Method for improving stress resistance of chrysanthemum through trans-CgHSP70 genes
A stress-resistant, transgenic technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of cut chrysanthemums such as high temperature, drought and high-salt stress tolerance are not strong
Inactive Publication Date: 2012-10-03
NANJING AGRICULTURAL UNIVERSITY
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[0012] The object of the present invention is to provide a method for improving the stress resistance of chrysanthemums by transfecting CgHSP70 gene in order to solve the problem that cut chrysanthemums are not highly resistant to high temperature, drought and high salinity in actual production
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Embodiment 1
[0054] 1. Construction of plant expression vector pCAMBIA 1301-CgHSP70
[0055] Using chrysanthemum'Zhongshan Zigui' as material, total RNA was extracted, 1μg of total RNA was reverse transcribed into cDNA according to M-MLV reverse transcription kit (TaKaRa), cDNA product was digested with RNase, and cDNA was used as template to design primers Carry out PCR reaction and introduce SmaI and XbaI restriction sites respectively upstream and downstream of CgHSP70 gene. The upstream primer is: CgHSP70-F: 5′-CCCCGGGATGGCTGGTAAAGGTGAA-3′, and the downstream primer is CgHSP70-R: 5′-AGTAGAAC AGATAAATATCGACCACA-3. '. 50μL reaction system: 10×RCR Buffer 5.0μL, CgHSP70-F, CgHSP70-R primer each 1.0μL (20μmol·L -1 ), dNTP mix 4.0μL(2.5mmol·L -1 ), PrimeSTAR TM HS DNAPolymerase 0.2μL, cDNA template 1μL, ddH 2 O 37.8μL; reaction procedure: 95°C pre-denaturation for 4min, then 94°C melting for 30sec, 55°C annealing for 30sec, 72°C extension for 1min 30sec, reaction for 33 cycles, 72°C extensio...
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Abstract
The invention belongs to the field of plant gene engineering and transgenic breeding, and relates to a method for improving the stress resistance of cut chrysanthemum through trans-CgHSP70 genes. CgHSP70 established plant expression vectors obtained by cloning from Zhongshan purple cinnamon of the chrysanthemum are transferred into the cut chrysanthemum by using an agrobacterium-mediated method for cultivation so as to initially obtain resistant plants, and positively transferred plants are obtained through the screening of hygromycin resistance; PCR (polymerase chain reaction) and fluorescence quantitative PCR analysis is performed on converted plants to prove that endogenous genes are transferred into genome DNA of transgenic plants and transcription occurs; and the resistance analysis on the offsprings of the transgenic plants proves that the resistance to high temperature, drought and high salt is obviously improved. In the invention, the stress resistance of the cut chrysanthemumis improved through the conversion of the endogenous CgHSP70 genes and normal transcription expression as well as the induction of the expression of the genes at an adverse environment; a novel and practical method is provided for selecting and breeding stress resistance varieties of chrysanthemum by using a gene engineering technology; and the breeding progress of the biotechnology of the chrysanthemum can be effectively accelerated.
Description
Technical field [0001] The invention belongs to the field of plant genetic engineering and transgenic breeding, and relates to a method for improving the stress resistance of chrysanthemum by transducing CgHSP70 gene. Background technique [0002] Chrysanthemum grandiflorum (Chrysanthemum grandiflorum), a perennial herb of the Compositae family, is native to China, has a long cultivation history, is rich in varieties, has ever-changing flower types and colors, and has high ornamental value. It is one of China's top ten traditional famous flowers and is also popular among the general public Horticultural plants are widely used in potted flowers, cut flowers, etc., ranking first among the four major cut flowers in the world. However, the high temperature in summer affects its ornamental value and the time range of cultivation and production. In order to solve the annual stable supply of chrysanthemums, obtaining chrysanthemum materials suitable for the high temperature climate in ...
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IPC IPC(8): C12N15/82C12N15/29C12Q1/68G01N33/00G01N25/00
Inventor 陈素梅朱喜荣陈发棣高海顺陈煜管志勇蒋甲福
Owner NANJING AGRICULTURAL UNIVERSITY