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作物学报 ›› 2022, Vol. 48 ›› Issue (8): 1938-1947.doi: 10.3724/SP.J.1006.2022.14155

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

Bna.C02SWEET15通过光周期途径正向调控油菜开花时间

李胜婷1,**(), 徐远芳1,**(), 常玮1, 刘亚俊2, 谷嫄2, 朱红1, 李加纳1,3,4, 卢坤1,3,4,*()   

  1. 1西南大学农学与生物科技学院, 重庆 400715
    2云南省临沧市农业技术推广站, 云南临沧 677099
    3南方山地农业教育部工程研究中心, 重庆 400715
    4西南大学农业科学研究院, 重庆 400715
  • 收稿日期:2021-08-26 接受日期:2021-11-29 出版日期:2022-08-12 网络出版日期:2021-12-16
  • 通讯作者: 卢坤
  • 作者简介:李胜婷, E-mail: lishengting0123@163.com;
    徐远芳, E-mail: 1095245283@qq.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(B12006);重庆市自然科学基金项目(cstc2021ycjh-bgzxm0033);重庆市研究生科研创新项目(CYB21115);西南大学种质创制专项资助。

Bna.C02SWEET15 positively regulates the flowering time of rapeseed through photoperiodic pathway

LI Sheng-Ting1,**(), XU Yuan-Fang1,**(), CHANG Wei1, LIU Ya-Jun2, GU Yuan2, ZHU Hong1, LI Jia-Na1,3,4, LU Kun1,3,4,*()   

  1. 1College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2Agricultural Technology Extension Station in Lincang City, Lincang 677099, Yunnan, China
    3Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, China
    4Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China
  • Received:2021-08-26 Accepted:2021-11-29 Published:2022-08-12 Published online:2021-12-16
  • Contact: 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 Program of China(2018YFD0100500);Project of Intellectual Base for Discipline Innovation in Colleges and Universities(B12006);Key Project of Chongqing Natural Science Foundation(cstc2021ycjh-bgzxm0033);Chongqing Postgraduate Research Innovation Project(CYB21115);Germplasm Creation Special Program of Southwest University

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摘要：

开花时间是作物的重要农艺指标, 关系着作物的生育周期和产量。糖转运体作为植物重要的碳水化合物运输载体, 作用于油菜开花时间的研究鲜有报道。本研究在油菜FOX-Hunting文库中鉴定了一个与油菜开花相关的蔗糖转运蛋白Bna.C02SWEET15, 通过组织表达和亚细胞定位分析, 转基因和突变体表型观察, 开花关键基因表达水平检测等解析其生物学功能与调控机制。Bna.C02SWEET15在油菜各组织部位均能够表达, 在花后30 d种子中最显著。Bna.C02SWEET15定位于细胞膜, 启动子活性主要在花药和花后30 d种子中。在拟南芥中过表达Bna.C02SWEET15使植株开花提前, 利于植株开花的光周期关键基因CO、FT、LFY表达明显上升, 负调控基因FLC表达量降低。拟南芥突变体atsweet15a和RNAi-Bna.C02SWEET15转基因油菜均为晚花表型。推测Bna.C02SWEET15能够通过光周期途径正向调控油菜开花时间影响油菜的生育周期。研究结果对理解糖转运体在作物中的调控作用, 为油菜高产育种提供了基因资源, 奠定了理论基础。

关键词: 甘蓝型油菜, Bna.C02SWEET15, 光周期, 开花

Abstract:

Flowering time, an important agronomic index of crops, is related to the growth cycle and the yield of crops. Sugar transporters are important carbohydrate transporters in plants, whereas there are few reports on the effect of sugar transporters on flowering time in rapeseed (Brassica napus). In this study, sucrose transporter Bna.C02SWEET15 related to flowering in rapeseed were screened and identified from Fox-Hunting library, whose biological function and regulation mechanism were further characterized through expression pattern and subcellular localization analysis, phenotype analysis in transgenic plants and mutant, detection of marker genes expression related to flowering. Bna.C02SWEET15 could be identified in all tissues of Brassica napus, and the relative expression level was the most significant in seeds at 30 days after flowering. Bna.C02SWEET15 was located in cell membrane, and the promoter activity was mainly in anther and seed at 30 days after flowering. Overexpression of Bna.C02SWEET15 in Arabidopsis resulted in flowering in advance and increased the expression of photoperiod key genes CO, FT, and LFY, while decreased the expression of negative regulation gene FLC. In addition, the flowering time of atsweet15a and transgenic RNAi-Bna.C02SWEET15 were later. We proposed that Bna.C02SWEET15 could positively regulate the flowering time through photoperiod pathway, thus affecting the growth cycle and final yield in rapeseed. These results provide important basis for understanding the regulatory role of sugar transporters in crops and lay a theoretical foundation for understanding the regulatory role of sugar transporters in crops.

Key words: Brassica napus, Bna.C02SWEET15, photoperiod, flowering

附表1

本研究中所用引物"

引物名称
Prime name		 引物序列
Primer sequences (5'-3')
26S-F rRNA CACAATGATAGGAAGAGCCGAC
26S-R rRNA CAAGGGAACGGG CTTGGCAGAATC
BnaActin7-F TGGGTTTGCTGGTGACGAT
BnaActin7-R TGCCTAGGACGACCAACAATACT
qRT F-BnaSWEET15 TCCTTGTATTCCTCGCTCCC
qRT R-BnaSWEET15 CAGCCGAAAGAGTTGATGGT
OV-SWEET15 F CACCATGAGTTTGGCACACCAAAT
OV-SWEET15 R ATGCCTGAGAGCTTCTTGCAC
Bna.C02SWEET15-PRO-F acctgcaggcatgcaACAGACCTCGTTTCTATAGCTATCT
Bna.C02SWEET15-PRO-R taccctcagatctaccatggAAACTAATGAGTCTCAGGTGGTGT
F Hyg CTATTTCTTTGCCCTCGGACGA
R Hyg ATGAAAAAGCCTGAACTCACCGCG
F35S3ND GGAAGTTCATTTCATTTGGAGAG
OCS5ND CGATCATAGGCGTCTCGCATATCTC
F Bar CGACATCCGCCGTGCCACCGA
R Bar GTACCGGCAGGCTGAAGTCCAGC
Actin real F GGTAACATTGTGCTCAGTGGTGG
Actin real R AACGACCTTAATCTTCATGCTGC
qRT F-AtSWEET15 CATAGCCATGTTCTTCGCTTAC
qRT R-AtSWEET15 CTTTGTCTTTATCACACGAGCC
M13pF ACACAGGAAACAGCTATGACCAT
M13pR GGGTCCTAACCAAGAAAATGAA
RNAi-SWEET15-F GGATCCGACGTCTCTCCCATACCAACATTCATAACA
RNAi-SWEET15-R TCTAGACCATGGATAGTATAAGCTGTACTACT
BnaPAP2-F GGATCCACCTAAGCATGCATTTGAAAA
BnaPAP2-R ATTTAAATGACGTCAGGTTTACATTCAAGACACA
引物名称
Prime name		 引物序列
Primer sequences (5'-3')
F35S3N GGAAGTTCATTTCATTTGGAGAG
ROCST5N GCTCAGGTTTTTTACAACGTGCAC
CO real F GCCATCAGCGAGTTCCAATTCTAC
CO real R CCTTCCTCTTGATCCACCACCAG
FLC real F CGTCGCTCTTCTCGTCGTCTC
FLC real R TTCGGTCTTCTTGGCTCTAGTCAC
FT real R GCCTGCCAAGCTGTCGAAACAATA
FT real F TCCCTGCTACAACTGGAACAACCT
LFY real F TCCACTGCCTAGACGAAGAAGC
LFY real R TCCCAGCCATGACGACAAGC
N674321-LP CAGCCGAGTACAGAGACTTGG
N674321-RP TTTCCTCGCTTTTATCTTCGG
LBb1.3 ATTTTGCCGATTTCGGAAC

图1

甘蓝型油菜Bna.C02SWEET15的组织表达分析 A: qRT-PCR检测甘蓝型油菜中Bna.C02SWEET15的表达模式。R: 根; St: 茎; L: 叶; F: 花; MB: 主蕾; 5D-S、10D-S: 花后5 d和10 d角果; 15D-SE、21D-SE、30D-SE: 花后15、21和30 d的种子; 15D-SW、21D-SW、30D-SW: 花后15、21和30 d的种皮。B: proBna.C02WEET15::GUS的组织化学染色。a: 萌发种皮; b: 幼苗; c: 莲座; d: 茎叶; e: 花药; f: 角果; g: 根; h: 30 d种子。"

图2

Bna.C02SWEET15-YFP融合蛋白的亚细胞定位 标尺为20 μm。"

图3

Bna.C02SWEET15过表达转基因植株表型观察及标志基因表达量检测 A: Bna.C02SWEET15转基因植株表型观察; 标尺为1 cm。B: qRT-PCR检测转基因植株基因的表达量; C: Bna.C02SWEET15转基因植株开花时间统计; D: 开花途径标志基因表达量检测。*、**分别表示在0.05和0.01水平上显著差异。"

图4

拟南芥突变体atsweet15表型观察 A: AtSWEET15和Bna.C02SWEET15序列相似性; B: 拟南芥突变体T-DNA插入位点示意图; C: atsweet15a表型观察; D: qRT-PCR检测突变体中基因表达量。标尺为1 cm。**表示在0.01水平上显著差异。"

图5

RNAi-Bna.C02SWEET15转基因油菜表型观察 A: RNAi-Bna.C02SWEET15表型观察; B: qRT-PCR检测转基因植株基因表达量; C: RNAi-Bna.C02SWEET15转基因植株开花时间统计。标尺为10 cm。**表示在0.01水平上显著差异。"

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