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A kind of maize zmbhlh55 transcription factor and its application

A technology of transcription factor and maize, applied in the field of molecular biology, can solve the problem that there is no report on L-galactose regulatory factor yet

Active Publication Date: 2022-04-01
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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  • A kind of maize zmbhlh55 transcription factor and its application
  • A kind of maize zmbhlh55 transcription factor and its application
  • A kind of maize zmbhlh55 transcription factor and its application

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 bait, positive clones were screened by conventional yeast one-hybridization, and the sequence of ZmbHLH55 was compared in the maize genome by sequencing and alignment. The full-length primers of ZmBHLH55 gene were designed.

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

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

[0032] The extraction of RNA, cDNA synthesis, gene amplification and cloning are carried out according to the following steps:

[0033] A. For corn leaf RNA extraction, use the MiniBEST Plant RNA Extraction Kit extraction kit of Baobio to process leaf RNA extraction;

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

[0035] C, ZmbHLH55 gene full-length cDNA clone.

[0036] Use ExTaq (Bao Bio) for amplification and PCR amplification. The reaction sys...

Embodiment 2

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

[0039] Transcription factors need to enter the nucleus and bind to gene promoters to regulate gene expression. In order to clarify the subcellular localization of ZmbHLH55, the following experiments were carried out.

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

[0041] Through peer exchanges, our laboratory obtained the pCAMBIA-2300-35S-N-eGFP-OCS empty vector, 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 products were recovered by 1% agarose electrophoresis gel electrophoresis. After the pCAMBIA-2300-35S-N-eGFP-OCS empty vector was linearized with BamHI, it was connected with the one-step cloning kit (10911) of Shanghai Yishen...

Embodiment 3

[0047] Embodiment 3: the construction of prokaryotic expression vector pSart-I-ZmbHLH55 vector and protein expression

[0048] (1) pSart-ZmBHLH55 vector cloning

[0049] According to the sequence of the MCS of the 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 (about 5-10 ng / μL), amplify according to the PCR system and amplification procedure in Example 1, and recover the PCR product. The PCR product and the vector pSart-I were digested with BamHI and XhoI, and subjected to routine cloning identification to obtain the heavy vector.

[0053] (2) Protein induced expression

[0054] Transform BL21(DE3) bacteria with the pSart-ZmBHLH55 vector obtained in Example 3(1), and induce protein expression according to the pET system operation manual. The final concentration of IPTG is 0.5mM, the tem...

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Abstract

The invention relates to the fields of molecular biology and genetic engineering, in particular to a corn ZmbHLH55 transcription factor related to corn vitamin C synthesis and an application thereof. The maize ZmbHLH55 transcription factor promotes gene expression by binding to the promoter of maize GDP-mannose-3',5'-isomerase ZmGMEI. In maize, the expression of ZmbHLH55 transcription factor was down-regulated, the expression of ZmGMEI gene decreased, the content of vitamin C decreased, and the sensitivity of maize to salt stress increased; Stress resistance increased. The invention not only discloses a molecular mechanism for regulating vitamin C synthesis in plants, but also provides a genetic resource for improving the vitamin C content of plants (especially crops) and the salt stress ability of plants.

Description

technical field [0001] The invention relates to the fields 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 large number of saline-alkali soils (such as the coastlines in Northwest China and the eastern coastal areas) that are not suitable for planting common crop varieties. The ability of crops to tolerate salt and alkali can be improved through genetic improvement combined with cultivation measures. Genetic improvement will play a fundamental role. Corn is an important food and feed crop. Increasing the content of vitamin C (or ascorbic acid, AsA) 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, and increasing its content can also improve the plant's ability to resist salt...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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