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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (10): 1474-1484.doi: 10.3724/SP.J.1006.2020.04014

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

Mechanism research of Bna-novel-miR311-HSC70-1 module regulating heat stress response in Brassica napus L.

LU Hai-Qin1(), CHEN Li1,2, CHEN Lei1, ZHANG Ying-Chuan1, WEN Jing1, YI Bin1, TU Jing-Xing1, FU Ting-Dong1, SHEN Jin-Xiong1,*()   

  1. 1 National Key Laboratory of Crop Genetic Improvement / National Engineering Research Center of Rapeseed, Huazhong Agriculatural University, Wuhan 430070, Hubei, China
    2 School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Chongqing 408100, China
  • Received:2020-01-16 Accepted:2020-06-02 Online:2020-10-12 Published:2020-06-22
  • Contact: Jin-Xiong SHEN E-mail:857929189@qq.com;jxshen@mail.hzau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31571698)

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Abstract:

HSP70 (heat shock protein 70) participates in the response to heat stress, and can enhance plant heat tolerance, but there have been no reports of miRNA regulating HSP70 in rapeseed. In this study, a new miRNA, named novel-miR311, was identified in the shoot tip of Brassica napus by high-throughput technology. novel-miR311 was present in Brassica napus but not in Arabidopsis, and 5°-RACE technology confirmed that its two target genes, belonged to heat stress homologous protein gene HSC70-1 (HSP70 family), and could be cleavaged in Brassica napus. An overexpression vector of novel-miR311 was constructed and transformed into Arabidopsis and Brassica napus, and the expression of HSC70-1 in transgenic positive seedlings was decreased significantly. High temperature stress experiments showed that the growth potential and survival rates of Arabidopsis and Brassica napus positive seedlings were lower than those of their corresponding controls. The qPCR results showed that the expression of HSC70-1 gene in rapeseed increased after heat stress than before stress. In conclusion, the results suggest that Bna- novel-miR311 could reduce the heat resistance of Arabidopsis and Brassica napus by mediating cleavaged of HSC70-1.

Key words: Brassica napus, Arabidopsis thaliana, novel-miR311, HSC70-1, high temperature stress

Table 1

Ten candidate target genes predicted from the sequencing of novel-miR311 degradation group"

miRNA 名称
miRNA name		 靶基因
Target gene		 拟南芥同源基因
Arabidopsis homologous gene		 靶基因功能
Function of target gene
novel miR311 BnaC06g18840D AT3G63460.1 Transducin family protein
novel miR311 BnaA07g19590D AT3G63460.1 Transducin family protein
novel miR311 BnaC03g46700D AT5G02490.1 Heat shock protein 70 (Hsp 70) family protein
novel miR311 BnaA03g39360D AT5G02490.1 Heat shock protein 70 (Hsp 70) family protein
novel miR311 BnaA03g39350D AT5G02490.1 Heat shock protein 70 (Hsp 70) family protein
novel miR311 BnaC04g42010D AT5G02500.1 Heat shock cognate protein 70-1 (HSC70-1)
novel miR311 BnaA10g27080D AT5G02500.1 Heat shock cognate protein 70-1 (HSC70-1)
novel miR311 BnaA09g05850D AT5G02500.1 Heat shock cognate protein 70-1 (HSC70-1)
novel miR311 BnaCnng03470D AT5G02500.1 Heat shock cognate protein 70-1 (HSC70-1)
novel miR311 BnaA10g27060D AT5G02500.1 Heat shock cognate protein 70-1 (HSC70-1)

Fig. 1

Validation of target genes using 5°-RACE “:” represents the miRNA and its targets are labeled on the right, “.” represents mismatch, the arrow indicates the cleavage site, and the numbers above sequences represent the detected cleavage site of independent clones."

Fig. 2

Comparison of target gene sites between Brassica napus and Arabidopsis a: transduction protein gene; b: heat shock protein HSP70-2 gene; c: heat shock cognate protein HSC70-1 gene. The red part is the binding site of novel-miR311 and target gene."

Fig. 3

Expression levels of novel-miR311 and HSC70-1 in root, stem, leaf, flower, and pod of wild type and positive Arabidopsis thaliana Data are shown as mean ± SD of three biological replicates. * and ** represent P < 0.05 and P < 0.01, respectively."

Fig. 4

Response to high temperature stress on 1/2 MS in wild type and positive Arabidopsis thaliana a: phenotypic analysis; b: statistical analysis."

Fig. 5

Germination experiment under high temperature stress in Brassica napus positive seedlings and J572 seeds a: comparison chart before and after treatment at different temperatures; b: germination rate chart for two days."

Fig. 6

Phenotype and expression analysis under high temperature stress treatment of Brassica napus positive seedlings and J572 in soil a: comparison of positive seedlings and J572 before and after heat stress, the red arrow in figures refers to the surviving rapeseed. b: expression of novel-miR311 and HSC70-1 before and after heat stress. Data are shown as mean ± SD of three biological replicates. ** P < 0.01."

Fig. 7

HSP70 action pathway According to Jacob et al.[33] with slight modification, the results in the dashed box are for the introduction of this article."

Supplementary Fig. 1

novel-miR311 stem-loop structure"

Supplementary table 1

novel-miR311 precursor sequence and mature sequence"

类别Type 序列Sequence (5°-3°)
成熟体
Mature sequence (5′-3′)		 TTGGTGATAATTGGATTGGCA
前体
Precursor sequence (5′-3′)		 TATGTGGTTAGAGCCAATCCATTTATCACCAATTCGTTTAAGTTGGTATAAATGTCGGTTTAAGAGCGATCAGGCCTATCAAAAATAGGCCGGATCGAATTCTGTTCAGTTGGGCAAGGTGTGTTTGTGCTCTGATACCATGATAAATTTCTTAGTTTTACAATTAAAACTAATTGGTGATAATTGGATTGGCACGTCCCTAAC
引物
Primer (5′-3′)		 5' primer: AACTGCAGTTGTAGTTTTGAGAGATTAGAAGTGG
3' primer: CGGGATCCCTTTTATTAATCCCTCAGTAATACACC
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