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Populus deltoidesx populus nigra PdHSP70 gene and application thereof

A gene, European and American technology, applied in the field of PdHSP70 gene to achieve the effect of improving expression

Inactive Publication Date: 2015-01-21
DALIAN NATIONALITIES UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the HSP gene of many plants has been widely studied, but the study of this gene in Populus americana has not been reported

Method used

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  • Populus deltoidesx populus nigra PdHSP70 gene and application thereof
  • Populus deltoidesx populus nigra PdHSP70 gene and application thereof
  • Populus deltoidesx populus nigra PdHSP70 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The extraction of embodiment 1 total RNA

[0021] 1. The European and American Populus 107 material used in the present invention is taken from Baoding, Hebei. It is an annual cutting cuttage cultivated in the nursery of Beijing Forestry University. After the cuttage, it is watered once every three days in order to keep the soil moist. After new leaves grow, the growth similar (Growing for 3 months) European and American poplar seedlings were sprayed with 250mM NaCl, and the top leaves were removed at 0, 2, 4, and 6 hours respectively and placed in liquid nitrogen, and then stored in a -80°C ultra-low temperature refrigerator for later use. For total RNA extraction and cDNA synthesis.

[0022] 2. The process of extracting total RNA from the leaves of Populus americana under 250mM NaCl stress by CTAB method:

[0023] (1) Before extracting RNA, add DTT at a final concentration of 0.1 mM to 2×CTAB buffer, and preheat at 65°C.

[0024] (2) Put 0.5 g of liquid nitrogen gro...

Embodiment 2

[0030] Example 2 Detection of PdHSP70 Gene Expression of Populus americana in Stress by Fluorescent Quantitative PCR

[0031] According to the conserved sequence of Populus americana PdHSP70 gene, specific primers were designed: upstream primer: PdHSP701 (SEQ ID NO.1) and downstream primer: PdHSP702 (SEQ ID NO.2).

[0032] Primer name

sequence name

Base sequence (5'--3')

PdHSP701

SEQ ID NO.1

AGAATGCGGT CGTTACGGTG CCTGCTT

PdHSP702

SEQ ID NO.2

ACTTTGGGGA TTCTTGTTGA ACCAC

[0033]According to the method of Example 1, the cDNA samples prepared by extracting the apical leaves at 0, 2, 4, and 6 hours were used as templates, and PdHSP701 (SEQ ID NO.1) and PdHSP702 (SEQ ID NO.2) were used as templates. Primers were used for fluorescent quantitative PCR analysis.

[0034] The fluorescent quantitative PCR reaction system (20 μL):

[0035]

[0036] The fluorescent quantitative PCR reaction is divided into three steps, namely: den...

Embodiment 3

[0038] Example 3 Clone PdHSP70 Gene of Populus americana by PCR

[0039] According to the conserved sequence of Populus americana PdHSP70 gene, specific primers were designed: upstream primer: PdHSP701 (SEQ ID NO.1) and downstream primer: PdHSP702 (SEQ ID NO.2).

[0040] Primer name

sequence name

Base sequence (5'--3')

PdHSP701

SEQ ID NO.1

ATGGCATCAA AGCCCGAAGG CAAATTCATT

PdHSP702

SEQ ID NO.2

TCAATCAACT TCCTCAATCT TAGGTCCG

[0041] The synthetic cDNA prepared by the method described in Example 1 was used as a template, and PdHSP701 (SEQ ID NO.1) and PdHSP702 (SEQ ID NO.2) were used as primers for PCR reaction;

[0042] The PCR reaction system is (25 μl):

[0043]

[0044]

[0045] The PCR reaction program is as follows: 35 cycles of pre-denaturation at 95°C for 5 min, each cycle of denaturation at 95°C for 50 s, annealing at 63°C for 90 s, extension at 72°C for 2 min, and finally, the sample was extended at 72°C for ...

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PUM

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Abstract

The invention discloses a populus deltoidesx populus nigra resistance-related gene PdHSP70 which plays an important role in the plant salt resistance process. The provided HSP gene is named as PdHSP70, and the gene has a base sequence shown in SEQ ID NO:3 of a sequence table. When the resistance-related gene PdHSP70 is constructed to an expression vector pCAMBIA1304, a promoter is added in front of a transcription initiation nucleotide, a selective marker GFP is added to authenticate and screen transgenosis vegetable cells or plants, and the expression vector carrying the resistance-related gene PdHSP70 can convert plant hosts through multiple methods and is used for cultivating salt-resistant plant varieties. The gene has wide application prospects in cultivation of salt-resistance plants.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a PdHSP70 gene from Populus deltoides×Populus nigra and its application. Background technique [0002] European and American poplar (Populus deltoides×Populus nigra) is one of the most suitable tree species for short-rotation industrial timber intensive management in mid-latitude regions. In recent years, many excellent European and American poplar clones have been introduced into our country to create large-scale fast-growing and high-yielding forests and have achieved good economic and social benefits. But high salt etc. limit its further popularization. Therefore, when it is introduced into high-salt and water-scarce areas, screening and cultivating salt-resistant strains is a prerequisite. With the development of molecular biology, it has become an important way to solve this problem by using molecular biology techniques to study the molecular mechani...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/11C12N15/82C12N1/21C07K14/415A01H5/00
Inventor 郭鹏董燕
Owner DALIAN NATIONALITIES UNIVERSITY
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