向日葵LACS家族鉴定及响应非生物胁迫表达分析

doi:10.16420/j.issn.0513-353x.2020-1013

摘要/Abstract

摘要：

利用生物信息学技术从向日葵（Helianthus annuus）基因组中鉴定了13个长链酰基辅酶A合成酶（long-chain acyl-CoA synthases,LACSs）家族基因,其分布在向日葵8条染色体上。系统发育树显示,HaLACS家族成员分为6个亚族,且同一亚族成员具有相似的基因结构和蛋白保守基序。基因复制分析发现HaLACS家族扩增的主要因素是片段复制。HaLACS的启动子区域富含逆境响应和植物激素调控相关元件。表达谱分析显示HaLACS在向日葵不同组织中的表达存在差异。比较2个向日葵品种（高/低亚油酸含量）中HaLACS在种子发育不同阶段的表达,HaLACS呈现差异表达。qRT-PCR结果表明,200 mmol · L^-1 NaCl处理24 h后,7个HaLACS在茎中的表达量与同期对照相比显著上调;100 μmol · L^-1 ABA 处理3 h后,8个HaLACS在根中的表达量显著上调;15% PEG 6000模拟干旱胁迫后,多数HaLACS在不同组织中呈现诱导表达。综上,向日葵LACS基因家族不仅参与了向日葵脂质的合成,而且在应对非生物胁迫时发挥重要作用。

关键词: 向日葵, LACS基因家族, 非生物胁迫, 基因表达

Abstract:

Based on bioinformatics analyses,13 long-chain acyl-CoA synthetases（LACSs）putative genes from LACS gene family were identified from the genome of sunflower（Helianthus annuus）and mapped to eight chromosomes. The phylogenetic analysis showed that HaLACSs clustered into six subgroups and HaLACS proteins within each subgroup share the similar structures and conserved motifs. The main factors for HaLACS family amplification in sunflower were segmental duplication. Bioinformatic analysis of HaLACS genes promoter regions showed multiple cis-elements related to environmental stresses and plant hormone response. Tissue expression profile analysis showed that the HaLACS genes exhibited differential expression profiles in various tissues. The comparison of gene expression profiles between two sunflower cultivars（high/low linoleic acid）found that HaLACSs showed differential expression at different seed development stages. The qRT-PCR analysis showed that seven HaLACS up-regulated significantly in sunflower stem treated with 200 mmol · L^-1 NaCl for 24 h compared with control. After three hours treatment with 100 μmol · L^-1 ABA,eight HaLACS synchronized especially in roots,and most HaLACS colud be induced by drought stress（15% PEG 6000）. The above results indicate that the HaLACS play essential roles in dealing with different abiotic stresses and lipid biosynthesis.

Key words: sunflower, LACS gene family, abiotic stress, gene expression

中图分类号:

S565.5

周至铭, 杨佳宝, 张程, 曾令露, 孟晚秋, 孙黎. 向日葵LACS家族鉴定及响应非生物胁迫表达分析[J]. 园艺学报, 2022, 49(2): 352-364.

ZHOU Zhiming, YANG Jiabao, ZHANG Cheng, ZENG Linglu, MENG Wanqiu, SUN Li. Genome-wide Identification and Expression Analyses of Long-chain Acyl-CoA Synthetases Under Abiotic Stresses in Helianthus annuus[J]. Acta Horticulturae Sinica, 2022, 49(2): 352-364.

图/表 12

表1 qRT-PCR引物序列

Table 1 Primers used for qRT-PCR

基因Gene	正向引物（5′-3′）Forward primer	反向引物（5′-3′）Reverse primer
18S rRNA	CTACCACATCCAAGGAAGGCAG	CGACAGAAGGGACGAGTAAACC
HaLACS1-1	GAGCTCGAAAACGGAACGTG	ATTGCAAGCCTCCATCGCTA
HaLACS1-2	TTGGACCATGCCGAAGTTGA	AAGGAACTCGTTCCACGCAT
HaLACS2	AGAAACAGACAAACCGGGCA	ACGCCTTCTTTACAGTGCCT
HaLACS4-1	GGTTATGAGCATGGAGGCGT	CCACGCCAAAATTTCCGACT
HaLACS4-2	GGTTATGAGCATGGAGGCGT	GGTTATGAGCATGGAGGCGT
HaLACS4-3	CCTCCGATTGACGGTTTGGA	CTCAACTCCACAAGCTCGGA
HaLACS4-4	CGGTGAAAGACGGAACTCCA	CGACGCCACATGAACGGATA
HaLACS4-5	GAAAACAATCGTGAGCTTTGGGA	AGTTCAACGATACTTTCGCGCT
HaLACS6	CACTGCCGGAGAGTTCTTCA	TGCTTCCCCGTAAGTTGTCC
HaLACS7	TCATCTTCTTCCCTCCACCGA	ATCTGGTGACAAGCTTCAGCG
HaLACS8	GCAGATGGATGTTGGGGGAT	CGCCAACTTCAACTGGAACG
HaLACS9-1	ACTCTCGCCCTTCGGAAATC	TTTCAAATGCCCTCCCGTCT
HaLACS9-2	ATTGCATTGCAGGCGTGTTT	TTTCCAACTGCTGCTTCCGT

表2 向日葵LACS基因基本信息

Table 2 Detailed information of LACS genes in sunflower genome

转录本ID Transcript ID	基因名称 Gene name	CDS/bp	大小/aa Size	蛋白分子量/kD Molecular weight	等电点 pI	拟南芥同源基因 Arabidopsis homologous gene
转录本ID Transcript ID	基因名称 Gene name	CDS/bp	大小/aa Size	蛋白分子量/kD Molecular weight	等电点 pI	登录号Accession No.	名称Name
OTG27997	HaLACS1-1	1 980	659	74.4	5.82	AT2G47240	AtLACS1
OTF93791	HaLACS1-2	1 986	661	74.8	7.23	AT2G47240	AtLACS1
OTG12244	HaLACS2	1 980	659	73.5	5.70	AT1G49430	AtLACS2
OTG32156	HaLACS4-1	1 986	661	73.5	6.07	AT4G23850	AtLACS4
OTG33063	HaLACS4-2	1 995	664	74.0	6.69	AT4G23850	AtLACS4
OTG20245	HaLACS4-3	1 980	659	73.5	6.50	AT4G23850	AtLACS4
OTG02621	HaLACS4-4	1 974	657	73.4	6.69	AT4G23850	AtLACS4
OTG02629	HaLACS4-5	1 983	660	73.8	8.49	AT4G23850	AtLACS4
OTG11238	HaLACS6	2 094	697	76.3	7.51	AT3G05970	AtLACS6
OTG31091	HaLACS7	2 097	698	77.0	6.58	AT5G27600	AtLACS7
OTG29824	HaLACS8	2 163	720	78.6	7.86	AT2G04350	AtLACS8
OTG15459	HaLACS9-1	2 091	696	76.2	6.10	AT1G77590	AtLACS9
OTG07054	HaLACS9-2	2 121	706	77.4	6.78	AT1G77590	AtLACS9

图1 向日葵HaLACS基因染色体定位黑色虚线连接为重复基因。

Fig. 1 Chromosome location of sunflower HaLACS genes Black dashed links are duplicate gene pairs.

表3 向日葵LACS家族成员基因复制事件和发散时间分析

Table 3 Gene duplication events and divergence time of the LACS genes in Helianthus annuus

重复基因对 Duplicated gene pairs	非同义替换率Ka	同义替换率 Ks	Ka/Ks	重复类型 Duplicated type	选择压力 Selective pressure	分化时间/ Mya Divergence time
HaLACS9-1/HaLACS9-2	0.0540	0.5316	0.1016	片段重复Segmental	纯化Purifying	17.72
HaLACS4-1/HaLACS4-5	0.0739	0.6713	0.1101	片段重复Segmental	纯化Purifying	22.38
HaLACS4-1/HaLACS4-4	0.0905	0.6463	0.1400	片段重复Segmental	纯化Purifying	21.54
HaLACS4-1/HaLACS4-3	0.1003	1.0273	0.0976	片段重复Segmental	纯化Purifying	34.24
HaLACS4-4/HaLACS4-5	0.0600	0.3516	0.1706	串联重复Tandem	纯化Purifying	11.72

图2 向日葵和其他物种LACS基因家族系统进化分析绿色圆圈代表单子叶植物,红色圆圈代表双子叶植物。 Ec：大肠杆菌（原核生物）;Sc：酿酒酵母（真菌）;Pt：三角褐指藻（藻类）;Pp：小立碗藓（藓）;Sm：江南卷柏（蕨）;Mp：地钱（苔）;At：拟南芥;Ha：向日葵;Ls：莴苣;Eg：油棕;Zm：玉米;Os：水稻;Hs：人;Mm：家鼠。

Fig. 2 Phylogenetic analysis of LACS gene family in sunflower and other species The green circles indicate monocotyledonous plants,the red circle indicates a dicot plant. Ec：Escherichia coli（prokaryote）;Sc：Saccharomyces cerevisiae（fungi）;Pt：Phaeodac tylumtricornutum（algae）;Pp：Physcomitrella patens（moss）;Sm：Selaginella moellendorffii（fern）;Mp：Marchantia polymorpha（moss）;At：Arabidopsis thaliana;Ha：Helianthus annuus;Ls：Lactuca sativa;Eg：Elaeis guineensis;Zm：Zea mays;Os：Oryza sativa;Hs：Homo sapiens;Mm：Mus musculus.

图3 HaLACS基因结构和编码蛋白保守基序

Fig. 3 The gene structure and predicted conserved motifs of HaLACS family gene encoding proteins

图4 向日葵HaLACS与其他同源蛋白的氨基酸多序列比对 Ec：大肠杆菌;At：拟南芥;Sc：酿酒酵母;Ha：向日葵。

Fig. 4 Multiple sequence alignment of the HaLACS amino acid sequences with other proteins Ec：Escherichia coli;At：Arabidopsis thaliana;Sc：Selaginella moellendorffii;Ha：Helianthus annuus.

图5 HaLACS基因启动子顺式作用元件分析

Fig. 5 Analysis of cis-acting elements in HaLACS promoters

图6 HaLACS蛋白互作网络图

Fig. 6 HaLACS protein interaction network diagram

图7 HaLACS在2个向日葵品系不同发育阶段种子（A）和不同组织（B）的表达模式

Fig. 7 Expression levels in different seed development stages（A）of two sunflower cultivars and different tissues（B）

图8 向日葵HaLACS在盐胁迫下的表达分析

Fig. 8 Expression analysis of sunflower HaLACS under salt stresses

图9 向日葵HaLACS在干旱和外源ABA处理下的表达分析

Fig. 9 Expression analysis of sunflower HaLACSs under drought and ABA treatments

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