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作物学报 ›› 2023, Vol. 49 ›› Issue (5): 1211-1221.doi: 10.3724/SP.J.1006.2023.24079

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

Bna-miR43-FBXL调控模块参与甘蓝型油菜铝胁迫的功能分析

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. 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, 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:2022-04-02 接受日期:2022-07-21 出版日期:2023-05-12 网络出版日期:2022-08-19
  • 通讯作者: *沈金雄, E-mail: jxshen@mail.hzau.edu.cn
  • 作者简介:E-mail: yczhang612@163.com
  • 基金资助:
    国家自然科学基金项目(31871654)

Functional analysis of Bna-miR43-FBXL regulatory module involved in aluminum stress in Brassica napus

ZHANG Ying-Chuan1(), WU Xiao-Ming-Yu1, TAO Bao-Long1, CHEN Li1,2, LU Hai-Qin1, ZHAO Lun1, WEN Jing1, YI Bin1, TU Jing-Xing1, FU Ting-Dong1, SHEN Jin-Xiong1,*()   

  1. 1National Key Laboratory of Crop Genetic Improvement/National Engineering Research Center of Rapeseed, Huazhong Agricultural University, Wuhan 430070, Hubei, China,
    2School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Chongqing 408100, China
  • Received:2022-04-02 Accepted:2022-07-21 Published:2023-05-12 Published online:2022-08-19
  • Contact: *E-mail: jxshen@mail.hzau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31871654)

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摘要：

在酸性土壤中, 铝是制约作物生长和产量的一个重要因素, 如何利用酸性土地意义重大。本研究的对象是前期本课题鉴定到的一个未被报道的miRNA——Bna-miR43。系统地对甘蓝型油菜中Bna-miR43及其靶基因进行生物信息学分析以及表达模式鉴定, 并构建Bna-miR43过表达载体探讨了Bna-miR43-FBXL模块在甘蓝型油菜响应铝胁迫的分子机制。5°-RACE结果表明Bna-miR43在甘蓝型油菜中真实切割BnaA09g03940D、BnaCnng24950D。生物信息学分析表明, BnaA09g03940D、BnaCnng24950D在拟南芥中的同源基因为AT5G27950, 编码含F-box的E3泛素连接酶。qRT-PCR结果显示, Bna-miR43及其靶基因在甘蓝型油菜不同组织以及铝胁迫瞬时处理下的表达水平存在此消彼长的现象。油菜转基因试验表明, Bna-miR43的过表达株系地上部分长势良好, 体内积累了较少的MDA和H2O2, 同时转基因植株根系的铝积累量较对照少。本研究结果为甘蓝型油菜中铝胁迫响应提供参考。

关键词: 甘蓝型油菜, Bna-miR43, F-box, 铝胁迫

Abstract:

Aluminum is one of the main factors that limited crop growth and yield in acid soil and how to utilize acid soil is of great significance. This study is based on Bna-miR43, an unreported miRNA identified in the previous study. We analyzed the molecular characteristics, conserved structure, phylogenetic analysis, and the expression level of Bna-miR43 and its target genes in Brassica napus. The Bna-miR43 over expression vector was constructed to explore the molecular mechanism of Bna-miR43- FBXL module in Brassica napus in aluminum stress. 5'-RACE showed that Bna-miR43 actually cleaved BnaA09g03940D and BnaCnng24950D. Bioinformatics analysis revealed that the homologous gene of BnaA09g03940D and BnaCnng24950D in Arabidopsis was AT5G27950, encoding E3 ubiquitin ligase contained F-box. The qRT-PCR indicated that the relative expression levels of Bna-miR43 and its target genes had a phenomenon of trade-off in different tissues of Brassica napus and under transient treatment of aluminum stress. Aluminum treatment showed that the entire aerial part of the transgenic plant grew much better than control and accumulated less MDA and H2O2. Meanwhile, the aluminum accumulation in the root of transgenic plants was less than the control. In this study, these results may provide reference for the response of aluminum stress in Brassica napus.

Key words: Brassica napus L., Bna-miR43, F-box, aluminum stress

附表1

Bna-miR43的前体和成熟体序列"

Bna-miR43 序列 Sequence (5°-3°)
前体序列
Precursor sequence		 AGTTTAGGTCGAAGATTGGAACATTGGGACCGTGCCCTATTACAAACTTACACACATTGCAAGCATATTTCTATGCTCATACATATGATCAATATAGCCTTCTATCTTAAGTCAAGTATTGTATAATTTAATTTTGTTATACACTAATTTAGATCATGAAGAGTCTGGATTTTTAGTTTTTTTTTCTACAAT
成熟体序列
Mature sequence		 GAAGATTGGAACATTGGGACC

图1

Bna-miR43前体结构预测图 箭头所指处为Bna-miR43成熟体序列。"

表1

Bna-miR43降解组预测靶基因情况概述"

靶基因
Target gene		 拟南芥同源基因
Arabidopsis homologous gene		 靶基因功能
Function of target genes		 类别
Category
BnaA09g03940D AT5G27920 F-box蛋白家族 F-box family protein 0
BnaCnng24950D AT5G27920 F-box蛋白家族 F-box family protein 0
BnaA06g06260D AT5G27920 F-box蛋白家族 F-box family protein 0
BnaC05g08010D AT5G27920 F-box蛋白家族 F-box family protein 0

图2

RLM-RACE验证靶基因定向切割位点 图中箭头表示切割位点, 数字表示挑斑克隆验证目标mRNA切割位点的克隆频率。"

图3

油菜靶基因和拟南芥同源基因序列比对结果"

图4

油菜FBXL基因家族进化分析 A: 油菜FBXL基因家族系统发育进化树; B: F-box家族的基因结构与motif。"

图5

FBXL亚族基因序列同一性分析"

图6

Bna-miR43及其靶基因在甘蓝型油菜不同组织中的表达分析 每次试验3次生物学重复, 3次技术重复。*和**分别表示显著水平(P < 0.05)与极显著水平(P < 0.01)。"

图7

Bna-miR43及其靶基因表达量分析 A: 铝胁迫下甘蓝型油菜J572中Bna-miR43及其靶基因的表达模式; B: 过表达Bna-miR43转基因油菜基因表达量分析。每次试验3次生物学重复, 3次技术重复。*和**分别表示显著水平(P < 0.05)与极显著水平(P < 0.01)。"

图8

铝处理J572和Bna-miR43过表达转基因植株表型鉴定 A: 未进行铝处理油菜植株地上部分表型分析; B: 铝处理后的油菜植株地上部分表型分析, 随机取5株同一株系不同油菜植株的倒一叶照相, 其中地上部分表型图标尺为5 cm, 倒一叶图标尺为2 cm; C: 苏木精染色鉴定根中铝分布。-Al3+: J572在0.5 mmol L-1 CaCl2 (pH 4.5)的培养液中培养1周后, 使用苏木精染色, 体式显微镜观察拍照。+Al3+: 转基因油菜与J572在0.5 mmol L-1 CaCl2, 300 μmol L-1 AlCl3 (pH 4.5)培养液中处理1周后, 使用苏木精染色, 体式显微镜观察照相, 左侧图标尺为1 mm, 右侧图标尺为0.5 mm。"

图9

过表达Bna-miR43油菜根系铝胁迫后生理生化指标分析 A: 过表达Bna-miR43油菜根系铝胁迫后MDA含量分析; B: 过表达Bna-miR43油菜根系铝胁迫后过氧化氢含量分析。每次试验3次生物学重复, 3次技术重复。*和**分别表示显著水平(P < 0.05)与极显著水平(P < 0.01)。"

图10

Bna-miR43的可能作用途径"

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[2] 王兰珍;米国华;陈范骏;张福锁. 不同产量结构小麦品种对缺磷反应的分析[J]. 作物学报, 2003, 29(06): 867 -870 .
[3] 杨建昌;张亚洁;张建华;王志琴;朱庆森. 水分胁迫下水稻剑叶中多胺含量的变化及其与抗旱性的关系[J]. 作物学报, 2004, 30(11): 1069 -1075 .
[4] 袁美;杨光圣;傅廷栋;严红艳. 甘蓝型油菜生态型细胞质雄性不育两用系的研究Ⅲ. 8-8112AB的温度敏感性及其遗传[J]. 作物学报, 2003, 29(03): 330 -335 .
[5] 王永胜;王景;段静雅;王金发;刘良式. 水稻极度分蘖突变体的分离和遗传学初步研究[J]. 作物学报, 2002, 28(02): 235 -239 .
[6] 王丽燕;赵可夫. 玉米幼苗对盐胁迫的生理响应[J]. 作物学报, 2005, 31(02): 264 -268 .
[7] 田孟良;黄玉碧;谭功燮;刘永建;荣廷昭. 西南糯玉米地方品种waxy基因序列多态性分析[J]. 作物学报, 2008, 34(05): 729 -736 .
[8] 胡希远;李建平;宋喜芳. 空间统计分析在作物育种品系选择中的效果[J]. 作物学报, 2008, 34(03): 412 -417 .
[9] 王艳;邱立明;谢文娟;黄薇;叶锋;张富春;马纪. 昆虫抗冻蛋白基因转化烟草的抗寒性[J]. 作物学报, 2008, 34(03): 397 -402 .
[10] 郑希;吴建国;楼向阳;徐海明;石春海. 不同环境条件下稻米组氨酸和精氨酸的胚乳和母体植株QTL分析[J]. 作物学报, 2008, 34(03): 369 -375 .
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