Soybean bHLH transcription factor and encoding gene and application thereof

A technology that encodes genes and transcription factors, applied in the field of genetic engineering, can solve problems that affect plant growth and development, endanger human health, etc., and achieve the effects of reducing cadmium content, reducing harm, and improving tolerance

Active Publication Date: 2016-07-13
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, soybeans are very easy to accumulate heavy metals. The accumulation of heavy metals in plants not only seriously affects the growth and development of plants, but also endangers human health through the food chain.

Method used

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  • Soybean bHLH transcription factor and encoding gene and application thereof
  • Soybean bHLH transcription factor and encoding gene and application thereof
  • Soybean bHLH transcription factor and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: soybean GmORG3 Gene cloning and subcellular localization

[0032] Cloning of Soybean by Combining Bioinformatics and Conventional Polymerase Chain Reaction (PCR) GmORG3 The DNA sequence, the specific method is:

[0033] Soybean roots were used as experimental materials, and total RNA was extracted using SVTotalRNAlysis Kit (Promega, USA). The first strand of cDNA was synthesized according to the method provided by TaKaRa Company (PrimeScriptTMReverseTranscriptase), and used as a template for PCR amplification. According to known in NCBI GmORG3 Sequence design a pair of primers (primers were synthesized by Shanghai Invitrogen Company):

[0034] P1: 5'-ATGGTTGCTTTGTTTTTCCCCT-3',

[0035] P2: 5'-TTAGAAAATCCTTTGCTTCTC-3'.

[0036] Amplified by RT-PCR GmORG3 The complete ORF fragment of the gene. 25μl PCR reaction system: 2.5μl containing MgCl 210×PCR buffer, 1.0 μl forward and reverse 10 μM primers, 1.0 μl 10 mM dNTP (deoxynucleotide mixture), 2.0 μl cDN...

Embodiment 2

[0038] Example 2: GmORG3 Transcript spatiotemporal distribution and Cd stress on GmORG3 Effects on Gene Expression

[0039] The tissue distribution of GmORG3 in soybean plants was detected by semi-quantitative PCR, and the following measures were taken to ensure the reliability of the detection results: the contamination of a small amount of genomic DNA that may exist in the total RNA extracted by digestion with amplification grade DNaseI was To check whether the genomic DNA is completely eliminated, a part of the total RNA sample digested with DNaseI can be taken out for conventional PCR reaction. When no amplified band is found, the reverse transcription step is carried out. At the same time, in order to further verify the band amplified by real-time quantitative PCR belt is GmORG3 , PCR products were gel-cut and recovered for sequencing. The specific method is as follows: extract total RNA from soybean roots, stems, leaves, flowers and pods in different growth stages as ...

Embodiment 3

[0051] Example 3: GmORG3 Construction of yeast expression vector and detection of Cd tolerance in transgenic yeast

[0052] (1) Construction of expression vector

[0053] by GmORG3 The full-length sequence of the cDNA was used as a template, and the primer (synthesized by Shanghai Biological Engineering Co., Ltd.) containing a restriction site: P5: 5′-TCC CCCGGG ATGGTTGCTTTGTTTTTCCCCT-3′ (underlined bases are restriction endonuclease SmaI recognition sites) and P6: 5′-CG GAATTC Under the guidance of TTAGAAAAATCCTTTGCTTCTC-3′ (the underlined base is the restriction endonuclease EcoRI recognition site), PCR amplified the 5’ end with SmaI recognition site and the 3’ end with EcoRI recognition site GmORG3 cDNA full-length sequence. After the reaction, the PCR amplification product was detected by 1.0% agarose gel electrophoresis, and the target fragment of about 800bp was recovered and purified, digested with SmaI and EcoRI, and then combined with the vector p424GPD ( Figu...

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Abstract

The invention discloses a bHLH transcription factor and an encoding gene and application thereof. The protein has an amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence which is formed by replacing, missing or adding one or more amino acids in the sequence and has the same function. The encoding gene GmORG3 of the bHLH transcription factor shows resistance on Cd stress when in over-expression, so that the GmORG3 gene in the soybean is separated and cloned; the anti-Cd mechanism of the soybean is disclosed; and the anti-Cd molecular breeding of plants can be carried out through gene operation means to improve the Cd resistance of the plants.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a soybean bHLH transcription factor and its coding gene and application. Background technique [0002] With the development of society, the rapid expansion of non-agricultural construction land, the increase of waste discharge and the use of agricultural chemical fertilizers, soil heavy metal pollution has increasingly become a worldwide environmental problem. According to statistics from the Ministry of Land and Resources in 2012, more than 10% of the country's cultivated land area has been polluted by heavy metals. Cadmium (Cd) is one of the heavy metals with strong biological toxicity. The half-life of Cd is very long. Excessive intake of Cd can lead to kidney and bone lesions. In the 1970s, "Itai-Itai disease" appeared in Japan, which was caused by the pollution of cadmium to the living environment of human beings. The cadmium-contaminated rice is the main source of cadm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/81C12N15/82C12N5/10A01H5/00C12R1/93
CPCC07K14/415C12N15/81C12N15/8271C12N2800/102C12N2800/60
Inventor 徐照龙刘晓庆张大勇许玲何晓兰黄益洪邵宏波
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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