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Application of herba saussureae involucratae sikCOR gene in cultivating cold resistant plant

A gene, the technology of snow lotus, applied in the application field of Tianshan snow lotus sikCOR gene in cultivating cold-resistant plants

Active Publication Date: 2010-09-01
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, between different plants and within the same plant, the amino acid composition, sequence, repeat unit, repeat number and spatial structure of different cold-regulated proteins show a certain degree of difference, and their composition and structure show diversity and complexity. sex

Method used

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  • Application of herba saussureae involucratae sikCOR gene in cultivating cold resistant plant
  • Application of herba saussureae involucratae sikCOR gene in cultivating cold resistant plant
  • Application of herba saussureae involucratae sikCOR gene in cultivating cold resistant plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Extraction of monoclonal plasmid from Tianshan snow lotus cDNA library

[0050] The glycerol tube of the cDNA library of Tianshan Xuelian was stored in 10 ml of LB liquid medium (Cm 50 μg / ml) and cultured overnight at 37°C with shaking at 220 rpm. Dip the bacterial liquid into streak culture on LB solid medium (Cm 50μg / ml) plate, and cultivate in dark at 37°C for 12-16hr. Pick a single clone in 20ml LB liquid medium (Cm 50μg / ml), shake and culture at 37°C at 220rpm for 14hr, and extract the plasmid. The specific method is as follows:

[0051] 1) The bacterial liquid was dispensed into 1.5ml Ep tubes, centrifuged at 12000rpm for 3min, and the supernatant was discarded;

[0052] 2) Add 400ml STE solution, resuspend, centrifuge at 12000rpm for 3min, discard the supernatant;

[0053] 3) Resuspend the pellet with 150 μL of alkaline lysis solution I, and mix well;

[0054] 4) Add 300 μL of freshly prepared alkaline lysis solution II and mix gently until the solu...

Embodiment 2

[0059] Example 2: Cloning of sikCOR gene from Tianshan snow lotus

[0060] The monoclonal plasmid of the Tianshan snow lotus cDNA library was used as the template, and the sequence of CP1 was 2 and the sequence of CP2 was 3 for amplification, and deionized water was used as the template for amplification as a negative control.

[0061] CP 1 For: 2

[0062] 5′ ATG ATG AAG GGA GTAAAGAAC TA 3′

[0063] BamHI

[0064] CP 2 For: 3

[0065] 5′ TCTAATAAC ACGAAATCTAAAATA 3′

[0066] SalI

[0067] PCR reaction system (20μl) is:

[0068] 10×PCR Buffer 2.0μl

[0069] dNTPs (2.5mM each) 0.5μl

[0070] MgCl2 (25mM) 1.0μl

[0071] Upstream primer (25μM) 0.5μl

[0072] Downstream primer (25μM) 0.5μl

[0073] Template DNA (ddH 2 O) 0.5μl

[0074] Taq DNA Polymerase (2.5U / μl) 0.3μl

[0075] ddH 2 O 14.7μl

[0076] Total 20.0μl

[0077] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30s, renaturation at 57°C for 30s, e...

Embodiment 3

[0079] Example 3: Construction of siikCOR gene plant expression vector

[0080] Construction of plant expression vector: pBin438-sikCOR. The plant expression vector pBin438 was double digested with BamHI / SalI to obtain the vector fragment. Recover target gene fragments and vector fragments. The target gene fragment and the vector fragment were connected in vitro, and the correct recombinant plasmids were named pBin438-sikCOR respectively. In this embodiment, we choose tobacco as the transgenic plant material, and other plants can also be used as the transgenic material. like Figure 4 , 5 .

[0081]In this embodiment, the sikCOR gene and pBin438 constitute a plant expression vector for plant transformation. According to this embodiment of the present invention, in addition to pBin438, other plant expression vectors can also be selected as the selected plant expression vector for construction.

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Abstract

The invention relates to application of a herba saussureae involucratae sikCOR gene in cultivating a cold resistant plant. In the invention, a sikCOR gene is cloned from a herba saussureae involucratae, and a plant expression vector is constructed by utilizing the gene to obtain the cold resistant transgenic plant through genetic transformation. The sikCOR gene is cloned from the herba saussureae involucratae and then is expressed in the transgenic plant to improve the cold resistance of the transgenic plant, improve the low temperature stress resistance of the plant through excessive expression of the gene and finally obtain the plant with obviously enhanced low-temperature resistant capability. By screening and cloning genes directly related to cold resistance from the herba saussureae involucratae, the low-temperature adaptability mechanism of the saussureae involucratae is revealed. The herba saussureae involucratae sikCOR gene is used in breed improvement of crops, thus having great academic and economic value.

Description

Technical field: [0001] The invention relates to a method for obtaining a sikCOR gene by cloning from a plant of the genus Asteraceae, Saussurea involucrata Kar.et Kir, constructing a constitutive plant expression vector, transforming the plant, evaluating the cold resistance effect, and increasing the resistance of the plant. cold performance. Background technique: [0002] Low temperature is one of the main natural disasters that endanger agricultural production. Low temperature stress can reduce plant photosynthesis, enhance respiration, block energy production and material synthesis, increase consumption, and put plants in a state of starvation, which seriously affects the normal growth and development of plants, and even leads to death (Guo Ziwu, Li Xianli, etc., Plant low temperature stress response Advances in Biochemical and Molecular Biology Mechanisms of Chinese Journal of Ecological Agriculture, 2004, 12(2): 54-57). [0003] Cold acclimation proteins (CORPs) ref...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/84
Inventor 祝建波王爱英张煜星沈海涛刘红玲
Owner SHIHEZI UNIVERSITY
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