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HsCIPK2 gene of Hordeum spontoneum C. Koch on Tibetan Plateau

A barley, wild technology, applied in the field of genetic engineering

Inactive Publication Date: 2011-08-31
ZHEJIANG NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This makes the CBL-CIPK signaling system more complex and diversified

Method used

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  • HsCIPK2 gene of Hordeum spontoneum C. Koch on Tibetan Plateau
  • HsCIPK2 gene of Hordeum spontoneum C. Koch on Tibetan Plateau
  • HsCIPK2 gene of Hordeum spontoneum C. Koch on Tibetan Plateau

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Obtaining of the full sequence of the HsCIPK2 gene

[0025] 1.1 Design primers (the underlined sequence is the restriction site):

[0026] HsCIPK2-SacI-F:TCC GAGCTC ATGGGGGAGCAGAAGGGGAATATTCTGAT

[0027] HsCIPK2-XbaI-R:ACT TCTAGA TCAACAAGATTGCTGCTGCGGCTGCGGC

[0028] Table 1 reaction system

[0029]

[0030] Table 2PCR reaction parameters

[0031] number of cycles

temperature(℃)

time(s)

temperature(℃)

time(s)

temperature(℃)

time(s)

1

94

180

-

-

-

-

30

94

30

60

15

72

60

1

72

600

-

-

-

-

[0032] 1.2 Experimental results

[0033] 1.2.1PCR results

[0034] Get PCR product 5ul electrophoresis, obtain a clear band about 1.3kb, its size is similar to the fragment size of the CIPK2 gene of known rice and Arabidopsis ( figure 1 ), it can be preliminarily considered that the desired target DNA fragment h...

Embodiment 2

[0037] Example 2 Obtaining of HsCIPK2 Gene Regulatory Sequence

[0038] 2.1 Design primers:

[0039] SP1: ACGGTGATAGACGCCCCTGCTGTGACA

[0040] SP2: AAGTACTTCCTTGCAGCATCTTCCTTG

[0041] SP3: TGACATGCTCCAACACAAAGTAAATCTTGG

[0042] AD1 Primer: NTCGASTWTSGWGTT

[0043] AD2 Primer: NGTCGASWGANAWGAA

[0044] AD3 Primer: WGTGNAGWANCANAGA

[0045] AD4 Primer: TGWGNAGSANCASAGA

[0046] AD5 Primer: AGWGNAGWANCAWAGG

[0047] AD6 Primer: STTGNTASTNCTNTGC

[0048] 2.2 Thermal asymmetric PCR (thermal asymmetric interlaced PCR, TAIL-PCR)

[0049] TAIL-PCR is a kind of random primer PCR. Random primer PCR is to bind a specific primer to the known gene region, and the random primer is combined to a certain region next to the known region, thereby triggering the gene including the known region. Amplification of fragments. The principle of TAIL-PCR technology is: using genomic DNA as a template, using three nested specific primers (special primers, SP) with higher annealing temperatur...

Embodiment 3

[0064] Embodiment 3 The technical route adopted by the present invention is as Figure 6 Shown:

[0065] 1. According to the relevant literature, search and obtain the known CIPK2 gene sequence in other species of Gramineae (such as rice, wheat, sorghum, corn) from various databases, and according to the coding rules and arrangement of exons and introns Search and locate the open reading frame ORF (open read frame), and then obtain the complete coding sequence CDS (codingsequence).

[0066] 2. Use the complete or partially conserved sequence of OsCIPK2 as a probe to search and compare (nBlast or tBlastn) various nucleotide sequence libraries of barley, and speculate that a sequence with higher homology may be part of HsCIPK2 in wild barley sequence, the sequence is listed as the seed sequence for the next step of electronic assembly and extension.

[0067] 3. Using the seed sequence as a probe, repeatedly search the above database, save the retrieved homologous sequence to th...

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Abstract

The invention discloses an HsCIPK2 gene of Hordeum spontoneum C. Koch on Tibetan Plateau. The gene is defined by a nucleotide sequence of SEQ ID NO:1 and has a regulatory sequence defined by SEQ ID NO:2. A method of acquiring the gene comprises the steps of designing an electron probe based on an OsCIPK (CIPK, CBL-interacting protein kinases) homologous gene sequence of paddy rice; searching homologous fragments from an existed nucleotide sequence database of various barley such as an EST (expressed sequence tags) database; obtaining a coding sequence of a targeted gene by methods of cluster analysis, electronic splicing, etc.; conducting composition analysis and function prediction of the targeted gene by the bioinformatics; extracting a total RNA of the Hordeum spontoneum C. Koch; preparing a cDNA and designing a PCR primer; cloning the targeted gene of HsCIPK2 via a PCR technology; conducting TA clone sequencing and sequence analysis on the obtained targeted gene; and on the above basis, designing three primers in a 5'non-coding region of an HsCIPK2cDNA and further cloning the HsCIPK2 gene by utilizing six arbitrary degenerate primers and a Tail-PCR technology. The regulatory sequence is of significance to the research on tissue-specific expression of the HsCIPK2 gene and on response to stresses.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a wild barley HsCIPK2 gene on the Qinghai-Tibet Plateau. Background technique [0002] Abiotic stress (drought, high salinity, low temperature, high temperature, etc.) is a common environmental factor in nature, which seriously restricts plant growth and development (Knight H et al., Trends Plant Sci, 6(6):262~267.2001). Plants grow in the soil and cannot move by themselves, so plants must form a set of self-protection mechanisms against drought, high salinity, low temperature, high temperature and other adversity stresses to adapt to or resist these adverse environments. [0003] Many studies have shown that when various stress factors act on plant cells, they first trigger intracellular Ca 2+ change in concentration. Ca 2+ The change of concentration in time and space represents a specific stress information, which is called calcium signal. Ca 2+ -Ca 2+ Sensor...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415
Inventor 潘建伟郑仲仲潘伟槐王文祥
Owner ZHEJIANG NORMAL UNIVERSITY
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