芥蓝二氢黄酮醇4-还原酶基因BoaDFR的克隆与功能鉴定

doi:10.16420/j.issn.0513-353x.2020-0378

摘要/Abstract

摘要：

以白花芥蓝‘四季粗条’为材料克隆了BoaDFR基因,GenBank登录号为MT472647,CDS序列长为1 158 bp,编码385个氨基酸,与甘蓝型油菜、甘蓝和白菜等具有高度同源性。BoaDFR表达水平在芥蓝不同发育时期、不同器官中存在显著差异,随植株发育总体呈现先下降后上升的趋势,在幼嫩种子中表达量最高。芥蓝原生质体的亚细胞定位结果显示BoaDFR定位在细胞核上。构建了农杆菌介导的芥蓝瞬时过表达体系,将BoaDFR转入白花芥蓝‘四季粗条’和黄花芥蓝‘福州黄花’中,发现BoaDFR在两种芥蓝中均高水平表达,转基因植株叶片颜色明显变紫,花青素苷显著积累,总含量最高达461.43 μg·g^-1 FW,而野生型和转空载体芥蓝叶片中几乎检测不到花青素苷。通过HPLC在转基因植株叶片中共检测到8种花青素苷,均为矢车菊花青素苷,其中矢车菊-3-阿魏酰-丙二酰-槐糖苷-5-葡萄糖苷占比最高。

关键词: 芥蓝, 二氢黄酮醇4-还原酶（DFR）, 花青素苷, 基因瞬时过表达, 基因功能

Abstract:

The BoaDFR was cloned from the cultivar‘Siji Cutiao’of white flower Chinese kale,and the GenBank accession number was MT472647. BoaDFR is 1 158 bp-long CDS sequence,encoding 385 amino acids. BoaDFR has high homology with DFR of Brassica napus,B. oleracea and B. rapa. The expression level of BoaDFR differed significantly at different stages and organs,and generally showed a trend of decreasing first and then rising during the plant growth. The highest expression level of BoaDFR was in young seeds,which was significantly higher than other organs. The subcellular localization showed that BoaDFR was localized in the nucleus. The Agrobacterium-mediated transient overexpression system in Chinese kale was constructed,and BoaDFR was transferred into‘Siji Cutiao’and‘Fuzhou Huanghua’. It was found that BoaDFR can be expressed at high levels in both two cultivars and the leaves of transgenic plants obviously turned purple,the anthocyanin accumulated significantly and the highest content was up to 461.43 μg·g ^-1FW,whereas the anthocyanin almost could not be detected in both wild-type and empty vector plants. Furthermore,a total of eight anthocyanins were detected in the transgenic plant leaves by HPLC,all of which were cyanidins,and cyanidin 3-feruloylmalonylso- phoroside-5- glucoside was predominant.

Key words: Chinese kale, dihydroflavonol 4-reductase（DFR）, anthocyanin, gene transient overexpression, gene function

中图分类号:

S635

郑好, 张芬, 简越, 黄文莉, 梁莎, 江敏, 袁巧, 汪俏梅, 孙勃. 芥蓝二氢黄酮醇4-还原酶基因BoaDFR的克隆与功能鉴定[J]. 园艺学报, 2021, 48(1): 73-82.

ZHENG Hao, ZHANG Fen, JIAN Yue, HUANG Wenli, LIANG Sha, JIANG Min, YUAN Qiao, WANG Qiaomei, SUN Bo. Cloning and Function Identification of Dihydroflavonol 4-Reductase Gene BoaDFR in Chinese Kale[J]. Acta Horticulturae Sinica, 2021, 48(1): 73-82.

图/表 9

参考文献 22

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用途 Use	引物名称 Primer name	序列（5′-3′） Sequence
CDS全长扩增 Amlification of CDS	DFR-CDS-F	ATGGTAGCTCACAAAGAGACCGTGT
CDS全长扩增 Amlification of CDS	DFR-CDS-R	CTAAGCACAGATCTGCTGTGCCGA
qRT-PCR	DFR-qPCR-F	CGCATCAGGATTCATTGGTTCAT
qRT-PCR	DFR-qPCR-R	CGTCTTCGCGTTTGGCAAATCA
定量内参引物 Primers for internal control	β-actin-F	CCACCAATCTTGTACACATCC
定量内参引物 Primers for internal control	β-actin-R	AGACCACCAAGTACTACTGCAC
亚细胞定位 Subcellular localization	DFR-GFP-XmaI-F	CCCCCCGGGATGGTAGCTCACAAAGA
亚细胞定位 Subcellular localization	DFR-GFP-XbaI-R	GCTCTAGAAGCACAGATCTGCTGTG
过表达酶切位点引物 Primers for overexpression sites	DFR-pCAM-XbaI-F	GGGGGATCCACTAGTTCTAGAATGGTAGCTCACAAAGAGACCGTG
过表达酶切位点引物 Primers for overexpression sites	DFR-pCAM-SalI-R	AAGCTTACGATACCGTCGACCTAAGCACAGATCTGCTGTGCC

用途 Use	引物名称 Primer name	序列（5′-3′） Sequence
CDS全长扩增 Amlification of CDS	DFR-CDS-F	ATGGTAGCTCACAAAGAGACCGTGT
CDS全长扩增 Amlification of CDS	DFR-CDS-R	CTAAGCACAGATCTGCTGTGCCGA
qRT-PCR	DFR-qPCR-F	CGCATCAGGATTCATTGGTTCAT
qRT-PCR	DFR-qPCR-R	CGTCTTCGCGTTTGGCAAATCA
定量内参引物 Primers for internal control	β-actin-F	CCACCAATCTTGTACACATCC
定量内参引物 Primers for internal control	β-actin-R	AGACCACCAAGTACTACTGCAC
亚细胞定位 Subcellular localization	DFR-GFP-XmaI-F	CCCCCCGGGATGGTAGCTCACAAAGA
亚细胞定位 Subcellular localization	DFR-GFP-XbaI-R	GCTCTAGAAGCACAGATCTGCTGTG
过表达酶切位点引物 Primers for overexpression sites	DFR-pCAM-XbaI-F	GGGGGATCCACTAGTTCTAGAATGGTAGCTCACAAAGAGACCGTG
过表达酶切位点引物 Primers for overexpression sites	DFR-pCAM-SalI-R	AAGCTTACGATACCGTCGACCTAAGCACAGATCTGCTGTGCC

品种 Cultivar	植株 Plant lines	L^*		a^*		b^*
品种 Cultivar	植株 Plant lines	叶正面 Leaf front	叶背面 Leaf back	叶正面 Leaf front	叶背面 Leaf back	叶正面 Leaf front	叶背面 Leaf back
四季粗条 Siji Cutiao	野生型Wild type	43.02 a	51.96 a	-10.29 b	-11.81 d	25.15 a	24.45 a
	空载体Empty vector	44.13 a	53.13 a	-9.55 b	-11.13 d	25.88 a	25.80 a
	过表达Overexpression #1	29.89 c	40.55 b	4.16 a	5.95 b	4.15 bc	2.35 c
	过表达Overexpression #2	35.31 b	53.13 a	3.67 a	-1.51 c	6.40 b	14.70 b
	过表达Overexpression #3	29.57 c	30.79 c	3.64 a	12.72 a	4.01 c	-6.47 d
福州黄花 Fuzhou Huanghua	野生型Wild type	45.76 a	53.77 a	-10.81 c	-10.49 b	26.64 b	23.66 a
	空载体Empty vector	47.72 a	55.99 a	-9.38 c	-12.27 b	32.23 a	27.45 a
	过表达Overexpression #1	31.49 c	54.10 a	4.61 a	-2.83 a	5.12 d	13.77 b
	过表达Overexpression #2	34.57 bc	54.03 a	-1.63 b	-3.32 a	9.50 c	11.71 bc
	过表达Overexpression #3	35.37 b	51.09 b	-1.22 b	-1.06 a	4.96 d	7.20 c

品种 Cultivar	植株 Plant lines	L^*		a^*		b^*
品种 Cultivar	植株 Plant lines	叶正面 Leaf front	叶背面 Leaf back	叶正面 Leaf front	叶背面 Leaf back	叶正面 Leaf front	叶背面 Leaf back
四季粗条 Siji Cutiao	野生型Wild type	43.02 a	51.96 a	-10.29 b	-11.81 d	25.15 a	24.45 a
	空载体Empty vector	44.13 a	53.13 a	-9.55 b	-11.13 d	25.88 a	25.80 a
	过表达Overexpression #1	29.89 c	40.55 b	4.16 a	5.95 b	4.15 bc	2.35 c
	过表达Overexpression #2	35.31 b	53.13 a	3.67 a	-1.51 c	6.40 b	14.70 b
	过表达Overexpression #3	29.57 c	30.79 c	3.64 a	12.72 a	4.01 c	-6.47 d
福州黄花 Fuzhou Huanghua	野生型Wild type	45.76 a	53.77 a	-10.81 c	-10.49 b	26.64 b	23.66 a
	空载体Empty vector	47.72 a	55.99 a	-9.38 c	-12.27 b	32.23 a	27.45 a
	过表达Overexpression #1	31.49 c	54.10 a	4.61 a	-2.83 a	5.12 d	13.77 b
	过表达Overexpression #2	34.57 bc	54.03 a	-1.63 b	-3.32 a	9.50 c	11.71 bc
	过表达Overexpression #3	35.37 b	51.09 b	-1.22 b	-1.06 a	4.96 d	7.20 c

品种 Cultivar	植株 Plant line	A	B	C	D	E	F	G	H	I
四季粗条 Siji Cutiao	野生型Wild-type	0 c	0 d	0 b	0 d	0 c	0 d	0 d	0 d	0 d
	空载体Empty vector	0 c	0 d	0 b	0 d	0 c	0 d	0 d	0 d	0 d
	过表达Overexpression #1	4.31 b	80.36 c	3.79 a	12.80 b	42.13 a	157.79 b	54.85 b	26.15 b	382.17 b
	过表达Overexpression #2	4.69 b	102.39 a	3.53 a	6.53 c	15.58 d	100.52 c	43.03 c	15.22 c	291.49 c
	过表达Overexpression #3	5.97 a	98.73 b	4.30 a	17.07 a	43.85 a	177.94 a	74.21 a	39.36 a	461.43 a
福州黄花 Fuzhou Huanghua	野生型Wild-type	0 c	1.58 d	0 c	0 c	0 d	4.40 d	0 d	0.98 b	6.97 d
	空载体Empty vector	0 c	0 e	0 c	0 c	0 d	0 e	0 d	0 c	0 e
	过表达Overexpression #1	2.47 a	71.20 a	12.06 a	4.80 a	15.61 a	202.46 a	72.35 a	3.51 a	384.47 a
	过表达Overexpression #2	1.14 b	12.41 c	0 c	0 c	5.32 c	42.76 c	18.32 c	0 c	79.95 c
	过表达Overexpression #3	2.41 a	33.63 b	6.56 b	2.04 b	7.43 b	86.70 b	55.44 b	0 c	194.20 b