Bna-miR43 Mediates the Response of Drought Tolerance in Brassica napus

doi:10.11983/CBB22223

Abstract

Abstract: miRNAs are involved in plant growth and response to stress by regulating the expression of target genes. We conducted a functional study on a single miRNA identified by our group, Bna-miR43. The function of Bna-miR43 under drought stress was investigated by constructing a Bna-miR43 overexpression vector. Degradation group sequencing has predicted that the four target genes of Bna-miR43 belonged to the F-box protein family. Under simulated drought conditions, the expression of Bna-miR43 in J572 roots decreased gradually, while the expression pattern of target genes was shown to be opposite to that of Bna-miR43. With the increaseing time of drought treatment, the expression of target genes increased gradually. Expression pattern analysis showed that Bna-miR43 could respond to drought stress by negatively regulating the expression of target genes. Transgenic experiment showed that under drought stress, the Bna-miR43 overexpressed lines were extremely sensitive to drought. It was shown that the germination rate of the overexpressed lines decreased significantly, the plant dehydration was serious, and more MDA and H₂O₂ were accumulated in the body. After drought treatment, three coding superoxide dismutase (SOD), catalase (CAT) and glycolate oxidase (GOX) were identified in the Bna-miR43 overexpressed lines, and their expressions were down-regulated in the Bna-miR43 overexpressed lines. The results showed that Bna-miR43 plays a key role in regulating plant drought tolerance by regulating the osmotic accumulation and ROS homeostasis in Brassica napus.

Key words: Brassica napus, Bna-miR43, F-box, drought stress

Zhang Yingchuan, Wu Xiaomingyu, Tao Baolong, Chen Li, Lu Haiqin, Zhao Lun, Wen Jing, Yi Bin, Tu Jinxing, Fu Tingdong, Shen Jinxiong. Bna-miR43 Mediates the Response of Drought Tolerance in Brassica napus[J]. Chinese Bulletin of Botany, 2023, 58(5): 701-711.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22223

https://www.chinbullbotany.com/EN/Y2023/V58/I5/701

Figures/Tables 8

Table 1 The primer sequences of qRT-PCR

Gene	Annotation	Primer sequence (5'-3')
BnaC05g39240D	COX1	F: CAAGAACAGGAAAGTGGTTGAG R: GCAACGTGAACCTGTTCTTAAT
BnaA07g11360D	CAT1	F: AATCGTCTTTGCATCATCCATG R: GTCAAAGAGGAGTTGTTGTTCC
BnaC08g42970D	SOD1	F: GATCACAAAACTATGGCCAAGG R: AAACAGTTCCTGTCACAGTAGT
BnaC09g53650D	ABI2	F: GATGAGTTTGATCCGAGATCGA R: TGAACTCGAACAAGCTTCTACT
BnaC01g04330D	ABF	F: GCCGATTTGACTAGATCAACAC R: CGTCCTAGAAAGCAACATCAAG
BnaA10g24440D	ABCG22	F: TTGATGATCTTGATTGACACGC R: CCAAACGCACAACTGTAACTAA

Figure 1 Basic information of Bna-miR43 and its target gene (A) Bna-miR43 precursor’s secondary structure prediction; (B) Base pairing characterization between Bna-miR43 and its target genes; (C) The Pfam domain annotation of BnaA09g03940D; (D) The Pfam domain annotation of BnaCnng24950D

Figure 2 Prediction of cis acting elements of target genes and Arabidopsis homologous gene

Figure 3 Expression patterns of target genes in Brassica napus (A) The expression pattern of BnaA09g03940D; (B) The expression pattern of BnaCnng24950D

Figure 4 Expression patterns of Bna-miR43 and its target genes of Brassica napus under drought stress (A) The expression pattern of Bna-miR43; (B) The expression pattern of BnaA09g03940D; (C) The expression pattern of BnaCnng24950D. Three biological replicates and three technical replicates per experiment. * P<0.05; ** P<0.01

Figure 5 Germination rate of Brassica napus under drought stress (A) and phenotypic analysis of Bna-miR43 overexpressed transgenic plants under PEG simulated drought treatment (B) J572: Control; OE-2, OE-3, and OE-5 indicate different transgenic individuals; ** P<0.01; Bars=1 cm

Figure 6 Analysis of malondialdehyde (MDA) (A) and hydrogen peroxide (H2O2) (B) content in roots of Bna-miR43 overexpressed transgenic rapeseed after drought stress Before: Before drought treatment; After: After drought treatment. Three biological replicates per experiment. J572, OE-2, OE-3, and OE-5 are the same as shown in Figure 5. * P<0.05

Figure 7 Analysis of the expression of oxidative stress-responsive genes and genes involved in ABA signaling pathway in Bna-miR43 overexpressed transgenic rapeseed WT: Wild type; OE-2, OE-3, and OE-5 are the same as shown in Figure 5. * P<0.05; ** P<0.01

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