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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 low temperature, drought and high-salt stress tolerance

Inactive Publication Date: 2011-09-07
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[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

Method used

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  • Method for improving stress resistance of chrysanthemum through trans-CgHSP70 genes
  • Method for improving stress resistance of chrysanthemum through trans-CgHSP70 genes
  • Method for improving stress resistance of chrysanthemum through trans-CgHSP70 genes

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Embodiment 1

[0054] 1. Construction of plant expression vector pCAMBIA 1301-CgHSP70

[0055] Using chrysanthemum 'Zhongshan Zigui' as material, extract total RNA, take 1 μg of total RNA to reverse transcribe into cDNA according to M-MLV reverse transcription kit (TaKaRa), digest cDNA product with RNase, use cDNA as a template, and design primers Carry out PCR reaction, introduce SmaI and XbaI restriction sites respectively in the upstream and downstream of CgHSP70 gene, the upstream primer is: CgHSP70-F: 5′-CCCCGGGATGGCTGGTAAAGGTGAA-3′, the downstream primer is CgHSP70-R: 5′-AGTAGAAC AGATAAATATCGACCACA-3 '. 50 μL reaction system: 5.0 μL 10×RCR Buffer, 1.0 μL each of CgHSP70-F and CgHSP70-R primers (20 μmol L -1 ), dNTP mix 4.0μL (2.5mmol·L -1 ), PrimeSTAR TM HS DNAPolymerase 0.2 μL, cDNA template 1 μL, ddH 2 O 37.8 μL; reaction program: pre-denaturation at 95°C for 4 min, then melting at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min and 30 sec, 33 cycles o...

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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 chrysanthemum is 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 chrysanthemums through transgenic CgHSP70 gene. Background technique [0002] Chrysanthemum (Chrysanthemum grandiflorum), a perennial herb of Compositae, originates in China, has a long history of cultivation, rich varieties, ever-changing flower shapes and colors, and high ornamental value. It is one of the top ten traditional famous flowers in China and is also loved by 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 affected its ornamental value and cultivation and production time range. In order to solve the annual stable supply of chrysanthemums, obtain chrysanthemum materials adapted to the high temperature climate in summer, and improve the heat resi...

Claims

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Application Information

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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
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