金银花花形基因的发掘及生物信息学分析

doi:10.19586/j.2095-2341.2022.0071

生物技术进展 ›› 2023, Vol. 13 ›› Issue (1): 90-101.DOI: 10.19586/j.2095-2341.2022.0071

金银花花形基因的发掘及生物信息学分析

李凤梅(), 汤雪媛, 焦高洋, 齐玉亭, 杜献婷, 鲍倩倩, 张梦, 徐小博, 徐鑫, 张艳芳()

新乡学院生命科学与基础医学学院，河南新乡 453003

收稿日期:2022-05-09 接受日期:2022-08-01 出版日期:2023-01-25 发布日期:2023-02-07
通讯作者: 张艳芳
作者简介:李凤梅 E-mail: lfm3714167@163.com；
基金资助:
河南省高等学校重点科研项目计划项目(21B180009);2021年度新乡市科技攻关计划项目(GG2021014)

Identification and Analysis of Genes Controlling Flower Shape in Lonicerajaponica Thunb.

Fengmei LI(), Xueyuan TANG, Gaoyang JIAO, Yuting QI, Xianting DU, Qianqian BAO, Meng ZHANG, Xiaobo XU, Xin XU, Yanfang ZHANG()

School of Life Sciences & Basic Medicine，Xinxiang University，Henan Xinxiang 453003，China

Received:2022-05-09 Accepted:2022-08-01 Online:2023-01-25 Published:2023-02-07
Contact: Yanfang ZHANG

摘要/Abstract

摘要：

金银花作为我国重要的中药材，具有消炎、抗菌、抗病毒、抗氧化、防癌等多种功效。随着金银花市场供需矛盾日益加剧，通过分子标记辅助选择育种方法来培育高产优质品种势在必行。通过NCBI的Blast工具扫描金银花蛋白组数据发掘花形候选基因，并执行候选基因的亲缘关系分析、结构域分析、表达模式分析、理化性质分析、蛋白质结构预测等一系列生物信息学分析。依据拟南芥调控花形的ABE类基因，通过NCBI-Blast工具扫描金银花氨基酸序列，筛选出包含MADS结构域的8个调控花形的金银花候选基因。经LjaFGD表达模式分析发现，金银花的花中GWHGAAZE016592和GWHGAAZE014905表达量显著高于其他部位，可能正向调控金银花花形。GWHGAAZE014905是一个包含MADS结构域的调控花器官发育的B类基因；GWHGAAZE016592是AP3同源基因。生物信息学分析发现，GWHGAAZE016592和GWHGAAZE014905均是稳定的亲水蛋白，属于非分泌蛋白，包括Motif1、Motif3、Motif4、Motif2、Motif6和Motif5，蛋白质三级结构模板为6byy.2.A和4ox0.2.C。GWHGAAZE014905被定位到细胞核上，而GWHGAAZE016592被定位到叶绿体上，且包含1个位于151~173 bp的跨膜螺旋区域，属于膜蛋白。研究结果为分子标记辅助选择方式培育道地高产优质金银花品种提供了基因资源和分子标记。

关键词: 金银花, 花形基因, 表达模式分析, 生物信息学分析

Abstract:

Lonicera japonica Thunb. is an important Chinese medicinal material in our country， which has many functions such as anti-inflammatory， anti-bacterial， anti-virus， anti-oxidation and anti-cancer. With the increasing contradiction between supply and demand in markets of Lonicera japonica Thunb.， it is essential to breed high yield and high quality varieties by marker-assisted selection. The amino acid sequence of Lonicera japonica Thunb. was scanned with NCBI-Blast tool， and a series of bioinformatics analyses including phylogenetic analysis， conserved domain analysis， expression pattern analysis， physical properties and chemical analysis and protein structure prediction were performed. In this study， the amino acid sequence of Lonicera japonica Thunb. was scanned with NCBI-Blast tool， and 8 candidate genes containing MADS domain were selected， based on ABE genes regulating petal in Arabidopsis. Expression pattern analysis in LjaFGD found that the expression in flower of GWHGAAZE014905 and GWHGAAZE016592 was significantly higher than that in other parts， which might positively regulate flower shape of Lonicera japonica Thunb.. GWHGAAZE014905 was a class B gene containing MADS domain that regulating the development of floral organs； GWHGAAZE016592 was an AP3 homologous gene. Bioinformatics analysis found that GWHGAAZE014905 and GWHGAAZE016592 belonged to stable hydrophilic proteins and non-secreted proteins， both including Motif1， Motif3， Motif4， Motif2， Motif6 and Motif5， 3D structure template of proteins were both 6byy.2.A and 4ox0.2.C. Subcellular localization analysis indicated that GWHGAAZE014905 was targeted to the chloroplast； GWHGAAZE016592 was targeted to the chloroplast， which belonged to membrane protein， contained a transmembrane helix region in 151~173 bp. This research provided genetic resources and molecular markers for the cultivation of top quality and high varieties in Lonicera japonica Thunb. by molecular marker-assisted selection.

Key words: Lonicera japonica Thunb., gene of flower shape, expression pattern analysis, bioinformatics analysis

中图分类号:

Q343.1

李凤梅, 汤雪媛, 焦高洋, 齐玉亭, 杜献婷, 鲍倩倩, 张梦, 徐小博, 徐鑫, 张艳芳. 金银花花形基因的发掘及生物信息学分析[J]. 生物技术进展, 2023, 13(1): 90-101.

Fengmei LI, Xueyuan TANG, Gaoyang JIAO, Yuting QI, Xianting DU, Qianqian BAO, Meng ZHANG, Xiaobo XU, Xin XU, Yanfang ZHANG. Identification and Analysis of Genes Controlling Flower Shape in Lonicerajaponica Thunb.[J]. Current Biotechnology, 2023, 13(1): 90-101.

图/表 16

参考文献 33

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类型	调控部位	基因
其他调控花器官大小的因子		ARGOS、OSR1、ANT、AILS、KLU、GRFs、GIFs、SWP、ARL、MED8、BB、DA1、DAR1、TCP4、MED25、BPEp/ARF8等
A类基因	花瓣、萼片	AP1、AGL79、AP2、CLF、CAL、LUG、FUL、AGL79等
B类基因	花瓣、雄蕊	PI、APETALA3（AP3）、NAP、PIS、B类基因表达相关基因（LFY、UFO）等
E类基因	花瓣	SEP1、SEP2、SEP3、SEP4、AGL3等

类型	调控部位	基因
其他调控花器官大小的因子		ARGOS、OSR1、ANT、AILS、KLU、GRFs、GIFs、SWP、ARL、MED8、BB、DA1、DAR1、TCP4、MED25、BPEp/ARF8等
A类基因	花瓣、萼片	AP1、AGL79、AP2、CLF、CAL、LUG、FUL、AGL79等
B类基因	花瓣、雄蕊	PI、APETALA3（AP3）、NAP、PIS、B类基因表达相关基因（LFY、UFO）等
E类基因	花瓣	SEP1、SEP2、SEP3、SEP4、AGL3等

基因名称 (蛋白名称)		染色体	位置	编码区 CDS	正(+)反(-)向	结构域	拟南芥同源基因 (同一性)
同一基因簇	GWHGAAZE001422 (GWHPAAZE001424)	1	37616176~37616658	37616176~37616658	-	MADS(1~61), low complexity(101~114), coiled coil(129~160)	AP1(40.000), AP3(34.177), SEP3(34.343)
同一基因簇	GWHGAAZE001424 (GWHPAAZE001426)	1	37773949~37774431	37773949~37774431	-	MADS(1~61), coiled coil(89~160）,	AP1(38.667), AP3(34.177), SEP3(32.653), SEP4(47.158)
同一基因簇	GWHGAAZE003600 (GWHPAAZE003603）	1	100741719~100742564	100741719~100742564	-	MADS(1~61), coiled coil(125~156),	AP1(47.059), AP3(37.500), SEP3(54.167)
同一基因簇	GWHGAAZE003601 (GWHPAAZE003604)	1	100748839~100749663	100748839~100749663	-	MADS(1~61)	AP1(46.154), AP3(38.889), SEP3(56.250), SEP4(54.167)
GWHGAAZE014905 (GWHPAAZE014912)		4	21322982~21326145	21323004~21323191, 21324258~21324324, 21324428~21324489, 21324610~21324709, 21325160~21325201, 21325317~21325361, 21325548~21325733	+	MADS(1~60), low complexity(111~121), coiled coil(144~164)	AP1(34.659), AP3(52.913), SEP3(35.859), SEP4(38.217)
GWHGAAZE016592 (GWHPAAZE016602)		4	79255018~79260192	79255018~79255245, 79255659~79255718, 79257047~79257091, 79257197~79257238, 79258143~79258317, 79258437~79258531, 79259395~79259461, 79260005~79260192	-	MADS(1~60), coiled coil(91~122), transmembrane region(151~173)	AGL79(37.662) AP1(38.272) AP3(50.345) SEP3(43.243)
GWHGAAZE031567 (GWHPAAZE031586)		9	61028167~61028670	61028167~61028670	-	MADS(1~60), coiled coil(78~117,140~167)	AP1(42.857) AP3(26.627) SEP3(45.833)
GWHGAAZE032544 (GWHPAAZE032564)		21	4431033~4436189	4435923~4436104 4434534~4434609 4434055~4434122 4433812~4433911 4432855~4432896 4432353~4432394 4432092~4432216 4431316~4431415	-	MADS(1~60), coiled coil(154~177)	AP1(42.286) AP3(36.559) SEP3(45.349) SEP4(39.429)

基因名称 (蛋白名称)		染色体	位置	编码区 CDS	正(+)反(-)向	结构域	拟南芥同源基因 (同一性)
同一基因簇	GWHGAAZE001422 (GWHPAAZE001424)	1	37616176~37616658	37616176~37616658	-	MADS(1~61), low complexity(101~114), coiled coil(129~160)	AP1(40.000), AP3(34.177), SEP3(34.343)
同一基因簇	GWHGAAZE001424 (GWHPAAZE001426)	1	37773949~37774431	37773949~37774431	-	MADS(1~61), coiled coil(89~160）,	AP1(38.667), AP3(34.177), SEP3(32.653), SEP4(47.158)
同一基因簇	GWHGAAZE003600 (GWHPAAZE003603）	1	100741719~100742564	100741719~100742564	-	MADS(1~61), coiled coil(125~156),	AP1(47.059), AP3(37.500), SEP3(54.167)
同一基因簇	GWHGAAZE003601 (GWHPAAZE003604)	1	100748839~100749663	100748839~100749663	-	MADS(1~61)	AP1(46.154), AP3(38.889), SEP3(56.250), SEP4(54.167)
GWHGAAZE014905 (GWHPAAZE014912)		4	21322982~21326145	21323004~21323191, 21324258~21324324, 21324428~21324489, 21324610~21324709, 21325160~21325201, 21325317~21325361, 21325548~21325733	+	MADS(1~60), low complexity(111~121), coiled coil(144~164)	AP1(34.659), AP3(52.913), SEP3(35.859), SEP4(38.217)
GWHGAAZE016592 (GWHPAAZE016602)		4	79255018~79260192	79255018~79255245, 79255659~79255718, 79257047~79257091, 79257197~79257238, 79258143~79258317, 79258437~79258531, 79259395~79259461, 79260005~79260192	-	MADS(1~60), coiled coil(91~122), transmembrane region(151~173)	AGL79(37.662) AP1(38.272) AP3(50.345) SEP3(43.243)
GWHGAAZE031567 (GWHPAAZE031586)		9	61028167~61028670	61028167~61028670	-	MADS(1~60), coiled coil(78~117,140~167)	AP1(42.857) AP3(26.627) SEP3(45.833)
GWHGAAZE032544 (GWHPAAZE032564)		21	4431033~4436189	4435923~4436104 4434534~4434609 4434055~4434122 4433812~4433911 4432855~4432896 4432353~4432394 4432092~4432216 4431316~4431415	-	MADS(1~60), coiled coil(154~177)	AP1(42.286) AP3(36.559) SEP3(45.349) SEP4(39.429)

基因名称	GWHGAAZE014905	GWHGAAZE016592
化学方程式	C₁₁₇₀H₁₈₆₀N₃₄₆O₃₅₆S₉	C₁₄₉₈H₂₄₁₅N₄₂₅O₄₄₄S₁₂
氨基酸数量	229	296
分子量	26 758.28	33 867.95
等电点pI	8.56	8.92
氨基酸组成	Ala(2.6%)，Arg(8.3%)，Asn(4.8%)，Asp(7.0%)，Cys(1.7%)，Gln(3.9%)，Glu(8.3%)，Gly(7.4%)，His(4.4%)，Ile(4.8%)，Leu(10.0%)，Lys(8.3%)，Met(2.2%)，Phe(3.9%)，Pro(1.3%)，Ser(4.4%)，Thr(7.4%)，Trp(0.4%)，Tyr(3.9%)，Val(4.8%)，Pyl(0.0%)，Sec(0.0%)	Ala(3.0%)，Arg(6.4%)，Asn(5.4%)，Asp(3.0%)，Cys(2.0%)，Gln(5.4%)，Glu(8.4%)，Gly(6.8%)，His(3.0%)，Ile(6.1%)，Leu(12.5%)，Lys(7.1%)，Met(2.0%)，Phe(3.7%)，Pro(3.4%)，Ser(6.4%)，Thr(6.4%)，Trp(0.3%)，Tyr(3.0%)，Val(5.4%)，Pyl(0.0%)，Sec(0.0%)
负电子总数	35	34
正电子总数	38	40
原子组成	C(1170)、H(1860)、N(346)、O(356)、S(9)	C(1498)、H(2415)、N(425)、O(444)、S(12)
不稳定系数	26.1	38.96
脂肪系数	74.45	91.18
综合亲水性平均GRAVY	-0.828	-0.44

金银花花形基因的发掘及生物信息学分析

Identification and Analysis of Genes Controlling Flower Shape in Lonicerajaponica Thunb.

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参考文献 33

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同源蛋白	α螺旋	延伸链	β转角	无规则卷曲
GWHGAAZE014905	56.33%	13.10%	8.30%	22.27%
GWHGAAZE016592	44.26%	20.61%	6.42%	28.72%
At AGL79	48.19%	7.63%	4.42%	39.76%
AP1	57.65%	7.45%	2.75%	32.16%
AP3	52.16%	17.67%	7.33%	22.84%
SEP3	52.19%	9.96%	4.78%	33.07%
SEP4	49.81%	7.78%	3.89%	38.52%

同源蛋白	模板	描述	同一性	覆盖度	范围
GWHGAAZE014905	6byy.2.A	MEF2 CHIMERA/ MEF2 CHIMERA/DNA Complex	42.67%	0.33	2~77
GWHGAAZE014905	4ox0.2.C	Developmental protein SEPALLATA 3/ Crystal structure of the keratin-like domain from the MADS transcription factor Sepallata 3	29.76%	0.37	86~169
GWHGAAZE016592	6byy.2.A	同上	44.59%	0.25	2~76
GWHGAAZE016592	4ox0.2.C	同上	29.76%	0.28	97~205
At AGL79	6byy.2.A	同上	46.67%	0.30	2~76
At AGL79	4ox0.2.C	同上	36.47%	0.34	90~174
AP1	3kov.1.A	Myocyte-specific enhancer factor 2A/ Structure of MEF2A bound to DNA reveals a completely folded MADS-box/MEF2 domain that recognizes DNA and recruits transcription co-factors	56.34%	0.28	2~74
AP1	4ox0.2.C	同上	41.86%	0.34	89~174
AP3	6byy.2.A	同上	49.33%	0.32	2~77
3-Sep	6byy.2.A	同上	48.24%	0.34	2~88
3-Sep	4ox0.2.C	同上	100.00%	0.41	83~177
4-Sep	6byy.2.A	同上	53.85%	0.30	2~84
4-Sep	4ox0.2.C	同上	54.65%	0.33	89~174

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