Cloning and Function Identification of Dihydroflavonol 4-Reductase Gene BoaDFR in Chinese Kale

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

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (1): 73-82.doi: 10.16420/j.issn.0513-353x.2020-0378

• Research Papers • Previous Articles Next Articles

Cloning and Function Identification of Dihydroflavonol 4-Reductase Gene BoaDFR in Chinese Kale

ZHENG Hao¹, ZHANG Fen¹, JIAN Yue¹, HUANG Wenli¹, LIANG Sha¹, JIANG Min¹, YUAN Qiao¹, WANG Qiaomei², SUN Bo¹^,^**()

¹College of Horticulture,Sichuan Agricultural University,Chengdu 611130,China
²Department of Horticulture,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,China

Received:2020-05-19 Revised:2020-11-05 Online:2021-01-25 Published:2021-01-29
Contact: SUN Bo E-mail:bsun@sicau.edu.cn

Abstract

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

CLC Number:

S635

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.

Figures/Tables 9

References 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

Cloning and Function Identification of Dihydroflavonol 4-Reductase Gene BoaDFR in Chinese Kale

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