换锦花<em>LsMYB5</em>基因的克隆与表达分析

doi:10.3969/j.issn.1674-7968.2019.12.008

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摘要换锦花(Lycoris sprengeri)属石蒜科石蒜属多年生草本植物,花色为红蓝复色,是良好的鲜切花、盆花、地被和园林造景材料,具有很高的商业价值。为丰富培育花色多样的换锦花品种,本研究以换锦花花瓣转录组信息为基础,采用反转录-PCR (reverse transcription PCR, RT-PCR)和cDNA末端快速扩增(rapid-amplification of cDNA ends, RACE)技术相结合成功克隆了长871 bp的换锦花R2R3-MYB转录因子5基因(R2R3-MYB transcription factor 5 gene, LsMYB5) cDNA序列(GenBank No. MK779710),开放阅读框681 bp,编码226个氨基酸,LsMYB5蛋白C端含有保守的乙烯类响应元件结合因子相关的两亲性抑制(ethylene-responsive element binding factor-associated amphiphilic repression, EAR)抑制结构域(pdLNLD/ELxiG/s)和锌指结构域(CX-(1-2) CX-(7-12) CX-(1-2)C),可能具有转录抑制功能。qRT-PCR分析表明,LsMYB5基因具组织特异性表达,主要在叶片中表达;在花瓣的凋谢期的表达量最高,在颜色不同的无性系中表达量具有显著性差异(P<0.05),推测LsMYB5的表达与换锦花花色形成有关。为了进一步分析LsMYB5基因功能,本研究构建了原核表达载体pET-30(a)-LsMYB5,转化大肠杆菌(Escherichia coli) BL21 (DE3),成功获得异丙基硫代半乳糖苷(isopropyl β-D-thiogalactoside, IPTG)诱导的表达重组蛋白,并进行了His标签蛋白纯化。该研究结果可为进一步研究LsMYB5转录因子调控的换锦花花色形成相关结构基因的筛选提供理论依据。

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	侯朔
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关键词 ：换锦花, 原核表达, 换锦花R2R3-MYB转录因子5基因(LsMYB5), 表达分析

Abstract：Lycoris sprengeri is perennial herbaceous plants of Amaryllidaceae, Lycoris with the red and blue multicolor flower, which is a good material for cut flower, potted flower and landscape gardening, so L. sprengeri has the very high commercial value. To enrich the germplasm resources and cultivate varieties in L. sprengeri, a R2R3-MYB transcription factor gene LsMYB5 cDNA sequence (GenBank No. MK779710) was successfully screened by using reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). The LsMYB5 gene length was 871 bp, and the open reading frame (ORF) was 681 bp, which encoded 226 amino acids. The c-terminal of LsMYB5 protein contained conservative EAR inhibition domain (pdLNLD/ELxiG/s) and zinc finger domain (CX-(1-2), CX-(7-12), CX-(1-2)C), which may have transcriptional inhibition function. LsMYB5 gene mainly expressed in leaves and the fading period of petals, and there were significant differences in the expression of LsMYB5 in clones with different colors (P<0.05). To further analyze LsMYB5 gene function, prokaryotic expression vector of pET-30(a)-LsMYB5 was constructed and transformed into Escherichia coli BL21 (DE3), the expression of recombinant proteins was induced under the induction of isopropyl glucosinolates galactose glucoside (IPTG), and the His label protein was purified. The results will provide the theory basis for screening structural genes regulated by LsMYB5 transcription factor. which related to flower color formation in Lycoris sprengeri.

Key words： Lycoris sprengeri Prokaryotic expression Lycoris sprengeri MYB transcription factor gene 5 (LsMYB5) Expression analysis

收稿日期: 2019-04-15

ZTFLH:

S682.2

基金资助:国家自然科学基金(No. 31670696)和浙江省“十三五”林木新品种选育项目(No. 2016C02056-13)

通讯作者: * gaoyanhui408@126.com

引用本文:

侯朔, 高燕会, 童再康. 换锦花LsMYB5基因的克隆与表达分析[J]. 农业生物技术学报, 2019, 27(12): 2164-2174.
HOU Shuo, GAO Yan-Hui, TONG Zai-Kang. Cloning and Expression Analysis of LsMYB5 Gene in Lycoris sprengeri. 农业生物技术学报, 2019, 27(12): 2164-2174.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.12.008 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I12/2164

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