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Zea mays ZmbHLH55 transcription factor and application thereof

A transcription factor, corn technology, applied in the field of molecular biology, can solve the problems that have not yet been reported on L-galactose regulatory factors

Active Publication Date: 2019-11-29
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Maize is an important food and feed plant in the world, but there is no report on the regulatory factors in the L-galactose / GDP-mannose pathway

Method used

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  • Zea mays ZmbHLH55 transcription factor and application thereof
  • Zea mays ZmbHLH55 transcription factor and application thereof
  • Zea mays ZmbHLH55 transcription factor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Obtaining and cloning of maize ZmbHLH55 transcription factor

[0029] Using the promoter of the maize ZmGME1 gene as a bait, the positive clones were screened by conventional yeast one-hybridization, and the ZmbHLH55 sequence was compared in the maize genome by sequencing and alignment. The full-length primer of ZmBHLH55 gene was designed.

[0030] ZmbHLH F: 5′-ATGAACTGCGGGCCGCCCGA-3′

[0031] ZmbHLH R: 5′-TCAAAGCTCCATTTTCATGTGGA-3′

[0032] RNA extraction, cDNA synthesis, gene amplification and cloning are carried out in the following steps:

[0033] A. Maize leaf RNA extraction, use Bao Biological's MiniBEST Plant RNA Extraction Kit to process leaf RNA extraction;

[0034] B. cDNA synthesis, using PrimerScript of Bio-Bio TM 1 st Strand cDNA Synthesis Kit, follow the instructions;

[0035] C. The full length cDNA clone of ZmbHLH55 gene.

[0036] ExTaq was used for PCR amplification. The reaction system was: cDNA template 3μL, primers ZmbHLH F and ZmbHLH R (10μmol / L) e...

Embodiment 2

[0038] Example 2: Research on subcellular localization of ZmbHLH55

[0039] Transcription factors need to enter the nucleus and combine with gene promoters to regulate gene expression. In order to clarify the subcellular localization of ZmbHLH55, the following experiment was carried out.

[0040] (1) Construction of plant expression vector p2300-eGFP-ZmbHLH55

[0041] Through peer communication, our laboratory obtained the empty vector pCAMBIA-2300-35S-N-eGFP-OCS, and designed the following primers:

[0042] ZmbHLH-GFP F:5′-GACGAGCTGTACAAGGGATCCATGAACTGCGGGCCGCCCGA-3′

[0043] ZmbHLH-GFP R: 5′-CTGCAGGTCGACTCTAGAtcaAAGCTCCATTTTCATGTGGA-3′

[0044] The clone obtained in Example 1 was used as a template, and the PCR amplification method was used. The obtained PCR product was recovered by 1% agarose electrophoresis gel electrophoresis. After the pCAMBIA-2300-35S-N-eGFP-OCS empty vector was linearized with BamHI, it was ligated with the one-step cloning kit (10911) of Shanghai Yisheng Biot...

Embodiment 3

[0047] Example 3: Prokaryotic expression vector pSart-I-ZmbHLH55 vector construction and protein expression

[0048] (1) pSart-ZmBHLH55 vector cloning

[0049] According to the sequence of MCS of pSmart-I vector, the following primers were designed:

[0050] ZmbHLH Bam F:5′- taaGGATCC ATGAACTGCGGGCCGCCCGA-3′

[0051] ZmbHLH Xho R2: 5′-cct CTCGAG AAGCTCCATTTTCATGTGGA-3′

[0052] After diluting the T vector plasmid of Seq NO.2 (approximately 5-10ng / μL), it was amplified according to the PCR system and amplification procedure in Example 1, and the PCR product was recovered. The PCR product and vector pSart-I were digested with BamHI and XhoI, and the heavy vector was obtained after routine cloning and identification.

[0053] (2) Protein induced expression

[0054] The pSart-ZmBHLH55 vector obtained in Example 3(1) was transformed into BL21(DE3) bacteria, and the protein was induced to express according to the pET system operation manual. The final concentration of IPTG was 0.5mM, the tem...

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Abstract

The invention relates to the field of molecular biology and the field of genetic engineering, in particular to a zea mays ZmbHLH55 transcription factor pertinent to zea mays vitamin C synthesis and anapplication of the zea mays ZmbHLH55 transcription factor. The zea mays ZmbHLH55 transcription factor is combined with a promoter of zea mays GDP-mannose-3',5'-isomerase ZmGMEI, so that the expression of a gene is started. In zea mays, the expression of the ZmbHLH55 transcription factor is adjusted downwards, ZmGMEI gene expression is declined, the content of vitamin C is declined, and the sensibility of the zea mays to salt stress is increased; and in arabidopsis thaliana, ZmbHLH55 is subjected to overexpression, the content of the vitamin C is raised, and the resistance of the arabidopsis thaliana to the salt stress is increased. The invention discloses a molecular mechanism for adjusting and controlling vitamin C synthesis, and a genetic resource is provided for increasing the contentof the vitamin C in plants (particularly crops) and improving the salt stress resistance of the plants.

Description

Technical field [0001] The invention relates to the field of molecular biology and genetic engineering, in particular to a corn ZmbHLH55 transcription factor and its application. Background technique [0002] my country’s land resources are relatively scarce, the population is huge, and there are a lot of saline-alkali soils (such as the coastline of my country’s northwestern and eastern coastal areas) that are not suitable for planting common crop varieties. Genetic improvement combined with cultivation measures can improve crops’ salt-tolerant capacity. Genetic improvement will play a fundamental role. Corn is an important crop for both food and feed. Increasing the vitamin C (or Ascorbic acid, AsA) content in corn can not only provide more nutrients for humans and livestock. At the same time, vitamin C is an important small molecule antioxidant substance in plants. Increasing its content can also improve the plant's own resistance to salt, drought and other stresses. [0003] T...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/46A01H6/20
CPCC07K14/415C12N15/8243
Inventor 余春梅严铭柯勇超罗杰梁璐陈艳红张健
Owner NANTONG UNIVERSITY
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