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作物学报 ›› 2020, Vol. 46 ›› Issue (10): 1474-1484.doi: 10.3724/SP.J.1006.2020.04014

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

Bna-novel-miR311-HSC70-1模块调控甘蓝型油菜响应热胁迫的机制

鲁海琴1(), 陈丽1,2, 陈磊1, 张盈川1, 文静1, 易斌1, 涂金星1, 傅廷栋1, 沈金雄1,*()   

  1. 1 华中农业大学作物遗传改良国家重点实验室 / 国家油菜工程技术研究中心, 湖北武汉 430070
    2 长江师范学院现代农业与生物工程学院, 重庆 408100
  • 收稿日期:2020-01-16 接受日期:2020-06-02 出版日期:2020-10-12 网络出版日期:2020-06-22
  • 通讯作者: 沈金雄
  • 作者简介:E-mail: 857929189@qq.com
  • 基金资助:
    国家自然科学基金项目(31571698)

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 Published:2020-10-12 Published online:2020-06-22
  • Contact: Jin-Xiong SHEN
  • Supported by:
    National Natural Science Foundation of China(31571698)

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摘要：

HSP70 (heat shock protein 70)参与植物热胁迫应答, 增强植物耐热性, 但目前油菜中尚无miRNA调控HSP70基因的报道。本研究novel-miR311是利用高通量技术在甘蓝型油菜茎尖中筛选出的新miRNA。novel-miR311存在于油菜而不存在于拟南芥中, 5°-RACE技术证实其2个靶基因属热应激同源蛋白基因HSC70-1 (HSP70家族), 在甘蓝型油菜体内被剪切。构建novel-miR311超表达载体, 转化拟南芥和甘蓝型油菜, 其转基因阳性苗中HSC70-1基因表达量显著下降。高温胁迫试验表明, 拟南芥和甘蓝型油菜热胁迫后, 其阳性苗的生长势和存活率显著低于其对应的对照。qPCR结果显示, 油菜中HSC70-1基因表达量热胁迫后较热胁迫前上升。上述结果表明, 油菜novel-miR311介导HSC70-1基因的剪切降低了拟南芥和甘蓝型油菜耐热性。

关键词: 甘蓝型油菜, 拟南芥, novel-miR311, HSC70-1, 高温胁迫

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

表1

novel-miR311降解组测序预测到的10个候选靶基因"

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)

图1

5°-RACE验证靶基因 冒号表示碱基配对, 点号表示错配, 箭头表示切割位点, 数字表示挑斑克隆。"

图2

油菜和拟南芥靶基因作用位点序列对比 a: 转导蛋白基因; b: 热激蛋白HSP70-2基因; c: 热应激同源蛋白HSC70-1基因。红色部分为novel-miR311与靶基因结合部位。"

图3

野生型和阳性拟南芥中novel-miR311和HSC70-1分别在根、茎、叶、花和角果中的表达量 每次试验3次生物学重复。*和**分别表示P < 0.05和P < 0.01。"

图4

野生型和阳性拟南芥在1/2MS上的高温胁迫实验 a: 表型分析; b: 统计学分析。"

图5

甘蓝型油菜阳性苗与J572的种子高温胁迫后的发芽试验 a: 不同温度处理前后对比图; b: 2 d发芽率统计图。"

图6

甘蓝型油菜阳性苗与J572在土壤中的高温胁迫表型及表达分析 a: 热胁迫前后阳性苗与J572的对比图, 图中红色箭头指存活的油菜。b: 热胁迫前后novel-miR311和HSC70-1的表达量。每次试验3次生物学重复。**表示P < 0.01。"

图7

HSP70作用途径 根据Jacob等[33]并略作修改, 虚框中为引入本文结果。"

附图1

novel-miR311茎环结构"

附表1

novel-miR311的前体和成熟体序列"

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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