus

[0087] (1) Take mature rice seeds of Kongyu 131 (purchased from Jiansanjiang Branch of Heilongjiang Agricultural Reclamation Bureau), and disinfect them with 75% ethanol for 1 minute after shelling;

[0088] (2) Disinfect with 40% (V / V) sodium hypochlorite solution (NaClO) for 10-15 minutes;

[0089] (3) Rinse with sterilized water for 3 times;

[0090] (4) Inoculate the sterilized seeds on the induction medium

[0091] (5) Culture in dark at 28°C for 30 days.

[0092] 2. Subculture of callus

[0093] The calli with light yellow appearance, dry surface and firm texture were selected and placed on the subculture medium, cultured in the dark at 28°C, and subcultured every 2 weeks as a cycle.

[0094] 3. Preparation of Agrobacterium.

[0095] 1) On a sterile workbench, take out the Agrobacterium strains transformed with pZZ00011 and pZZ00013 vector plasmids prep...

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Abstract

The invention provides a preparation method of glyphosate resistant transgenic rice. The method comprises the following steps: converting an expression vector containing glyphosate resistant gene into Agrobacterium tumefaciens, invading rice calluses by the Agrobacterium tumefaciens, transferring the calluses to a selective aluminum containing glyphosate for screening, choosing resistant calluses, differentiating, rooting, hardening-seedling, and transplanting to obtain glyphosate resistant transgenic rice. A molecular identification result shows that an exogenous gene is stably expressed. The obtained glyphosate resistant transgenic rice contains glyphosate herbicide resistant gene, contains no other screening marker genes, and can be used as a commercial herbicide resistant rice kind to reduce the reduce the later process and accelerate the breeding pace. The method has the advantages of simple operation, low copy number of transgenic plants, stable heredity properties, and good market application prospect.

Description

technical field [0001] The invention relates to the field of plant transgenic technology, in particular to a preparation method of glyphosate-resistant transgenic rice. Background technique [0002] Weeds in paddy fields compete with rice in water, fertilizer and growth space, which directly affect the yield of rice. In recent years, the use of cultivation methods such as direct seeding and throwing seedlings has made the damage of weeds in paddy fields more and more serious; especially the emergence of a new type of weed in paddy fields—weedy rice—has posed a problem for weeding in paddy fields. In addition, with the acceleration of the migration of rural population to cities, the scale and mechanization of rice planting is a foreseeable trend, which makes the traditional method of manual weeding unrealistic. Herbicide application is one of the best solutions to these emerging problems, therefore, herbicide-resistant rice will have a very large actual demand and market pot...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 张琳马崇烈魏晶夏秀云王维鹏李晨章旺根
Owner CHINA NAT SEED GRP
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