藜麦FLS基因家族的鉴定、表达及DNA变异分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0094

摘要/Abstract

摘要：

【目的】 黄酮醇合成酶（FLS）是石竹目植物中多酚类次生代谢的关键酶，为探究FLS基因在藜麦生长发育中的功能，对FLS基因家族进行鉴定和表达分析。【方法】 利用生物信息学分析网站，鉴定出CqFLS家族成员，分析其基因结构、蛋白的理化性质、二级结构、三级结构、启动子顺式元件以及系统进化关系等，并通过基因克隆、构建表达载体的方法分析其蛋白表达情况。【结果】 共鉴定出3个CqFLSs基因，不均匀分布在2条染色体上，CqFLSs启动子区域包含水杨酸、脱落酸、茉莉酸甲酯和干旱诱导等元件；CqFLS2.1g发现多处InDel和SNP变异，在ch01 28871893、28871125、28872881处编码核苷酸删除，且均注释为上游效应，未检测到移码突变；FLS家族系统进化树分析可知，与其他两个基因相比，CqFLS2.1g与CqFLS1.1g、CqFLS3.10g处于不同分支，表达水平也存在差异，CqFLS2.1g可能发生分化；同时，基因表达分析表明，3个CqFLSs基因在青白1号籽粒中整体表达量高于青黑1号和贡扎4号。对克隆出的CqFLS1.1g用0.3 mmol/L的IPTG诱导，在20℃和37℃的条件下均可成功表达。【结论】 CqFLS1.1g 在花的形成以及籽粒发育过程中发挥作用，而CqFLS2.1g则主要参与藜麦籽粒的形成，CqFLS的表达具有组织特异性，在藜麦生长发育过程中发挥重要的作用。

关键词: 藜麦, 黄酮醇合酶, 荧光定量PCR, DNA变异, 原核表达

Abstract:

【Objective】 This study is to identify and analyze the flavonol synthetase FLS gene family as a key enzyme in the secondary metabolism of polyphenols in Caryophyllum plants and to explore the function of FLSs in Chenopodium quinoa. 【Method】 Bioinformatics analysis website was used to identify the CqFLS family members, and analyze their gene structure, protein physicochemical properties, secondary structure, tertiary structure, promoter cis element and phylogenetic relationship. The gene cloning and expression vector construction was to analyze their protein expression. 【Result】 The 3 CqFLSs genes were identified, and they were unevenly distributed on two chromosomes, and the promoter region of CqFLSs contained salicylic acid, abscisic acid, methyl jasmonate and drought-induced elements. There were multiple InDel and SNP variations in CqFLS2.1g,the coding nucleotides were deleted at ch01 28871893, 28871125, and 28872881, and all of them were annotated as upstream effects, and no frameshift mutations were detected. Phylogenetic tree analysis showed that CqFLS2.1g were in different branches from CqFLS1.1g and CqFLS3.10g, their expressions varied, and CqFLS2.1g were also differentiated. Meanwhile gene expression analysis revealed that the overall expressions of the three CqFLSs genes in “Qingbai 1” grains were higher than those of “Qinghei 1” and “Gongzha 4”. The cloned CqFLS1.1g was induced with 0.3 mmol/L IPTG and was successfully expressed at both 20℃ and 37℃. 【Conclusion】 CqFLS1.1g plays a role in the formation of flowers and grain development, while CqFLS2.1g is mainly involved in the formation of quinoa grains, and the expression of CqFLS is tissue-specific and plays an important role in the growth and development of quinoa.

Key words: Chenopodium quinoa, flavonol synthase, qRT-PCR, DNA variation, prokaryotic expression

孙慧琼, 张春来, 王锡亮, 徐宏申, 窦苗苗, 杨博慧, 柴文婷, 赵珊珊, 姜晓东. 藜麦FLS基因家族的鉴定、表达及DNA变异分析[J]. 生物技术通报, 2024, 40(7): 172-182.

SUN Hui-qiong, ZHANG Chun-lai, WANG Xi-liang, XU Hong-shen, DOU Miao-miao, YANG Bo-hui, CHAI Wen-ting, ZHAO Shan-shan, JIANG Xiao-dong. Identification, Expression and DNA Variation Analysis of FLS Gene Family in Chenopodium quinoa[J]. Biotechnology Bulletin, 2024, 40(7): 172-182.

图/表 15

表1 引物序列

Table 1 List of primer sequences

引物名称Primer name	引物序列Primer sequence（5'-3'）
CqFLS1.1g-qF	GGTCTTTGGTACAATGTCCGC
CqFLS1.1g-qR	GGGCCATGACATCCTTGTTTTT
EF1a-F	GACAAGCGTGTGATCGAGAG
EF1α-R	TCGGCCTTAAGTTTGTCGAGA
F1	ATGGAGGTAGAAAAAGTGCAA
R1	TTAACAAGCTCCCTCAGTGA

表2 藜麦染色体位置和基因结构

Table 2 Chenopodium quinoa chromosome location and gene structure

基因编号Gene code	基因ID Gene ID	染色体Chromosome	染色体位置Genome location	长度Length/ bp
CqFLS1.1g	AUR62004717	Chr01	122 518 204-122 519 817	1 613
CqFLS2.1g	AUR62014672	Chr01	28 868 426-28 870 600	2 174
CqFLS3.10g	AUR62023632	Chr10	243 450-244 961	1 511

图1 CqFLSs 基因结构

Fig. 1 Gene structure of CqFLSs

表3 藜麦FLS基因家族理化性质

Table 3 Physical and chemical characteristics of CqFLSs

蛋白名称 Protein name	氨基酸数量 Number of amino acids	分子量 Molecular weight	等电点 Theoretical pI	不稳定指数Instability index	脂肪族指数 Aliphatic index	亲疏水性（GRAVY） Grand average of hydropathicity
CqFLS3.10g	346	39496.05	6.04	44.05	85.61	-0.544
CqFLS2.1g	339	38423.03	6.28	42.27	85.40	-0.492
CqFLS1.1g	346	39409.91	6.04	46.50	84.48	-0.549

表4 CqFLSs蛋白二级结构和亚细胞定位

Table 4 Secondary structure and subcellular localization of CqFLSs

蛋白名称 Protein name	α-螺旋 Alpha helix/%	延伸链 Extended strand/%	β-折叠 Beta turn/%	无规则卷曲 Random coil/%	亚细胞定位 Intracellular location/%	可能性 Probabily/%
CqFLS3.10g	31.79	18.21	5.78	44.22	Cytoplasmic	65.2
CqFLS2.1g	33.92	17.99	5.60	42.48	Cytoplasmic	65.2
CqFLS1.1g	32.95	19.65	6.36	41.04	Cytoplasmic	65.2

图2 CqFLS蛋白三级结构预测

Fig. 2 Tertiary structure prediction of CqFLS proteins

图3 藜麦CqFLS蛋白分析 A：CqFLS蛋白结构域分析; B：CqFLS蛋白保守基序分析; C：CqFLS蛋白序列比对分析

Fig. 3 Analysis of CqFLS proteins A: Domain analysis of CqFLS proteins; B: conserved motif analysis of CqFLS proteins; C: protein sequence alignment analysis of CqFLS

图4 藜麦与甜菜、大豆、油菜和拟南芥FLS系统进化树分析

Fig. 4 Phylogenetic tree of FLSs in C. quinoa, Beta vulgaris, Glycine max, Brassica napus and Arabidopsis thaliana

图5 STRING预测CqFLS蛋白质的相互作用伙伴

Fig. 5 STRING prediction of CqFLS interactional partners

图6 CqFLSs基因启动子区的顺式作用调控元件核心序列及功能

Fig. 6 Core sequences and function of the cis-acting regulatory elements in promoter regions of CqFLSs

表5 藜麦品系CqFLSs的DNA变异检测

Table 5 DNA variation of CqFLSs among quinoa lines

基因编号Gene code	位置POS	参考REF	变异ALT	品系Q3	品系Q6	品系Q8	品系Q9	影响Effect
CqFLS2.1g	28871893	CT	C	C	N	C	C	UPSTREAM
CqFLS2.1g	28871125	TA	T	N	T	T	T	UPSTREAM
CqFLS2.1g	28872881	TTA	T	N	N	N	T	UPSTREAM
CqFLS2.1g	28872134	T	A	A	A	A	N	UPSTREAM
CqFLS2.1g	28871765	C	T	T	N	T	T	UPSTREAM
CqFLS2.1g	28871453	T	G	G	G	G	G	UPSTREAM
CqFLS2.1g	28870264	C	T	T	T	T	N	SYNONYMOUS_CODING
CqFLS2.1g	28871914	A	T	T	N	T	T	UPSTREAM
CqFLS2.1g	28871369	G	A	A	A	A	A	UPSTREAM
CqFLS2.1g	28870706	T	G	G	N	G	N	UPSTREAM
CqFLS2.1g	28872268	T	C	C	C	N	C	UPSTREAM
CqFLS2.1g	28872086	G	A	A	A	A	N	UPSTREAM
CqFLS2.1g	28871206	G	T	T	N	T	T	UPSTREAM

表6 CqFLSs家族成员进化选择参数

Table 6 Evolutionary selection parameters for members of the CqFLSs

基因编号 Gene code	非同义突变率 K_a	非同义突变率 K_s	比值 K_a/K_s
CqFLS3.10g	0.104881	0.726108	0.144443
CqFLS2.1g	0.140285	0.742346	0.188976
CqFLS1.1g	0.102561	0.767349	0.133656

图7 藜麦FLS在不同品种籽粒中的表达水平 10 d、20 d、30 d分别指授粉后10 d、20 d、30 d

Fig. 7 Expressions of FLS in different varieties of quinoa seeds 10 d, 20 d, 30 d refer to 10 days 20 days and 30 days after pollination, respectively

图8 CqFLS1.1g基因PCR扩增电泳 M：Trans2K Plus II DNA maker; 1：菌液PCR产物

Fig. 8 PCR amplification electrophoresis of CqFLS1.1g M: Trans2K Plus II DNA maker; 1: PCR results of bacterial solution

图9 CqFLS1.1g 原核表达（A）及蛋白纯化（B）的SDS-PAGE 分析图（A）M：Protein marker; 1：pET-28a（+）诱导前总蛋白; 2：pET-28a（+）载体对照; 3：PET-28a-CqFLS1.1g未加IPTG诱导; 4：20℃下IPTG诱导的上清; 5：20℃下IPTG诱导的沉淀; 6：37℃下IPTG诱导的上清; 7：37℃下IPTG诱导沉淀。（B）M：Protein marker; 1：最终纯化目的蛋白

Fig. 9 SDS-PAGE analysis of CqFLS1.1g prokaryotic expression（A）and protein purification（B）（A）M: Protein marker; 1: pET-28a（+）pre-induction total protein; 2: pET-28a（+）vector control; 3: PET-28a-CqFLS1.1g was induced without IPTG; 4: IPTG-induced supernatant at 20℃; 5: IPTG-induced precipitation at 20℃; 6: IPTG-induced supernatant at 37℃; 7: IPTG induces precipitation at 37℃.（B）M: Protein marker; 1: final purified target protein

参考文献 36

[1]	丰扬, 郭凤根, 王仕玉, 等. 藜麦Cq6GT基因的克隆与表达分析[J]. 植物生理学报, 2022, 58(10): 2017-2024.
	Feng Y, Guo FG, Wang SY, et al. Cloning and expression analysis of Cq6GT gene from Chenopodium quinoa[J]. Plant Physiol J, 2022, 58(10): 2017-2024.
[2]	Nowak V, Du J, Charrondière UR. Assessment of the nutritional composition of quinoa(Chenopodium quinoa Willd.)[J]. Food Chem, 2016, 193: 47-54.
[3]	Li XH, Kim YB, Kim Y, et al. Differential stress-response expression of two flavonol synthase genes and accumulation of flavonols in Tartary buckwheat[J]. J Plant Physiol, 2013, 170(18): 1630-1636.
[4]	Kou M, Li C, Song WH, et al. Identification and functional characterization of a flavonol synthase gene from sweet potato[Ipomoea batatas(L.) Lam.][J]. Front Plant Sci, 2023, 14: 1181173.
[5]	Kimura S, Nakatsuka T, Yamada E, et al. A flavonol synthase gene GtFLS defines anther-specific flavonol accumulation in gentian[J]. Plant Biotechnol, 2010, 28(2): 211-221.
[6]	Harborne JB, Williams CA. Advances in flavonoid research since 1992[J]. Phytochemistry, 2000, 55(6): 481-504. doi: 10.1016/s0031-9422(00)00235-1 pmid: 11130659
[7]	Deis L, Cavagnaro B, Bottini R, et al. Water deficit and exogenous ABA significantly affect grape and wine phenolic composition under in field and in-vitro conditions[J]. Plant Growth Regul, 2011, 65(1): 11-21.
[8]	Iwashina T. The structure and distribution of the flavonoids in plants[J]. J Plant Res, 2000, 113(3): 287-299.
[9]	Ishikura N, Yoshitama K. Anthocyanin-flavonol co-pigmentation in blue seed Coats of Ophiopogon jaburan[J]. J Plant Physiol, 1984, 115(2): 171-175.
[10]	Hichri F, Ben Salah N, Omri A, et al. New antioxidant C-glycosyl flavone and flavonol derivatives from the Tunisian Achille acretica L[J]. S Afr N J Bot, 2018, 116: 1-5.
[11]	Marzouk MS, Moharram FA, Haggag EG, et al. Antioxidant flavonol glycosides from Schinus molle[J]. Phytother Res, 2006, 20(3): 200-205.
[12]	Corsino J, Silva DHS, Zanoni MVB, et al. Antioxidant flavan-3-ols and flavonol glycosides from Maytenus aquifolium[J]. Phytother Res, 2003, 17(8): 913-916.
[13]	Dias TA, Duarte CL, Lima CF, et al. Superior anticancer activity of halogenated chalcones and flavonols over the natural flavonol quercetin[J]. Eur J Med Chem, 2013, 65: 500-510. doi: 10.1016/j.ejmech.2013.04.064 pmid: 23771043
[14]	Britton RG, Horner-Glister E, Pomenya OA, et al. Synthesis and biological evaluation of novel flavonols as potential anti-prostate cancer agents[J]. Eur J Med Chem, 2012, 54: 952-958. doi: 10.1016/j.ejmech.2012.06.031 pmid: 22789812
[15]	Li SG, Dong P, Wang JW, et al. Icariin, a natural flavonol glycoside, induces apoptosis in human hepatoma SMMC-7721 cells via a ROS/JNK-dependent mitochondrial pathway[J]. Cancer Lett, 2010, 298(2): 222-230. doi: 10.1016/j.canlet.2010.07.009 pmid: 20674153
[16]	Ortega YH, Foubert K, Vanden Berghe W, et al. Flavonol glycosides from the leaves of Boldoa purpurascens and their anti-inflammatory properties[J]. Phytochem Lett, 2017, 19: 71-76.
[17]	Tahiri O, Atmani-Kilani D, Sanchez-Fidalgo S, et al. The flavonol-enriched Cistus albidus chloroform extract possesses in vivo anti-inflammatory and anti-nociceptive activity[J]. J Ethnopharmacol, 2017, 209: 210-218.
[18]	Granica S, Czerwińska ME, Żyżyńska-Granica B, et al. Antioxidant and anti-inflammatory flavonol glucuronides from Polygonum aviculare L[J]. Fitoterapia, 2013, 91: 180-188.
[19]	Şöhretoğlu D, Sari S, Barut B, et al. Discovery of potent α-glucosidase inhibitor flavonols: insights into mechanism of action through inhibition kinetics and docking simulations[J]. Bioorg Chem, 2018, 79: 257-264. doi: S0045-2068(18)30332-8 pmid: 29778797
[20]	Sendrayaperumal V, Iyyam Pillai S, Subramanian S. Design, synthesis and characterization of zinc-morin, a metal flavonol complex and evaluation of its antidiabetic potential in HFD-STZ induced type 2 diabetes in rats[J]. Chem Biol Interact, 2014, 219: 9-17.
[21]	Sun YJ, He JM, Kong JQ. Characterization of two flavonol synthases with iron-independent flavanone 3-hydroxylase activity from Ornithogalum caudatum Jacq[J]. BMC Plant Biol, 2019, 19(1): 195.
[22]	Britsch L, Dedio J, Saedler H, et al. Molecular characterization of flavanone 3 beta-hydroxylases. Consensus sequence, comparison with related enzymes and the role of conserved histidine residues[J]. Eur J Biochem, 1993, 217(2): 745-754. pmid: 8223617
[23]	Kim YB, Kim K, Kim Y, et al. Cloning and characterization of a flavonol synthase gene from Scutellaria baicalensis[J]. Sci World J, 2014, 2014: 980740.
[24]	Kim BG, Joe EJ, Ahn JH. Molecular characterization of flavonol synthase from poplar and its application to the synthesis of 3-O-methylkaempferol[J]. Biotechnol Lett, 2010, 32(4): 579-584.
[25]	Falcone Ferreyra ML, Rius S, Emiliani J, et al. Cloning and characterization of a UV-B-inducible maize flavonol synthase[J]. Plant J, 2010, 62(1): 77-91.
[26]	Li CL, Bai YC, Li SJ, et al. Cloning, characterization, and activity analysis of a flavonol synthase gene FtFLS1 and its association with flavonoid content in Tartary buckwheat[J]. J Agric Food Chem, 2012, 60(20): 5161-5168.
[27]	Xu F, Li LL, Zhang WW, et al. Isolation, characterization, and function analysis of a flavonol synthase gene from Ginkgo biloba[J]. Mol Biol Rep, 2012, 39(3): 2285-2296.
[28]	Artimo P, Jonnalagedda M, Arnold K, et al. ExPASy: SIB bioinformatics resource portal[J]. Nucleic Acids Res, 2012, 40(Web Server issue): W597-W603.
[29]	Geourjon C, Deléage G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J]. Comput Appl Biosci, 1995, 11(6): 681-684.
[30]	Almagro Armenteros JJ, Tsirigos KD, Sønderby CK, et al. SignalP 5.0 improves signal peptide predictions using deep neural networks[J]. Nat Biotechnol, 2019, 37(4): 420-423. doi: 10.1038/s41587-019-0036-z pmid: 30778233
[31]	姜晓东, 李新凤, 郝艳平, 等. 藜麦β-香树酯醇合酶和鲨烯合酶基因的克隆与表达[J]. 土壤, 2018, 50(6): 1214-1221.
	Jiang XD, Li XF, Hao YP, et al. Gene cloning and express of squalene synthase and β-amyrin synthase from Chenopodium quinoa[J]. Soils, 2018, 50(6): 1214-1221.
[32]	Dooner HK, Robbins TP, Jorgensen RA. Genetic and developmental control of anthocyanin biosynthesis[J]. Annu Rev Genet, 1991, 25: 173-199. pmid: 1839877
[33]	Balakrishnan G, Schneider RG. Quinoa flavonoids and their bioaccessibility during in vitro gastrointestinal digestion[J]. J Cereal Sci, 2020, 95: 103070.
[34]	Pelletier MK, Murrell JR, Shirley BW. Characterization of flavonol synthase and leucoanthocyanidin dioxygenase genes in Arabidopsis. Further evidence for differential regulation of “early” and “late” genes[J]. Plant Physiol, 1997, 113(4): 1437-1445. pmid: 9112784
[35]	Liu YJ, Liu JN, Kong ZY, et al. Transcriptomics and metabolomics analyses of the mechanism of flavonoid synthesis in seeds of differently colored quinoa strains[J]. Genomics, 2022, 114(1): 138-148.
[36]	宋奇琦, 张小秋, 宋修鹏, 等. 甘蔗HSP20基因克隆、原核表达及逆境胁迫响应[J]. 植物生理学报, 2022, 58(2): 371-380.
	Song QQ, Zhang XQ, Song XP, et al. Cloning and prokaryotic expression of sugarcane HSP20 gene and its responses to adversity stress[J]. Plant Physiol J, 2022, 58(2): 371-380.