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Method for cloning reference gene of kentucky bluegrass aquaporin

A technology of aquaporin and bluegrass, applied in the field of biotechnology breeding, to achieve the effect of stable expression

Inactive Publication Date: 2012-01-18
NORTHEAST AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0005] At present, the acquisition and application of internal reference genes at home and abroad are only reported in some crop research (Zheng Xiaoya et al., 2011, Study on the water use efficiency and aquaporin expression differences of waxy maize under adversity conditions), but for the water channel of bluegrass grass The internal reference gene for protein gene expression has not been reported at home and abroad

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  • Method for cloning reference gene of kentucky bluegrass aquaporin
  • Method for cloning reference gene of kentucky bluegrass aquaporin
  • Method for cloning reference gene of kentucky bluegrass aquaporin

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Embodiment Construction

[0031] The present invention carries out according to the following steps:

[0032] (1) Primer design: According to the EF1α gene of wheat (Wheat) / potato (Solanum tuberosum) / tobacco (Nicotiana tabacum) / pepper (Capsicum annuum) / tomato (Tomato) / wingless pig hair vegetable (Salsola komarovii) registered in Genbank mRNA sequence (Accession Number.M90077.1 / AB061263.1 / D63396.1 / AF242732.1 / X14449.1 / AB073631.1), design a pair of degenerate primers EF1αF1 / EF1αR1, the primer sequence is as follows:

[0033] EF1αF1: 5'-GTTGCHGTTGCHTBAAGCGTGG-3'

[0034] EF1αR1: 5'-TCWGCAAACTTRACAGCAATGTG-3'

[0035] Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0036](2) Poa annua EF-1α gene cloning: using total RNA as a template, use the kit HaiGene, cDNA 1st Synthesis Kit for reverse transcription to obtain the first strand of cDNA; prepare the following mixture in a PCR tube:

[0037]

[0038] The reverse transcription reaction was carried out on the PCR instrument accordi...

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Abstract

The present invention discloses a method for cloning reference gene of kentucky bluegrass aquaporin. The method comprises: (1) adopting the total RNA of the kentucky bluegrass as a template to carry out a reverse transcription reaction to obtain a first strand of the cDNA; (2) adopting the cDNA as a template, and adopting EF1alphaF1 as the primer to carry out PCR amplification; (3) carrying out recovery for the amplification product to recover the target gene band; (4) ligating the resulting target gene to a pMD18-T vector, and transforming the resulting vector into competent cells, then carrying out DNA sequencing for the cultured bacteria liquid to obtain the reference gene sequence of the kentucky bluegrass aquaporin, wherein the reference gene sequence comprises 176 bp. Compared to the traditional reference gene, the obtained reference gene EF-1alpha of the kentucky bluegrass is prevented from the influence due to the cell growth state, can undergo the influence due to external non-biological factors, and can be expressed stably in the tissue.

Description

technical field [0001] The invention belongs to the field of biotechnology breeding, in particular to a method for cloning an internal reference gene of bluegrass aquaporin. Background technique [0002] With the rapid development of my country's economic construction, lawns have played more and more roles in improving, beautifying and protecting the environment on which people live, and have become an important part of the living environment. In densely populated cities in my country, a living and working environment with green grass and fragrant flowers will bring immeasurable ecological, social and economic benefits. However, the scarcity of water resources and the increase of lawn area are the most prominent contradictions restricting the application of lawn. While people are increasingly relying on the functionality, entertainment and ornamental of lawn, they are also faced with the problem of solving the water needed for the growth of lawn grass. . Especially in urba...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/29C12N15/10
Inventor 陈雅君张璐刘威王艳彬陈阳
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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