CRISPR/dCas9-KRAB mediated transcriptional suppression of NtbHLH47 enhances tolerance to iron stress and modulates iron content in tobacco

IF 4.2 2区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Plant Science Pub Date : 2025-02-25 DOI:10.1016/j.plantsci.2025.112449

Anshu Alok , Hanny Chauhan , Biswaranjan Rout , Ashutosh Pandey , Kashmir Singh

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引用次数: 0

Abstract

Iron homeostasis is a multifaceted regulatory process that needs to be studied to elucidate iron distribution, uptake, and storage in plants. NtbHLH47, a homologue to AtbHLH47, is a negative regulator of iron. The current study deploys CRISPR interference-dCas9-KRAB (Krüppel-associated box) in the transcriptional suppression of NtbHLH47 and its effect on iron uptake by plants. The pHSN6I01 harbouring dCas9-KRAB and gRNA targeting NtbHHLH47 was constructed. Four gRNAs were designed, G1, G2, G3, and G4, located at + 19, + 111, + 232, and + 335 bp upstream from the ATG start codon in the promoter region of NtbHLH47. The NtbHLH47 was repressed in the developed transgenic lines of tobacco and the qRT-PCR analysis showed that target sites G1 and G2 suppressed NtbHLH47 effectively. The transgenic pHSN6I01 +G1 plants were tolerant to the elevated levels of iron, copper, zinc, and magnesium. The root Ferric chelate reductase activity of pHSN6I01 +G1 lines was reduced against wild type. The Perl staining showed high iron content in the roots of the pHSN6I01 +G1 plants. ICP-MS analysis showed increased Fe content in the roots of pHSN6I01 +G1 line suggesting that NtbHLH47 modulates it. The expression of NtbHLH38, NtbHLH100, NtbHLH101, and NtFIT was found to be upregulated in the pHSN6I01 +G1 line. This is the first report of using CRISPRi based on dCas9-KRAB in tobacco and its application in the functional validation of a gene. Using this, NtbHLH47 was transcriptionally suppressed and the generated lines expressed increased levels of iron in the roots of N. tabacum and gave insight in the iron homeostasis.

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来源期刊

Plant Science 生物-生化与分子生物学

CiteScore

9.10

自引率

1.90%

发文量

322

审稿时长

33 days

期刊介绍： Plant Science will publish in the minimum of time, research manuscripts as well as commissioned reviews and commentaries recommended by its referees in all areas of experimental plant biology with emphasis in the broad areas of genomics, proteomics, biochemistry (including enzymology), physiology, cell biology, development, genetics, functional plant breeding, systems biology and the interaction of plants with the environment. Manuscripts for full consideration should be written concisely and essentially as a final report. The main criterion for publication is that the manuscript must contain original and significant insights that lead to a better understanding of fundamental plant biology. Papers centering on plant cell culture should be of interest to a wide audience and methods employed result in a substantial improvement over existing established techniques and approaches. Methods papers are welcome only when the technique(s) described is novel or provides a major advancement of established protocols.