Bna-miR43介导甘蓝型油菜响应干旱胁迫

doi:10.11983/CBB22223

植物学报 ›› 2023, Vol. 58 ›› Issue (5): 701-711.DOI: 10.11983/CBB22223

Bna-miR43介导甘蓝型油菜响应干旱胁迫

张盈川¹, 吴晓明玉¹, 陶保龙¹, 陈丽¹^,², 鲁海琴¹, 赵伦¹, 文静¹, 易斌¹, 涂金星¹, 傅廷栋¹, 沈金雄¹^,^*()

1.华中农业大学作物遗传改良全国重点实验室/国家油菜工程技术研究中心, 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:2022-09-16 接受日期:2023-02-25 出版日期:2023-09-01 发布日期:2023-09-21
通讯作者: *E-mail: jxshen@mail.hzau.edu.cn
基金资助:
湖北省重点研发计划(2022ABA001)

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

Zhang Yingchuan¹, Wu Xiaomingyu¹, Tao Baolong¹, Chen Li¹^,², Lu Haiqin¹, Zhao Lun¹, Wen Jing¹, Yi Bin¹, Tu Jinxing¹, Fu Tingdong¹, Shen Jinxiong¹^,^*()

1. National Engineering Research Center of Rapeseed/National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
2. School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Chongqing 408100, China

Received:2022-09-16 Accepted:2023-02-25 Online:2023-09-01 Published:2023-09-21
Contact: *E-mail: jxshen@mail.hzau.edu.cn

摘要/Abstract

摘要： miRNA通过调控靶基因的表达参与植物生长发育和响应逆境胁迫等多个方面。对前期鉴定到的1个miRNA——Bna-miR43进行功能研究, 通过构建过表达载体探讨了Bna-miR43在甘蓝型油菜(Brassica napus)响应干旱胁迫中的功能。降解组测序预测到Bna-miR43的4个靶基因均属于F-box蛋白家族。在模拟干旱条件下, 甘蓝型油菜J572根系Bna-miR43的表达量逐渐下降; 靶基因则呈现与Bna-miR43相反的表达模式, 且随着干旱处理时间的增加, 靶基因的表达量逐渐上升。转基因实验表明, 在干旱胁迫下, 油菜Bna-miR43过表达株系表现为对干旱极度敏感, 转基因株系种子在干旱条件下发芽率显著下降, 植株脱水严重, 体内积累了更多的丙二醛(MDA)和过氧化氢(H₂O₂)。干旱处理后, Bna-miR43过表达株系中编码超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和乙醇酸氧化酶(GOX)的3个基因下调表达。上述结果表明, Bna-miR43通过调控靶基因的表达调节甘蓝型油菜渗透物质积累和细胞活性氧(ROS)稳态, 在调控植物耐旱性中发挥重要作用。

关键词: 甘蓝型油菜, Bna-miR43, F-box, 干旱胁迫

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

张盈川, 吴晓明玉, 陶保龙, 陈丽, 鲁海琴, 赵伦, 文静, 易斌, 涂金星, 傅廷栋, 沈金雄. Bna-miR43介导甘蓝型油菜响应干旱胁迫. 植物学报, 2023, 58(5): 701-711.

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. Chinese Bulletin of Botany, 2023, 58(5): 701-711.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22223

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

图/表 8

表1 荧光定量PCR引物序列

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

图1 Bna-miR43及其靶基因的基本信息 (A) Bna-miR43前体二级结构预测; (B) Bna-miR43及其靶基因之间的碱基互补配对; (C) BnaA09g03940D的结构域注释; (D) BnaCnng24950D的结构域注释

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

图2 靶基因及拟南芥同源基因启动子顺式作用元件预测

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

图3 靶基因在甘蓝型油菜中的表达模式 (A) BnaA09g03940D的表达模式; (B) BnaCnng24950D的表达模式

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

图4 干旱胁迫下甘蓝型油菜J572中Bna-miR43及其靶基因的表达模式 (A) Bna-miR43的表达模式; (B) BnaA09g03940D的表达模式; (C) BnaCnng24950D的表达模式。实验设3次生物学重复, 3次技术重复。* P<0.05; **P<0.01

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

图5 干旱胁迫下甘蓝型油菜种子发芽率统计(A)及PEG模拟干旱处理下过表达Bna-miR43转基因植株地上部表型(B) J572: 对照; OE-2、OE-3和OE-5为不同转基因株系; ** P<0.01; Bars=1 cm

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

图6 过表达Bna-miR43油菜根系干旱胁迫后丙二醛(MDA) (A)和过氧化氢(H2O2) (B)含量分析 Before: 干旱胁迫处理前; After: 干旱胁迫处理后。实验设3次生物学重复。J572、OE-2、OE-3和OE-5同图5。* P<0.05

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

图7 过表达Bna-miR43油菜中氧化应激反应和ABA信号通路基因表达量分析 WT: 野生型; OE-2、OE-3和OE-5同图5。* P<0.05; ** P<0.01

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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Bna-miR43介导甘蓝型油菜响应干旱胁迫

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

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