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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (5): 1211-1221.doi: 10.3724/SP.J.1006.2023.24079

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Functional analysis of Bna-miR43-FBXL regulatory module involved in aluminum stress in Brassica napus

ZHANG Ying-Chuan1(), WU Xiao-Ming-Yu1, TAO Bao-Long1, CHEN Li1,2, LU Hai-Qin1, ZHAO Lun1, WEN Jing1, YI Bin1, TU Jing-Xing1, FU Ting-Dong1, SHEN Jin-Xiong1,*()   

  1. 1National Key Laboratory of Crop Genetic Improvement/National Engineering Research Center of Rapeseed, Huazhong Agricultural University, Wuhan 430070, Hubei, China,
    2School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Chongqing 408100, China
  • Received:2022-04-02 Accepted:2022-07-21 Online:2023-05-12 Published:2022-08-19
  • Contact: *E-mail: jxshen@mail.hzau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31871654)

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Abstract:

Aluminum is one of the main factors that limited crop growth and yield in acid soil and how to utilize acid soil is of great significance. This study is based on Bna-miR43, an unreported miRNA identified in the previous study. We analyzed the molecular characteristics, conserved structure, phylogenetic analysis, and the expression level of Bna-miR43 and its target genes in Brassica napus. The Bna-miR43 over expression vector was constructed to explore the molecular mechanism of Bna-miR43- FBXL module in Brassica napus in aluminum stress. 5'-RACE showed that Bna-miR43 actually cleaved BnaA09g03940D and BnaCnng24950D. Bioinformatics analysis revealed that the homologous gene of BnaA09g03940D and BnaCnng24950D in Arabidopsis was AT5G27950, encoding E3 ubiquitin ligase contained F-box. The qRT-PCR indicated that the relative expression levels of Bna-miR43 and its target genes had a phenomenon of trade-off in different tissues of Brassica napus and under transient treatment of aluminum stress. Aluminum treatment showed that the entire aerial part of the transgenic plant grew much better than control and accumulated less MDA and H2O2. Meanwhile, the aluminum accumulation in the root of transgenic plants was less than the control. In this study, these results may provide reference for the response of aluminum stress in Brassica napus.

Key words: Brassica napus L., Bna-miR43, F-box, aluminum stress

Table S1

Bna-miR43 precursor sequence and mature sequence"

Bna-miR43 序列 Sequence (5°-3°)
前体序列
Precursor sequence		 AGTTTAGGTCGAAGATTGGAACATTGGGACCGTGCCCTATTACAAACTTACACACATTGCAAGCATATTTCTATGCTCATACATATGATCAATATAGCCTTCTATCTTAAGTCAAGTATTGTATAATTTAATTTTGTTATACACTAATTTAGATCATGAAGAGTCTGGATTTTTAGTTTTTTTTTCTACAAT
成熟体序列
Mature sequence		 GAAGATTGGAACATTGGGACC

Fig. 1

Bna-miR43 precursor’s secondary structure Arrow indicates Bna-miR43 mature sequence."

Table 1

Overview of Bna-miR43 target genes predicted from degradome sequencing"

靶基因
Target gene		 拟南芥同源基因
Arabidopsis homologous gene		 靶基因功能
Function of target genes		 类别
Category
BnaA09g03940D AT5G27920 F-box蛋白家族 F-box family protein 0
BnaCnng24950D AT5G27920 F-box蛋白家族 F-box family protein 0
BnaA06g06260D AT5G27920 F-box蛋白家族 F-box family protein 0
BnaC05g08010D AT5G27920 F-box蛋白家族 F-box family protein 0

Fig. 2

RLM-RACE verify the cleavage site in target genes The arrow indicates the cleavage site in the target genes, the number represents the frequency of clones to validate the cleavage sites of target mRNA."

Fig. 3

Alignment of B. napus target genes and Arabidopsis homologous gene"

Fig. 4

Evolutionary of FBXL gene family in B. napus A: phylogenetic tree of FBXL gene family in B. napus; B: the gene structure and motif of F-box family."

Fig. 5

FBXL subfamily sequence identity"

Fig. 6

Relative expression profile of Bna-miR43 and its target genes in different tissues of B. napus Three biological replicates and three technical replicates per experiment. *: P < 0.05; **: P < 0.01."

Fig. 7

Relative expression level of Bna-miR43 and its target genes A: the expression level of Bna-miR43 and its target genes of B. napus under Al stress; B: gene expression of over expression Bna-miR43 transgenic rapeseed. Three biological replicates and three technical replicates per experiment, *: P < 0.05; **: P < 0.01."

Fig. 8

Phenotype identification of J572 and transgenic rapeseed in aluminum stress A: the untreated rapeseed material. B: rapeseed material treated with Al. The bottom leaves of the same strain were randomly taken for pictures, and each strain choose five rapeseed, in the pictures of above ground portion, bar: 5 cm; the bottom leaf, bar: 2 cm. C: the distribution of aluminum in root by hematoxylin dyeing, -Al3+: J572 was cultured in 0.5mmol L-1 CaCl2 (pH 4.5) for one week, then stained with hematoxylin, observed with stereo microscope and photographed. +Al3+: transgenic rapeseed and J572 were treated in 0.5 mmol L-1 CaCl2, 300 μmol L-1 AlCl3 (pH 4.5) for one week, then stained with hematoxylin, observed and photographed by stereo microscope, on the left side of the figure, bar: 1 mm, on the right side of the figure, bar: 0.5 mm."

Fig. 9

Physiological and biochemical indexes of over expression Bna-miR43 under aluminum stress A: MDA content in roots of Bna-miR43-overexpressed transgenic rapeseed after Al stress; B: hydrogen peroxide content in roots of Bna-miR43-overexpressed transgenic rapeseed after Al stress. Three biological replicates and three technical replicates per experiment, *: P < 0.05; **: P < 0.01."

Fig. 10

Pathway of Bna-miR43"

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