Chrysanthemum aquaporins coding gene CmAQP, plant expressing vector thereof and construction method

A plant expression vector and gene technology, applied in the field of molecular biology, can solve problems such as huge water consumption, and achieve the effect of improving plant varieties and increasing germination rate.

Active Publication Date: 2013-09-25
江苏南京农大科技开发有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are many deficiencies in the current soil improvement measures. At the same time, the huge water resource consumption has become the bottleneck of the current chrysanthemum industry development.

Method used

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  • Chrysanthemum aquaporins coding gene CmAQP, plant expressing vector thereof and construction method
  • Chrysanthemum aquaporins coding gene CmAQP, plant expressing vector thereof and construction method
  • Chrysanthemum aquaporins coding gene CmAQP, plant expressing vector thereof and construction method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Cloning of embodiment 1.CmAQP:

[0041] Chrysanthemum 'Shenma' was selected as the material, and 0.05 g of young leaves were taken, and total RNA was extracted according to the instructions of the Trizol RNA extraction kit (TaKaRa), and 1 μg of total RNA was reversed according to the M-MLV reverse transcription kit (TaKaRa). Transcribe into cDNA, digest the cDNA product with RNase, refer to the chrysanthemum CmAQP sequence information, analyze and design primers to amplify CmAQP by Primer 5 software;

[0042] Upstream primer CmAQP-F: 5'-ATGACTAAAGATGTTGAAGTAGC-3' (SEQ ID NO.2)

[0043] Downstream primer CmAQP-R: 5'-CAAGGCATAAATAAATATAGCCA-3' (SEQ ID NO.3)

[0044] Using the extracted leaf cDNA as a template, carry out PCR reaction,

[0045] 50μL reaction system: 5.0μL of 10×RCR Buffer, 4.0μL of dNTP mix (2.5mmol·L -1 ), CmAQP-F, CmAQP-R primers each 1.0μL (20μmol L -1 ), Taq DNA Polymerase 0.2 μL, cDNA template 1 μL, ddH 2O37.8 μL; reaction program: pre-denaturatio...

Embodiment 2

[0046] Embodiment 2. Construction of plant expression vector pCAMBIA 1301-CmAQP

[0047] Design primers for PCR reaction, introduce restriction sites BamH I and Xba I at the upstream and downstream of the target gene CmAQP respectively, connect the PCR product to the pMD19-T Simple vector, transform TOP10 competent cells, and extract positive plasmids, BamHI and Xba I The double-enzyme-digested CmAQP fragment was ligated with BamH I and Xba I double-digested pCAMBIA 1301 for transformation, the positive plasmid was extracted, detected by enzyme digestion electrophoresis and sequenced for verification ( figure 1 ).

[0048] Upstream primer CmAQP-TF: 5'-CGGGATCCATGACTAAAGATGTTGAAGTAGC-3' (SEQ ID NO.4)

[0049] Downstream primer CmAQP-TR: 5'-GCTCTAGACAAGGCATAAATAAATATAGCCA-3' (SEQ ID NO.5)

[0050] ①Using the cDNA of chrysanthemum 'Shenma' leaves as a template, high-fidelity enzyme (PrimeSTAR TM HS DNA Polymerase, TaKaRa) for PCR reaction, 50 μL reaction system: 10×HS RCR Buf...

Embodiment 3

[0052] Embodiment 3. Genetic transformation of Arabidopsis thaliana with plant expression vector pCAMBIA 1301-CmAQP and identification of its salt tolerance

[0053] ① Competent preparation and freeze-thaw transformation of Agrobacterium strain EHA105

[0054] Pick a single colony of EHA105 from the YEB (50ug / mL rifampicin) plate, inoculate it in 50mL YEB liquid medium containing 50ug / mL rifampicin, culture at 200rpm, 28°C until the OD value is 0.5, and then ice-bath the bacteria solution 30min, centrifuge to collect the bacteria, suspend in 2mL pre-cooled 100mM CaCl 2 (20% glycerol) solution, 200uL / tube aliquoted for use.

[0055] Take 10uL pCAMBIA 1301-CmAQP vector plasmid, add 200uL Agrobacterium EHA105 competent cells, bathe in ice for 30min, freeze in liquid nitrogen for 5min, 37℃ for 5min, add 800uLYEB liquid medium, pre-cultivate at 28℃200rpm for 4h, spread the bacterial solution on YEB (50ug / mL rifampicin + 50ug / mL kanamycin) on solid medium, cultured in the dark at...

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Abstract

The invention belongs to the field of molecular biology, and discloses a chrysanthemum aquaporins coding gene CmAQP, a plant expressing vector thereof and a constructing method. The constructed expression vector pCAMBIA1301-CmAQP is recombinant plasmid obtained by means of inserting CmAQP genes into a simple cloning vector pMD19-T, and connecting the CmAQP genes to BamH I locus and Xba I locus of a vector pCAMBIA 1301 after double digestion by the aid of the BamH I and the Xba I. The plant expression vector is used for plant genetic transformation, the CmAQP genes are promoted by CaMV35S promoter to perform excess expression, a great quantity of CmAQP protein is synthesized, and accordingly germination percentage under salt stress of arabidopsis seeds is increased.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a chrysanthemum aquaporin coding gene CmAQP, a plant expression vector and a construction method thereof. Background technique [0002] Chrysanthemum is one of the four major cut flowers in the world. Cut chrysanthemum accounts for about 30% of the world's total cut flowers and occupies an important position in the flower industry. With the rapid development of chrysanthemum industrialization, its protected cultivation area is increasing, and soil salinization has gradually become the main limiting factor for the annual supply of chrysanthemum. However, there are many deficiencies in the current soil improvement measures, and the huge water resource consumption has become the bottleneck of the current chrysanthemum industry development. Stress-resistant breeding of chrysanthemum has always been one of the important topics that horticulturalists at home and abroad are committed to....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/66A01H5/00
Inventor 陈素梅王红宾陈发棣蒋甲福李佩玲刘浦生管志勇房伟民滕年军刘兆磊
Owner 江苏南京农大科技开发有限责任公司
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