Method for creating cotton apolygus lucorum-resistant germplasm by using plant-mediated RNAi

A technology of Lygus and cotton, applied in the field of plant genetic engineering, can solve the problems of affecting growth and development, abnormal molt of fruit flies, etc., and achieve the effect of improving resistance

Pending Publication Date: 2020-06-16
HUAZHONG AGRI UNIV
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Problems solved by technology

After silencing the Mlp84B gene in Drosophila, it will lead to abnorm

Method used

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  • Method for creating cotton apolygus lucorum-resistant germplasm by using plant-mediated RNAi
  • Method for creating cotton apolygus lucorum-resistant germplasm by using plant-mediated RNAi
  • Method for creating cotton apolygus lucorum-resistant germplasm by using plant-mediated RNAi

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Experimental program
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Embodiment 1

[0053] The construction of embodiment 1 Lygus chlorophylla LIM gene plant interference vector

[0054] According to the obtained nucleotide sequence of SEQ ID NO: 1, primers were designed to construct the expression vector, that is, linker bases for BP reaction were added to both ends of the designed primers, and the primer sequences were as follows:

[0055] LIM-BP-F:5'-ggggacaagtttgtacaaaaaagcaggcttgCTGGAATCGTGCCGTGTCT-3',

[0056] LIM-BP-R:5'-ggggaccactttgtacaagaaagctgggttCGCTGGCTGTCTCAGTATGG-3';

[0057] The pGEM-T-LIM plasmid was used as a template for PCR amplification, and the PCR conditions were 95°C for 5 min; 95°C for 30 sec, 58°C for 30 sec, 72°C for 30 sec, and 30 cycles; 72°C for 5 min. The correct LIM fragment containing BP adapter was obtained by PCR amplification. The PCR product was connected to the pHellsgate 4 carrier by BP reaction (BP enzyme was purchased from Invitrogen, USA; 25°C, 4 hours) and then transformed into Escherichia coli Top10 competent cell...

Embodiment 2

[0058] Example 2 Genetic Transformation and Positive Detection of Lygus LIM Gene

[0059] (1) Preparation of sterile vaccine

[0060] The test material is cotton strain J668, (patent application number: 2015108336180, invention name: a method and application for improving cotton regeneration and transformation efficiency, notice of substantive examination of the invention patent application at the public level 2017-06-14). Sowing J668 cotton aseptic seedlings under in vitro culture conditions, the specific steps are: sterilize the shelled J668 cotton seed kernels with 0.1% mercury liter on the ultra-clean workbench for 8-12 minutes, and then wash them with sterile water for 3- 4 times, inoculate the aseptic seed kernels (or explants) obtained in the aseptic seedling medium, support the seedlings one day later, remove the seed coat, and obtain the hypocotyls of the aseptic cotton seedlings after 5 days.

[0061] 2. Activation and infection of Agrobacterium

[0062] Take out t...

Embodiment 3

[0079] Example 3: Analysis of expression level of transgenic plants, establishment of expression pattern and detection of copy number

[0080] 1. Analysis of LIM gene expression:

[0081] Total RNA was extracted from leaves of transgenic T1 generation plants, and the extraction method of total RNA was referred to the improved guanidine isothiocyanate method (a conventional method).

[0082] The cDNA synthesis steps are as follows: add 3ug RNA to a new 0.5mL RNA centrifuge tube, add 1ul oligo(dT) primer (purchased from Promega), supplement DEPC water to 15μl, mix well, then denature at 70°C for 5min, place in Keep on ice for 10 minutes; add 5 μl M-MLV 5×Reaction Buffer, 1.25uL dNTP, 1uL RNasin (40U), 1uL M-MLV reverse transcriptase (purchased from Promega, USA), and rehydrate to 25uL. Gently flick the centrifuge tube to mix the solution, and incubate at 42°C for 1 hour to synthesize the first strand; after the reaction, treat at 70°C for 7 minutes to inactivate the reverse tra...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for creating cotton apolygus lucorum-resistant germplasm by using plant-mediated RNAi. According to the invention, a RNAi vector is constructed by taking a conserved sequence of apolygus lucorum LIM gene as a target sequence, and the apolygus lucorum LIM gene is successfully introduced into a cotton host by utilizing an agrobacterium-mediated genetic transformation method, so that transgenic cotton for expressing the double-stranded RNA of the apolygus lucorum LIM gene is obtained. The transgenic cotton is planted in an outdoor net room, and insect inoculation experiments show that the transgenic cotton can cause down-regulation of the expression quantity of the apolygus lucorumLIM gene, further cause death of apolygus lucorum due to incapability of completing normal eclosion, and finally cause reduction of the population quantity of apolygus lucorum, so that the insect-resistant effect is achieved. The transgenic cotton provided by the invention has resistance to apolygus lucorum. And the transgenic cotton can be hybridized with BT cotton to produce new germplasm of multi-insect-resistant cotton.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for creating cotton germplasm resistant to Lygus lucidum by using plant-mediated RNAi technology. The invention provides a breeding method of transgenic cotton expressing the double-stranded RNA of the LIM gene of the green ligus, and provides a new method for the prevention and control of the green ligus. Background technique [0002] The feeding of herbivorous insects is one of the important factors causing the yield loss of agricultural cotton (Ute et al., 2004). In the past few decades, transgenic BT cotton quilts have been widely used in the control of cotton pests, especially in the control of Lepidoptera pests, and have successfully reduced the use of pesticides and increased farmers’ income (Pray et al. al., 2010; Wu et al., 2009). However, with the extensive planting of BT cotton, its shortcomings are gradually exposed. First, lep...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H4/00A01H5/00A01H6/60
CPCC12N15/8218C12N15/8286C07K14/43563A01H4/001A01H4/008
Inventor 金双侠梁思佳罗静马伟华陈利珍张献龙
Owner HUAZHONG AGRI UNIV
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