Yunnan red pear-1 metallothionein gene PyMT1 and prokaryotic expression vector and application thereof

A metallothionein, prokaryotic expression technology, applied in metallothionein, application, genetic engineering and other directions, to achieve the effect of improving the ability to tolerate heavy metals and improving the tolerance of microorganisms to heavy metal ions

Inactive Publication Date: 2012-01-11
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are few reports on MT genes in fruit trees, and the identification and functional analysis of MT genes in Yunhongli No. 1 has not been done so far. Since microorganisms play an irrepl...

Method used

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  • Yunnan red pear-1 metallothionein gene PyMT1 and prokaryotic expression vector and application thereof
  • Yunnan red pear-1 metallothionein gene PyMT1 and prokaryotic expression vector and application thereof
  • Yunnan red pear-1 metallothionein gene PyMT1 and prokaryotic expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Prokaryotic expression vector pGEX-4t-1- PyMT1 And pET-32a- PyMT1 Build

[0039] Prokaryotic expression vector pGEX-4t-1- PyMT1 、 p ET-32a- PyMT1 The construction process is as figure 1 with figure 2 As shown, first, according to the full-length gene sequence of the exocarp EST of Yunhong Pear No. 1 in GenBank, a pair of primers was designed, and the first strand cDNA of Yunhong Pear No. 1 was used as a template to amplify. PyMT1 Full-length cDNA sequence, recovered and purified PyMT1 After the full-length gene fragment, connect it to the pMD18-T vector to obtain pMD18- PyMT1 ;use EcoR I and Xho I double digestion pMD18- PyMT1 , PGEX-4t-1 and pET32a- xyk , Recovery and purification PyMT1 Gene fragments and large vector fragments pET-32a and pGEX-4t-1, then ligate to obtain recombinant plasmid pGEX-4t-1- PyMT1 And pET32a-PyMT1, which contains the promoter lac, followed by PyMT1 gene. The specific construction process is as follows:

[0040] 1. Yunhongli No. 1 ...

Embodiment 2

[0063] Example 2: Prokaryotic expression vector pGEX-4t-1- PyMT1 , PET-32a- PyMT1 Prokaryotic expression of

[0064] Take 500μl of the fusion expression engineering bacteria solution and add it to 50ml LB liquid medium, and culture for 2 hours at 37℃, 180 rpm shaker; after the cultivation, take out 1 ml, measure the OD600 value of the bacterial solution, and take it out. 2 tubes (2ml / tube) of bacterial solution, as an uninduced sample, add 0.1M IPTG (final concentration of 1 mM and 0.5 mM) to the remaining bacterial solution in the Erlenmeyer flask for induction, and culture in a shaker at 37°C and 180 rpm At 2h, take out 2 tubes (2ml / tube) of bacterial solution, which are samples for 2h induction; also take 2 tubes of samples at induction 4h, 6h, 8h.

[0065] After sampling, one tube of samples for 0, 2, 4, 6, and 8 hours of induction was taken and used for total protein analysis. The specific steps are: centrifugation at 4°C, 12000 rpm for 2 min; discard the supernatant, add 10...

Embodiment 3

[0075] Example 3: Fusion expression engineering strain pGEX-4t-1- PyMT1 BL21 and control bacteria pGEX-4t-1 BL21 heavy metal tolerance experiment

[0076] The experiment was performed on the fusion expression engineering strain pGEX-4t-1- under 37℃, IPTG 0.25 mM / L induction conditions PyMT1 BL21 and the control strain pGEX-4t-1 BL21 carried out heavy metal tolerance experiments. The experimental results showed that the fusion expression engineering strain pGEX-4t-1- PyMT1 BL21 at 1.1 mM / L Zn 2+ When processing, PyMT11 There is no difference between the fusion expression engineering bacteria and the negative control bacteria of the empty vector at 0-5 h, but after 5 h PyMT11 Accelerated growth of transformants ( Picture 12 ). Using 2.25 mM CuCl 2 After treatment, within 0-4 h, PyMT11 Both the transformed bacteria and the negative control grow, within 5-9 h PyMT11 The transformed bacteria still grew slowly, while the negative control showed growth arrest ( Figure 13 ). ...

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Abstract

The invention discloses Yunnan red pear-1 metallothionein gene PyMT1 and a prokaryotic expression vector thereof, and the prokaryotic expression vector is a prokaryotic expression vector which contains the Yunnan red pear-1 metallothionein gene PyMT1. According to the invention, PyMT1 gene is cloned from Yunnan red pear-1; the over expression of the gene in bacterial cells is controlled by T7 promoter and lactose operon with the induction of IPTG; PyMT1 protein is synthesized and secreted into cytoplasm; the expression level is detected by SDS-PAGE; test results show that the expression level of the PyMT1 protein reaches a maximum when the expression is inducted by 1mM IPTG at 37 DEG C for 4 hours and when the expression is inducted by 0.5 mM IPTG at 37 DEG C for 4 hours; the vector adopted by the invention can successfully express a lot of Yunnan red pear-1 metallothionein; the test results also demonstrates that the Yunnan red pear-1 metallothionein gene PyMT1 of Yunnan red pear has a typical function of heavy metal ion tolerance of the metallothionein gene, which lays a foundation for further research of PyMT1 functions and the applications of the metallothionein gene in microbial tolerance to heavy metal.

Description

technical field [0001] The invention relates to Yunhongli No. 1 metallothionein gene PyMT1 The prokaryotic expression carrier thereof and its application in improving the tolerance of microorganisms to heavy metal ions belong to the field of genetic engineering. Background technique [0002] Metallothionein (MT) is a kind of polypeptide with low molecular weight (6000-9000Da), homocysteine ​​(Cys) content, and strong metal binding ability, and is widely distributed in the biological world; metallothionein Not only can it participate in the storage, transport and metabolism of trace elements in the body, but it can also antagonize ionizing radiation, scavenge free radicals and detoxify heavy metals. Studies have shown that plant metallothionein can chelate heavy metals and scavenge active oxygen through its large number of Cys residues, so as to protect plants from oxidative damage, and also participate in the growth, development, reproduction, aging, immunity, stress and ev...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/825C12N15/70C12R1/19
Inventor 李昆志樊磊张晓东陈丽梅
Owner KUNMING UNIV OF SCI & TECH
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