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作物学报 ›› 2018, Vol. 44 ›› Issue (03): 397-404.doi: 10.3724/SP.J.1006.2018.00397

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝转录因子BoLH27的克隆与转基因甘蓝的表型分析

梁云飞1,*, 张林成1,*, 蒲全明2, 雷镇泽1, 施松梅1, 姜宇鹏1, 任雪松1, 高启国1,*()   

  1. 1西南大学园艺园林学院 / 南方山地园艺学教育部重点实验室, 重庆 400716
    2南充市农业科学院蔬菜研究所, 四川南充 637000
  • 收稿日期:2017-06-06 接受日期:2017-11-21 出版日期:2018-03-12 网络出版日期:2017-12-11
  • 通讯作者: 梁云飞,张林成,高启国
  • 作者简介:

    18847123096@163.com

  • 基金资助:
    本研究由国家重点基础研究发展计划项目(973计划)(2012CB113900)和国家自然科学基金项目(30900986)资助

Cloning of BoLH27 Gene from Cabbage and Phenotype Analysis of Transgenic Cabbage

Yun-Fei LIANG1,**, Lin-Cheng ZHANG1,**, Quan-Ming PU2, Zhen-Ze LEI1, Song-Mei SHI1, Yu-Peng JIANG1, Xue-Song REN1, Qi-Guo GAO1,*()   

  1. 1 College of Horticulture and Landscape Architecture, Southwest University / Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education, Chongqing 400716, China
    2 Institute of Vegetables, Nanchong Academy of Agricultural Sciences, Nanchong 637000, Sichuan, China
  • Received:2017-06-06 Accepted:2017-11-21 Published:2018-03-12 Published online:2017-12-11
  • Contact: Yun-Fei LIANG,Lin-Cheng ZHANG,Qi-Guo GAO
  • Supported by:
    This study was supported by the National Program on Key Basic Research Project (973 program)(2012CB113900) and the National Natural Science Foundation of China (30900986).

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摘要：

bHLH转录因子对植物生长发育、形态调控有重要作用, 为了研究BoLH27基因在甘蓝叶片发育及形态建成中的调控功能, 本文以甘蓝品种519为材料, 克隆出转录因子BoLH27基因。序列分析表明, 该基因含有一个795 bp的开放阅读框, 编码264个氨基酸, 具有一个保守的典型bHLH结构域。以农杆菌介导的遗传转化方法将正义BoLH27基因导入甘蓝品种519中以增强表达, 通过PCR筛选出9株T0代转基因单株, 结合T2代单株的qRT-PCR分析表明, 筛选的转基因植株内BoLH27基因的表达积累量明显高于野生型。试验基地隔离网内种植的转基因甘蓝表型明显, 主要表现为莲座期茎叶间距拉长、叶柄伸长以及植株茎和叶柄显示紫色, 植株叶片平展, 无向上向内卷曲趋势, 表明BoLH27基因可能对甘蓝叶片发育有重要的调控作用。

关键词: 甘蓝, BoLH27, 基因克隆, 转基因, 表型

Abstract:

bHLH transcription factor plays an important role in plant growth, development and morphological control. In order to explore BoLH27 gene regulatory function for leaf development and morphologic formation, the BoLH27 gene was cloned from cabbage (Brassica oleracea L.) variety 519. Sequence analysis indicated that the length of BoLH27 gene was 795 bp, which encoded 264 amino acids. The BoLH27 protein contained conservative structure domains of the bHLH family. The sense BoLH27 gene was transformed into cabbage variety 519 by Agrobacterium mediated method, PCR analysis exhibited that BoLH27 was genetically transformed into nine individual plants, qRT-PCR analysis revealed that BoLH27 had higher expression level in T2 transgenic cabbage than in wild type. The phenotype at rosette stage of transgenic cabbage grown in the test base was obvious, showing elongated the stems and leaves, elongated petiole, purple stems and petiole, and flat leaves without upward inward curling trend. It suggested that BoLH27 may play important roles in the control of leaves development.

Key words: Brassica oleracea, BoLH27, gene cloning, transgene, phenotype

表1

试验使用的引物"

引物名称及序列
Primer name and sequence (5°→3°)		 退火温度
Annealing temperature (°C)
klBoLH27S: ATGGAAGACCTCGAAGATGAGTACAAG
klBoLH27AS: GGTTTTTGGTACAATGAAACAAACTAGAAG		 63
pcBoLH27S: GGATCCATGGAAGACCTCGAAGATGAGTACAAG
pcBoLH27AS: GGATCCGGTTTTTGGTACAATGAAACAAACTAGAAG
NOS: CCCGATCTAGTAACATAGATGACAC		 62
jcBoLH27S: GGATCCATGGAAGACCTCGAAGATGAGTACAAG
jcBoLH27AS: GGATCCGGTTTTTGGTACAATGAAACAAACTAGAAG		 56.8
qBoLH27S: AGATTAGAAGCAGAGATCCAAGAGC
qBoLH27AS: GAAGTATTGTAATCCATCTGCCTGAAC		 58

图1

甘蓝BoLH27 cDNA及其推导的氨基酸序列黑色阴影示bHLH结构域; 实线框示N-糖基化位点; 虚线框示cAMP磷酸位点; 下画线示蛋白激酶C磷酸化位点; 波浪线示酪蛋白II磷酸化位点; 椭圆框示N-豆蔻酰化位点。"

图2

BoLH27及其同源序列bHLH结构域比对分析星号表示bHLH结构域保守的氨基酸位点; 三角表示参与二聚体形成的位点。"

图3

BoLH27及其同源氨基酸序列进化分析"

图4

pC35S-BoLH27转基因植株的获得 A: 抗性芽的生长; B、C: 抗性苗生根; D: 抗性植株幼苗。"

图5

转基因植株PCR检测 M: DL2000; 1~10: 转基因甘蓝; +: 阳性对照; -: 非转基因甘蓝。"

图6

BoLH27在野生型和转基因甘蓝中的表达分析 WT: 野生型株系; S13, S14, S33: 超表达株系。"

图7

野生型株系和BoLH27超表达株系表型 A: 10片叶幼苗期; B: 24片叶莲座期侧视图; C: 24片叶莲座期俯视图; WT: 野生型; BoLH27: 超表达株系; partial: 超表达株系局部放大。"

表2

超表达株系平均叶柄长度和平均叶间距"

超表达株系
Overexpressed lines		 平均叶柄长度
Average petiole length (cm)		 平均叶间距
Average leaf
spacing (cm)
1 16.8 1.7
2 15.2 1.8
3 14.5 1.6
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