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作物学报 ›› 2022, Vol. 48 ›› Issue (12): 3071-3079.doi: 10.3724/SP.J.1006.2022.14227

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

Bna-miR171g提高甘蓝型油菜耐渗透胁迫能力的功能鉴定

杨文静(), 陆海芹, 陈吴钧, 曾蕾, 谢涛, 蒋金金(), 王幼平   

  1. 扬州大学生物科学与技术学院, 江苏扬州 225009
  • 收稿日期:2021-12-04 接受日期:2022-03-25 出版日期:2022-12-12 网络出版日期:2022-04-20
  • 通讯作者: 蒋金金
  • 作者简介:E-mail: wjyang1205@126.com
  • 基金资助:
    国家自然科学基金项目(31972963);国家自然科学基金项目(31771825);,国家重点基础研究发展计划项目(2018YFE0108000);江苏省研究生实践创新计划项目(SJCX21_1602)

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 Published:2022-12-12 Published online:2022-04-20
  • Contact: JIANG Jin-Jin
  • 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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摘要：

MicroRNA171 (miR171)是植物中非常保守的miRNA分子, 在调控植物生长发育方面具有重要的功能。关于其功能研究的报道多见于拟南芥、水稻、番茄等植物, 而甘蓝型油菜中miR171的功能研究还尚未报道。本研究发现Bna-miR171家族7个成员的序列高度保守, 其中miR171g受甘露醇诱导上调表达。在150 mmol L-1甘露醇模拟的渗透胁迫条件下, Bna-miR171g过表达油菜(OE-miR171g)的根长显著高于对照(J9712)。DAB组织化学染色结果表明渗透胁迫后OE-miR171g中过氧化氢的积累量低于J9712, 推测Bna-miR171g过表达材料的活性氧清除能力高于对照植株。渗透胁迫后OE-miR171g中脯氨酸含量、过氧化物酶和超氧化物歧化酶活性均高于对照, 而丙二醛含量显著低于对照。此外, 胁迫应答基因ABI5、ERD10、RAB18、OSR1、RD20、RD29B在OE-miR171g中的表达水平均高于对照植株。综上, 过表达Bna-miR171g可以提高甘蓝型油菜对渗透胁迫的耐受性, 本研究可以为甘蓝型油菜的抗逆性状改良提供理论依据。

关键词: 甘蓝型油菜, Bna-miR171g, 甘露醇, 渗透胁迫

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

表1

本研究所用引物序列"

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

图1

Bna-miR171序列的系统发生分析 A: miR171成熟序列的保守性分析(从上到下分别为拟南芥、水稻、玉米、白菜型油菜、甘蓝和甘蓝型油菜的序列); B: miR171前体序列的系统发生分析。Bna: 甘蓝型油菜; Ath: 拟南芥; Osa: 水稻; Zma: 玉米; Bra: 白菜型油菜; Bol: 甘蓝。"

图2

渗透胁迫条件下Bna-miR171g的表达分析"

图3

渗透胁迫下Bna-miR171g过表达植株的表型 A: 渗透胁迫下OE-miR171和J9712的表型; B: 根长; C: 下胚轴长; D: 鲜重; E: Bna-miR171g的表达水平; F: 子叶的DAB染色。数值表示为平均值±标准误, n = 20; *: P < 0.05; **: P < 0.01。"

图4

渗透胁迫下Bna-miR171g过表达植株和对照的生理指标 MDA: 丙二醛; Pro: 脯氨酸; POD: 过氧化物酶; SOD: 超氧化物歧化酶。*: P < 0.05; **: P < 0.01。"

图5

Bna-miR171g过表达植株和对照中胁迫应答基因的表达量分析"

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