Method for high-flux rapidly cloning of rape draught-resistant gene

A high-throughput, rapeseed technology, applied in the field of plant gene cloning and functional research, can solve the problems of long cycle and low efficiency, and achieve the effects of improving cloning efficiency, speeding up the research process, and prolonging the growth cycle.

Inactive Publication Date: 2015-07-01
HENAN UNIVERSITY
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Traditional gene cloning starts with a single gene, cloning and verifying its function

Method used

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  • Method for high-flux rapidly cloning of rape draught-resistant gene
  • Method for high-flux rapidly cloning of rape draught-resistant gene
  • Method for high-flux rapidly cloning of rape draught-resistant gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Obtaining a Total RNA Library of Rapeseed Drought Stress Response

[0046] Drought stress treatment on rapeseed seedlings: seeds of drought-resistant rapeseed inbred line 07Y17 germinated in 1 / 2 Hoagland nutrient solution for 7 days, then placed in Hoagland nutrient solution containing 20% ​​PEG-6000 for 3h, 6h, 12h, 24h and 48h, so that the genes that respond to different degrees of drought stress can be expressed as much as possible. After treatment, the whole plant was sampled, frozen in liquid nitrogen and stored at -80°C for later use. RNA was extracted from a single plant using the CTAB method, and the integrity of the RNA was detected by electrophoresis (results in figure 1 ), and use the Thermo nanodrop nucleic acid analyzer to detect the purity and content of RNA, so that the value of A260 / A280 is around 2.0, and the value of A260 / A280 of the four groups of samples in this experiment is 2.17-2.18. The extracted single-plant RNAs treated with diffe...

Embodiment 2

[0047] Example 2 Construction of full-length cDNA homogeneous library of rapeseed transcriptome under drought stress

[0048] The above-mentioned total RNA library was purified by the magnetic bead method to obtain a total amount of not less than 5 μg of mRNA, and reverse-transcribed to synthesize double-stranded cDNA. The synthesized double-stranded cDNA was normalized and then cloned into a Gateway-compatible vector to construct a normalized full-length cDNA library of rapeseed drought stress transcriptome.

[0049] The construction of the cDNA normalized library mainly includes: the synthesis of the first strand of cDNA, the enrichment of the first strand cDNA with a 5' cap structure, the addition of a 5' adapter, the synthesis of the second strand of cDNA, cDNA hybridization, normalization treatment, and BP recombination Steps such as transformation of Escherichia coli DH10B by electroporation and quality determination of the library can be performed with reference to th...

Embodiment 3

[0063] Example 3 Construction of full-length cDNA plant expression library of drought stress transcriptome

[0064] Using the high-throughput Gateway technology, the above-mentioned cDNA normalization library was cloned into the Gateway-compatible plant expression vector pEarleyGate100, and a drought stress-induced full-length cDNA plant expression library of rapeseed transcriptome was constructed, as follows:

[0065] Extract the above-mentioned homogenized library plasmid, and carry out LR reaction with the Gateway-compatible plant expression vector pEarleyGate100 at a molar ratio of 1:1, according to the instructions of the Gateway? LR Clonase? enzyme mix kit (Invitrogen), so that the canola in the homogenized library plasmid The cDNA target fragment was transferred to the plant expression vector pEarleyGate100, and a drought stress-induced full-length cDNA plant expression library of rapeseed transcriptome was constructed.

[0066] The LR reaction product was transformed...

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Abstract

The invention relates to a method for high-flux rapidly cloning of a rape draught-resistant gene. The method comprises the following steps: (1) performing drought stress treatment on a germinal rape seedling, and sampling the whole stalk to obtain a drought stress responded total RNA base; (2) obtaining mRNA and performing inverse transcription to synthesize the double chain cDNA, and constructing a drought stress transcriptome overall length cDNA homogenization library; (3) constructing a drought stress transcriptome overall length cDNA plant expression library; (4) constructing an over-expression rape drought stress transcriptome overall length cDNA arabidopsis mutant library; (5) screening the obtained arabidopsis mutant library by using a high-flux far-infrared thermal imagery technology, and performing drought stress phenotype identification to obtain an over-expression rape drought stress transcriptome arabidopsis transgenic plant; and (6) cloning a rape drought stress response key gene causing the drought stress phenotype from the obtained drought stress arabidopsis transgenic plant. The method is rapid and convenient and the cloning efficiency of the plant drought stress gene is increased.

Description

technical field [0001] The invention belongs to the technical field of plant gene cloning and function research, and in particular relates to a method for rapidly cloning drought-resistant genes of rapeseed with high throughput. Background technique [0002] Drought has the greatest impact on crop yield potential. The damage caused by drought is almost the sum of the losses caused by other natural disasters. Therefore, how to increase crop yield by improving drought tolerance and water use efficiency of crops has become a major demand for sustainable and efficient agricultural development. [0003] The sensitivity of main crops to water stress is as follows: rapeseed, potato>rice>cotton, wheat, soybean>sweet potato>corn>sorghum, millet. It can be seen that the drought tolerance of rapeseed is poor among the main crops, and it is extremely vulnerable to drought damage. In recent years, with global climate change, droughts have occurred frequently and the deg...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/84C12N15/10
Inventor 王道杰杨翠玲宋纯鹏苗雨晨周云
Owner HENAN UNIVERSITY
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