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作物学报 ›› 2022, Vol. 48 ›› Issue (7): 1635-1644.doi: 10.3724/SP.J.1006.2022.14106

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

Bna-novel-miR36421调节拟南芥株型和花器官发育的功能验证

戴丽诗1,2(), 常玮1,2(), 张赛1,2, 钱明超1,2, 黎小东1,2, 张凯1,2, 李加纳1,2,3, 曲存民1,2,3,*(), 卢坤1,2,3,*()   

  1. 1西南大学农学与生物科技学院, 重庆 400715
    2南方山地农业教育部工程研究中心, 重庆 400715
    3西南大学农业科学研究院, 重庆 400715
  • 收稿日期:2021-06-22 接受日期:2021-10-19 出版日期:2022-07-12 网络出版日期:2021-11-03
  • 通讯作者: 曲存民,卢坤
  • 作者简介:戴丽诗, E-mail: dls375684@163.com
    常玮, E-mail: changwei1919@163.com第一联系人：

    ** 同等贡献

  • 基金资助:
    国家自然科学基金项目(31871653);国家重点研发计划项目(2018YFD0100500);高等学校学科创新引智基地项目“111项目”(B12006);重庆市自然科学基金重点项目;西南大学种质创制专项资助

Functional validation of Bna-novel-miR36421 regulating plant architecture and flower organ development in Arabidopsis thaliana

DAI Li-Shi1,2(), CHANG Wei1,2(), ZHANG Sai1,2, QIAN Ming-Chao1,2, LI Xiao-Dong1,2, ZHANG Kai1,2, LI Jia-Na1,2,3, QU Cun-Min1,2,3,*(), LU Kun1,2,3,*()   

  1. 1College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, China
    3Academy of Agricultural Sciences, Southwest University, Chongqing, 400715, China
  • Received:2021-06-22 Accepted:2021-10-19 Published:2022-07-12 Published online:2021-11-03
  • Contact: QU Cun-Min,LU Kun
  • About author:First author contact:

    ** Contributed equally to this work

  • Supported by:
    National Natural Science Foundation of China(31871653);National Key Research and Development Plan(2018YFD0100500);Project of Intellectual Base for Discipline Innovation in Colleges and Universities “the 111 Project”(B12006);Key Project of Natural Science Foundation;Germplasm Creation Special Program of Southwest University

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摘要：

MicroRNA参与油菜种子发育、胁迫响应和胚胎发育等多种生物学过程, 但其在油菜株型和花器官发育方面的报道还较少。本研究以高/低收获指数油菜中显著差异表达的Bna-novel-miR36421为对象, 通过转基因植株表型、靶基因预测、表达量和双荧光素酶报告系统等解析其调控机制。Bna-novel-miR36421与植物miR167家族成员高度同源, 可能为油菜新的miR167成员。Bna-novel-miR36421能靶向并抑制Bna.C03ARF6、Bna.C06ARF8、Bna.A09PATL2和Bna.C03DUF581的表达。过表达拟南芥植株中, Bna-novel-miR36421的表达量显著上调, 而上述4个靶基因的拟南芥同源基因表达量极显著下降。过表达拟南芥植株株型矮小, 茎间缩短, 叶片高度卷曲; 花器官发育异常, 雌蕊膨大, 雄蕊花丝缩短, 花药不开裂, 花粉败育。推测Bna-novel-miR36421可能通过抑制Bna.C03ARF6、Bna.C06ARF8、Bna.A09PATL2和Bna.C03DUF581基因表达, 进而对油菜株型和花器官的发育起到重要的调控作用。研究结果对解析miRNA介导的植物株型和花器官发育分子机制, 挖掘重要发育性状关键基因奠定了重要基础。

关键词: 甘蓝型油菜, Bna-novel-miR36421, miR167, 株型, 花器官

Abstract:

MicroRNA is involved in the regulation of various biological processes such as development, stress response, and embryonic development in rapeseed, however, only few studies focused on the regulation of miRNA on the plant architecture and floral organ development of rapeseed. In this study, Bna-novel-miR36421 that differentially expressed between the high and low harvest index accessions of rapeseed was identified, whose biological function and regulation mechanism were further characterized through phenotype analysis in transgenic plants, target gene prediction, expression pattern comparison, and dual luciferase reporter system. The results showed that Bna-novel-miR36421 was highly homologous to miR167 family members, and might be a novel miR167 member in rapeseed. The qRT-PCR and dual luciferase reporter system results indicated that Bna-novel-miR36421 could inhibit the relative expression levels of Bna.C03ARF6, Bna.C06ARF8, Bna.A09PATL2, and Bna.C03DUF581. In the overexpressing Arabidopsis plants, the expression of Bna-novel-miR36421 was significantly increased, while the transcription levels of the Arabidopsis orthologs of its target genes were decreased significantly. Phenotypic observation showed that the plant height of overexpression Arabidopsis plants were reduced, with shortened stems and curled leaves. The development of floral organs was abnormal, with enlarged pistil, shortened stamen filaments, unbreakable anthers, and aborted pollens. Hence, it could be proposed that Bna-novel-miR36421 may regulate plant architecture and floral organ development by repressing Bna.C03ARF6, Bna.C06ARF8, Bna.A09PATL2, and Bna.C03DUF581. The results laid a solid foundation for understanding the molecular mechanism of miRNA-mediated plant architecture and flower organ development, and mining the key genes involved in plant developmental processes.

Key words: Brassica napus, Bna-novel-miR36421, miRNA167, plant architecture, flower organ

附表1

Bna-novel-miR36421的成熟体和前体序列"

类别
Type		 序列
Sequence (5'-3')
成熟体序列
Mature sequence		 TAAGCTGCCAGCATGATCTTG
前体序列-1
Precursor sequence-1		 TAAGCTGCCAGCATGATCTTGTCTTCCTCTCCTAAGCTTCATATATAT
AACTAAGCTAAGGAAATAAATAATTTTCTCGTTCTCATAAGATTATAT
GATAATAGCTTAGAGAGAGAGAGACTAGGTCATGCTGGTAGCTTCAC
前体序列-2
Precursor sequence-2		 TAAGCTGCCAGCATGATCTTGTCTTCCTCTCTTAAGCTTCATATATAAC
TAAGCTAAGGAATAATATAATTTTCTTGTTCTCATAAGAATATATGATAA
TAGCTTAGAGAGAGAGAGAGAGAGACTAGGTCATGCTGGTAGTTTCAC

附表2

本研究中所用引物"

引物名称
Prime name		 引物序列
Primer sequence (5'-3')
62sk-miR36421F taagcttgatatcgaattcCAATATGAGATTTCGCAGTGACT
62sk-miR36421R cgctctagaactagtggatccCAAACACAACTAACCTTC
0800-C03ARF6F ttctagagcggccgcggatccGGTACAATGACGACACCTTCTAG
0800-C03ARF6R actggtgatttcagcgaattcTCCCCAAGTCATCACAGTTC
0800-C06ARF8F ttctagagcggccgcggatccCTGGATTTCAGAACACTTTGC
0800-C06ARF8R actggtgatttcagcgaattcGAAATGGGTGAGGTTCTGTG
0800-C03DUF581F ttctagagcggccgcggatccATGACTAAAATCTCTGTTGG
0800-C03DUF581R actggtgatttcagcgaattcAGGAACTATAAATAGCTGGCGT
0800-A09PATL2F ttctagagcggccgcggatccTCAGGAGCTACATATTTGAATATGG
0800-A09PATL2R actggtgatttcagcgaattcGACACGTTGTGATCTACAGCTCGT
OVmiR36421F caccAGTGACTAAGAAAGTTACCGAGGG
OVmiR36421R AGTGACTAAGAAAGTTACCGAGGG
F35S3ND GGAAGTTCATTTCATTTGGAGAG
OCS5ND CGATCATAGGCGTCTCGCATATCTC
F BAR CGACATCCGCCGTGCCACCGA
R BAR GTACCGGCAGGCTGAAGTCCAGC
BnaActin7F TGGGTTTGCTGGTGACGAT
BnaActin7R TGCCTAGGACGACCAACAATACT

图1

Bna-novel-miR36421 前体成员二级茎环结构预测 A: Bna-novel-MIR36421-1二级茎环结构, dG = -26.500 kcal mol-1; B: Bna-novel-MIR36421-2二级茎环结构, dG = -63.90 kcal mol-1。"

图2

Bna-novel-miR36421 与甘蓝型油菜Bna-miR167家族成员成熟及前体miRNA的序列比对 A: 成熟miRNA序列比对; B: 前体miRNA序列比对。"

图3

过表达Bna-novel-miR36421转基因拟南芥表达水平及表型观察 A: 过表达Bna-novel-miR36421拟南芥中基因表达水平检测; B: 过表达Bna-novel-miR36421转基因拟南芥表型观察; C: 过表达Bna-novel-miR36421转基因拟南芥表型数据统计。*、**和****分别表示在0.05、0.01和0.0001水平上显著差异; NS表示差异不显著。"

图4

Bna-novel-miR36421及其靶基因在不同材料及不同组织部位中的表达水平分析 P130: 甘蓝型油菜高收获指数极端材料; P202: 甘蓝型油菜低收获指数极端材料; 14dSe: 14 d种子; 14dSp: 14 d角果皮; 28dSe: 28 d种子; 28dSp: 28 d角果皮。**表示在0.01水平上显著差异。"

图5

Bna-novel-miR36421靶向负调控其靶基因 A: 双荧光素酶系统验证Bna-novel-miR36421与靶基因的关系; B: Bna-novel-miR36421及其靶基因表达水平检测。**表示在0.01水平上显著差异。"

图6

甘蓝型油菜Bna-novel-miR36421调节株型及花器官发育的可能模型"

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