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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (12): 3071-3079.doi: 10.3724/SP.J.1006.2022.14227

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

Functional identification of Bna-miR171g on improving tolerance to osmotic stress in Brassica napus

YANG Wen-Jing(), LU Hai-Qin, CHEN Wu-Jun, ZENG Lei, XIE Tao, JIANG Jin-Jin(), WANG You-Ping   

  1. College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, Jiangsu, China
  • Received:2021-12-04 Accepted:2022-03-25 Online:2022-12-12 Published:2022-04-20
  • Contact: JIANG Jin-Jin E-mail:wjyang1205@126.com;jjjiang@yzu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31972963);National Natural Science Foundation of China(31771825);National Key Research and Development Program of China(2018YFE0108000);Graduate Training Program for Innovation and Entrepreneurship(SJCX21_1602)

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Abstract:

MicroRNA171 (miR171) as a conserved miRNA family in plants, plays important roles in regulating plant growth and development. Functional researches of miR171 are mainly reported in Arabidopsis, rice, and tomato. However, the function of miR171 in Brassica napus is unclear. In the present study, we found that the seven members of Bna-miR171 were highly conserved, of which, miR171g was strongly up-regulated by osmotic stress. Under the osmotic stress of 150 mmol L-1 mannitol, the root length of rapeseed overexpressing Bna-miR171g (OE-miR171g) was significantly longer than the control (J9712). The diaminobenzidine staining revealed less H2O2 accumulation in OE-miR171g than that of J9712 after osmotic stress, indicating OE-miR171g had higher reactive oxygen species-scavenging ability than the control. The proline content, peroxidase, and superoxide dismutase activity in OE-miR171g were higher than the control, while the malondialdehyde content in OE-miR171g was lower than the control. Besides, the relative expression level of stress responsive genes (ABI5, ERD10, RAB18, OSR1, RD20, and RD29B) in OE-miR171g were higher than the control. Generally, the overexpression of Bna-miR171g improved the rapeseed tolerance to osmotic stress, and could be helpful to the improvement of abiotic stress tolerance in B. napus.

Key words: Brassica napus, Bna-miR171g, mannitol, osmotic stress

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 引物名称
Primer name		 引物序列
Primer sequence (5'-3')
Q-Bna-miR171g TGATTGAGCCGCGCCAATATCT Q-RAB18-F ACTGAAACTTGCGATTTGGG
5.8S GTCTGCCTGGGTGTCACG Q-RAB18-R AGGTCGATTTCCTTAGCCC
miR171g-F GTGTCCCTCCTTTCTGCG Q-OSR1-F CAACGGCTGAGATTTGATGTC
miR171g-R GAAGGGTTGGGTGGAGGT Q-OSR1-R CGGAAGGAACGGTGGTAGA
miR171g-F (Spe I) GGACTAGTGTGTCCCTCCTTTCTGCG Q-RD20-F CACTTCCGAGTTGGTTGCC
miR171g-R (Asc I) TTGGCGCGCCGAAGGGTTGGGTGGAGGT Q-RD20-R TCCTTCGGTATCATAGGTGCTT
Q-BnABI5-F GATAACATAGGAGGACAGTAT Q-RD29B-F GTCCAAGGTTACTGATCCCACTC
Q-BnABI5-R CTCAACTACCTTCTCTACC Q-RD29B-R CTTCAGACCAAACTCATGGTTTC
Q-ERD10-F GGGAGAGGTTAAGGATCGTG BnActin-F TCTTCCTCACGCTATCCTCCG
Q-ERD10-R CTCGAGGAGAGTAGGCTTATGC BnActin-R AGCCGTCTCCAGCTCTTGC

Fig. 1

Phylogenetic analysis of Bna-miR171 sequences A: the conservation analysis of miR171 mature sequences (From top to bottom are sequences from Arabidopsis thaliana, Oryza sativa, Zea mays, Brassica rapa, Brassica oleracea, and Brassica napus); B: phylogenetic analysis of miR171 precursor sequences. Bna: Brassica napus; Ath: Arabidopsis thaliana; Osa: Oryza sativa; Zma: Zea mays; Bra: Brassica rapa; Bol: Brassica oleracea."

Fig. 2

Relative expression levels of Bna-miR171g under osmotic stress"

Fig. 3

Phenotype of Bna-miR171g overexpression plants under osmotic stress A: the phenotype of OE-miR171 and J9712 under osmotic stress; B: root length; C: hypocotyl length; D: fresh weight; E: the relative expression level of Bna-miR171g; F: DAB staining of cotyledons. Data represent the means ± standard errors, n = 20. *: P < 0.05; **: P < 0.01."

Fig. 4

Physiological indexes of Bna-miR171g overexpression plants and CK under osmotic stress MDA: malondialdehyde; Pro: proline; POD: peroxidase dismutase; SOD: superoxide dismutase. *: P < 0.05; **: P < 0.01."

Fig. 5

Relative expression level of stress-responsive genes in Bna-miR171g overexpression plants and CK"

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