Soybean photosynthesis related gene GmDeg2 and application thereof

A photosynthesis and soybean technology, applied in the field of genetic engineering, can solve problems such as unclear and unreported Deg gene family

Active Publication Date: 2013-03-27
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the mechanism of how the reduction of the activity of the PSII reaction center converts the excitation

Method used

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  • Soybean photosynthesis related gene GmDeg2 and application thereof
  • Soybean photosynthesis related gene GmDeg2 and application thereof
  • Soybean photosynthesis related gene GmDeg2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the cloning of soybean GmDeg2 gene

[0038] The total RNA of the leaves of soybean variety Kefeng 1 was extracted with a plant total RNA extraction kit (purchased from Tiangen Company), and the integrity of the RNA was detected by 1% agarose gel electrophoresis. The cDNA was synthesized according to the instructions of SYBR PrimeScript RT-PCRKit II kit from TaKaRa Company. Design primers:

[0039] GmDeg2-F:

[0040] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCACCTTTTCACTCA-3' (SEQ ID NO. 3)

[0041] GmDeg2-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTGATTCATCAGGCTTTGGC-3' (SEQ ID NO. 4).

[0042]Prepare PCR reaction solution (50μl system) according to the following component order

[0043]

[0044] Carry out the PCR reaction according to the following procedure

[0045]

[0046]

[0047] PCR products were detected by 1% agarose gel electrophoresis ( figure 1 ) and recovery and purification, the recovered fragment was TA-cloned into pMD19-T Simple Vector...

Embodiment 2

[0048] Embodiment 2, fluorescence quantitative analysis of soybean GmDeg2 gene

[0049] Using MV (methyl viologen) as simulated photooxidation treatment, 600 μmol L containing 0.01% Tween-20 -1 MV was sprayed on the leaves, and the light was set at 500 μmol m -2 ·s -1 , the leaves were quickly taken at 0h, 1h, 3h, 6h and 12h respectively, quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator. Using 18S rRNA as an internal reference gene, Primer Premier (V5.0) software was used to design primers. The primer sequences are as follows:

[0050] Specific primers for amplifying GmDeg2:

[0051] Upstream primer: 5'-TGAACTCGCCACCGTTCGCC-3' (SEQ ID NO.5)

[0052] Downstream primer: 5'-GTGCGTCAACACGCAGCACG-3' (SEQ ID NO.6)

[0053] Specific primers for amplifying 18S rRNA:

[0054] Upstream primer: 5'-CGGCTACCACATCCAAGGAA-3' (SEQ ID NO.7)

[0055] Downstream primer: 5'-GCTGGAATTACCGCGGCT-3' (SEQ ID NO.8)

[0056] Prepare the PCR reaction system (20μl system) acco...

Embodiment 3

[0061] Embodiment 3, expression analysis of soybean GmDeg2 gene in Escherichia coli

[0062] Design specific primers, add HindIII (AAGCTT), XhoI (CTCGAG) restriction sites and protective bases, use the GmDeg2 gene cDNA in Example 1 as a template, amplify the truncated GmDeg2 fragment, and construct the pET30a-GmDeg2 vector with specificity Primers are:

[0063] Upstream primer: 5'-AAGCTTTCGTCGTCACCTCCCCCA-3' (SEQ ID NO.9)

[0064] Downstream primer: 5'-CTCGAGTTCATCAGGCTTTGGCTCCAG-3' (SEQ ID NO.10)

[0065] After the PCR reaction procedure, the PCR product was recovered and TA cloned into pMD19-T Simple Vector, the bacterial solution with the correct sequencing result was selected to extract the plasmid, and then pMD19T-GmDeg2 and pET30a were double digested to recover the digested product. Transform Escherichia coli, extract the plasmid and detect pET30a-GmDeg2 by double enzyme digestion. Transform pET30a-GmDeg2 into BL21(DE3) competent cells.

[0066] Prokaryotic expressi...

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Abstract

The invention belongs to the field of gene engineering, and discloses a soybean photosynthesis related gene GmDeg2 and an application thereof. The soybean photosynthesis related gene GmDeg2 has a nucleotide sequence as shown in SEQ ID NO. 1; protein coded by the gene has an amino acid sequence shown in SEQ ID NO.2. By using any vector which can guiding expression of exogenous genes in a plant, the GmDeg2 gene of the invention can be introduced into a plant cell so as to obtain a transgenic plant with improved photoinhibition resistance. The gene of the invention has important significance on soybean photoinhibition resistance, especially on cultivation of soybean varieties with high photosynthetic efficiency.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a soybean photosynthesis-related gene GmDeg2 and an application thereof. Background technique [0002] Photosynthesis is the source and basis for the survival of the biological world. However, under strong light, the photosynthetic function of plants is easily inhibited, and the photosynthetic mechanism is destroyed, thereby reducing the photosynthetic efficiency. This process is called photoinhibition. During the long-term evolution process, plants have formed a variety of biophysical and biochemical mechanisms to protect them from strong light damage, flexibly resist complex and changeable light stress, and minimize photoinhibition damage. In addition to some morphological adaptations, several protective mechanisms exist in the photosynthetic apparatus, such as heat dissipation, photorespiration, and turnover of the D1 protein. The main target of light damage is the ...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/50C12N15/82A01H5/00
Inventor 杨守萍孔星张璟曜
Owner NANJING AGRICULTURAL UNIVERSITY
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