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Method for introducing RNA (ribonucleic acid) interfering gene resistant to root-knot nematode into cucumber

An RNA interference and anti-root knot nematode technology, applied in the field of RNA interference fragments

Inactive Publication Date: 2012-08-15
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many reports on the use of AS-assisted transformation in the genetic transformation of cucumber. When 100mg / L AS is added to the co-cultivation medium, the regeneration frequency is as high as 83.3%.

Method used

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  • Method for introducing RNA (ribonucleic acid) interfering gene resistant to root-knot nematode into cucumber
  • Method for introducing RNA (ribonucleic acid) interfering gene resistant to root-knot nematode into cucumber
  • Method for introducing RNA (ribonucleic acid) interfering gene resistant to root-knot nematode into cucumber

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1. Establishment of cucumber genetic transformation method

[0051] 1.1 Explant preparation

[0052] Pre-soak plump cucumber seeds for 2-3 hours, peel off the seed coat and disinfect with 75% alcohol for about 30 seconds, then disinfect with 6.5% sodium hypochlorite for 15 minutes, shake frequently during the disinfection period, and finally rinse with sterilized deionized water for 5 times, Seeds were inoculated on MS medium and cultured in the dark. After the seeds were grown in the culture medium for 1-2 days, the radicle was excised, and the cotyledons were cross-cut into 4 pieces to obtain cotyledon explants. When the cotyledon node is used as the recipient, it should be cultured under light for 4-5 days after the seeds germinate, cut off when the cotyledon is in an upright state, remove 1 / 2 of the cotyledon and only leave a 2mm hypocotyl, and culture it on the pre-medium PM for 2 days stand-by.

[0053] 1.2 Bacteria solution preparation

[0054] Tak...

Embodiment 2

[0075] Embodiment 2 hygromycin is for the impact experiment of transformant selection

[0076] The transformation method is the same as 1.1-1.5 of Example 1, wherein the hygromycin application scheme in steps (5) and (6) is: when the explants are cotyledons, the hygromycin concentrations are respectively 10 mg / L and 25 mg / L. When the explant is a cotyledon node: the concentration of hygromycin is 7mg / L and 15mg / L respectively

[0077] 2.1 Acquisition of regenerated cucumber plants

[0078] Cotyledon explants can develop small buds after about 3 weeks of culture in the primary selection medium. After being transferred to high-concentration antibiotic medium for selection, some false positive regenerated shoots will turn yellow and stop growing. After 1 month of gradient selection, regenerated shoots that always keep green can be obtained. Transfer the regenerated shoots to the elongation medium, and after 2-3 weeks of culture, the normally developed regenerated shoots will g...

Embodiment 3

[0095] Example 3. Effect of Lipoic Acid on Cucumber Genetic Transformation

[0096] The transformation method is the same as 1.1-1.5 of Example 1, wherein the hygromycin use scheme in steps (5) and (6) is: the hygromycin concentration is always 15 mg / L for screening. The addition scheme and concentration of LA and AS were adjusted as shown in Table 3 to obtain 14 treatments, among which treatment 2 is routinely used in the prior art and recognized to have a relatively good effect.

[0097] Table 3 Concentration ratio of LA and AS during infection

[0098]

[0099] 3.1 Regeneration process of cucumber cotyledon node resistant buds

[0100] Before seeing the light, the color of the cotyledon nodes cut from the seedlings remains light green, and it does not begin to darken until it is cultivated under the light, and the volume of the cotyledon nodes increases obviously after seeing the light, and the leaves thicken. About 1 week after inoculation, a small amount of callus be...

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Abstract

The invention relates to a method for introducing a RNA (ribonucleic acid) interfering gene resistant to root-knot nematodes into cucumber, which relates to the technology of cucumber transgene and is characterized in that by means of an exogenous gene contained gene pCAGC1341-dsMiMpkl, 100mg / L lipoic acid and 50mg / L acetosyringone are added in each neutral co-culture medium of Agrobacterium Tumefaciens suspension to increase conversion rate of Agrobacterium Tumefaciens. In addition, the invention further provides a hygromycin gradient screening method and corresponding medium schemes, so that the efficiency of obtaining cucumber positive transformants is increased further.

Description

technical field [0001] The invention relates to a cucumber transgenic technology, in particular to a cucumber transgenic method for obtaining an RNA interference fragment expressing an interference nematode MiMpk1 gene. Background technique [0002] In recent years, with the large-scale promotion of protected cultivation and heavy cropping, the harm of root-knot nematodes to cucumber production has become increasingly serious. However, there is a lack of germplasm resources resistant to root-knot nematode in the existing resources, and traditional breeding methods cannot solve this problem. Therefore, it is necessary to introduce new antigens through transgenic technology to broaden the genetic basis of cucumber cultivation. In the patent No.: ZL200810118726.X, the name is: "An RNA interference vector and its application" discloses the MiMpkl RNA interference vector constructed with the MiMpkl gene of Meloidogyne incognita as the target, and the vector is transferred into I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/113A01H4/00A01H5/00
Inventor 王烨顾兴芳张圣平苗晗陈国华谢丙炎
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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