多根紫萍SpLAR基因的克隆、表达分析及表达载体构建

doi:10.14088/j.cnki.issn0439-8114.2025.07.021

摘要/Abstract

摘要： 为探究多根紫萍(Spirodela polyrrhiza)中无色花青素还原酶编码基因SpLAR在原花青素生物合成中的作用,基于多根紫萍转录组数据获取SpLAR全长cDNA序列,成功克隆该基因;对SpLAR蛋白的特性进行预测,并检测了不同培养天数及营养胁迫条件下SpLAR基因的表达水平;通过测定相应条件下原花青素的积累量,进一步分析了SpLAR基因表达量与原花青素积累量之间的相关性;并采用同源重组技术构建了pCAMBIA1301-SpLAR植物表达载体。生物信息学分析结果显示,该基因全长1 059 bp,编码352个氨基酸,预测分子质量为38 ku,为亲水性不稳定蛋白,含有一个跨膜结构域,无信号肽,属于NADB_Rossmann超家族,SpLAR蛋白与芋(Colocasia esculenta)同源蛋白相似性最高;RT-qPCR及含量测定结果表明,SpLAR基因的表达量呈先升高后降低的动态变化趋势,且与原花青素积累量呈正相关,Datko处理下,在9 d时SpLAR基因表达量最高,此时原花青素积累量达到峰值20.6 mg/g。营养胁迫处理可显著促进SpLAR基因的表达及原花青素的生物合成,使积累量在9 d提升至24.8 mg/g。

关键词: 多根紫萍(Spirodela polyrrhiza), SpLAR基因, 生物信息学, 表达分析, 载体构建

Abstract: To investigate the role of the leucoanthocyanidin reductase-encoding gene SpLAR in proanthocyanidin biosynthesis in Spirodela polyrrhiza, the full-length SpLAR cDNA sequence was obtained from transcriptome data and the gene was successfully cloned. The characteristics of the SpLAR protein were predicted, and the expression levels of the SpLAR gene under different culture days and nutrient stress conditions were detected. Additionally, by measuring the accumulation of proanthocyanidins under corresponding conditions, the correlation between the expression level of the SpLAR gene and the accumulation of proanthocyanidins was further analyzed. The pCAMBIA1301-SpLAR plant expression vector was constructed using homologous recombination technology. Bioinformatics analysis results showed that the gene was 1 059 bp in length, encoding 352 amino acids, with a predicted molecular weight of 38 ku. It was a hydrophilic and unstable protein, containing one transmembrane domain, having no signal peptide, and belonging to the NADB_Rossmann superfamily. The SpLAR protein had the highest similarity with the homologous protein from Colocasia esculenta. RT -qPCR and content determination results indicated that the expression level of the SpLAR gene showed a dynamic trend of first increasing and then decreasing, and was positively correlated with the accumulation of proanthocyanidins. Under Datko treatment, the expression level of the SpLAR gene was the highest at 9 d, and at this time, the accumulation of proanthocyanidins reached a peak of 20.6 mg/g. In addition, the nutrient stress treatment could significantly promote the expression of the SpLAR gene and the biosynthesis of proanthocyanidins, increasing the accumulation to 24.8 mg/g at 9 d.

Key words: Spirodela polyrrhiza, SpLAR gene, bioinformatics, expression analysis, vector construction

中图分类号:

R932
Q812

刘文英, 左研熙, 何舒萍, 杨璞, 向蓓蓓. 多根紫萍SpLAR基因的克隆、表达分析及表达载体构建[J]. 湖北农业科学, 2025, 64(7): 120-127.

LIU Wen-ying, ZUO Yan-xi, HE Shu-ping, YANG Pu, XIANG Bei-bei. Cloning , expression analysis and expression vector construction of the SpLAR gene in Spirodela polyrrhiza[J]. HUBEI AGRICULTURAL SCIENCES, 2025, 64(7): 120-127.

参考文献

[1] 杨晶晶,赵旭耀,李高洁,等.浮萍的研究及应用[J].科学通报,2021,66(9):1026-1045.
[2] ZHANG Y M,JIA R,HUI T Y,et al.Transcriptomic and physiological analysis of the response of Spirodela Polyrrhiza to sodium nitroprusside[J].BMC plant biology,2024,24(1):95.
[3] KIM J,PARK S,LEE H.Antioxidant properties of Lemna minor extracts and their potential applications in food preservation[J].Journal of food science,2021,86(4):1650-1660.
[4] XU J W,SHEN Y T,ZHENG Y,et al.Duckweed (Lemnaceae) for potentially nutritious human food: A review[J].Food reviews international,2023,39(7):3620-3634.
[5] JUN J H,XIAO X R,RAO X L,et al.Proanthocyanidin subunit composition determined by functionally diverged dioxygenases[J].Nature plants,2018,4(12):1034-1043.
[6] 刘玟君,李金洲,陈子隽,等.原花青素的研究进展[J].湖北农业科学,2021,60(14):5-9.
[7] CHEN Q,LIANG X,WU C L,et al.Overexpression of leucoanthocyanidin reductase or anthocyanidin reductase elevates tannins content and confers cassava resistance to two-spotted spider mite[J].Frontiers in plant science,2022,13:994866.
[8] WANG Y,ZHANG X,LIU H.Health benefits of proanthocyanidins: A review of the evidence[J].Journal of nutritional biochemistry,2020,120:150-165.
[9] SEFERLI M,KOTANIDOU C,LEFKAKI M,et al.Bioactives of the freshwater aquatic plants, Nelumbo nucifera and Lemna minor, for functional foods, cosmetics and pharmaceutical applications, with antioxidant, anti-inflammatory and antithrombotic health promoting properties[J].Applied sciences,2024,14(15):6634.
[10] LIU Y,LI C T,YAN R T,et al.Metabolome and transcriptome analyses of the flavonoid biosynthetic pathway for the efficient accumulation of anthocyanins and other flavonoids in a new duckweed variety (68-red)[J].Journal of plant physiology,2022,275:153753.
[11] LIU C G,WANG X Q,SHULAEV V,et al.A role for leucoanthocyanidin reductase in the extension of proanthocyanidins[J].Nature plants,2016,2(12):16182.
[12] 贾展慧,贾晓东,许梦洋,等.薄壳山核桃原花青素合成关键酶基因的克隆与表达分析[J].南京林业大学学报(自然科学版),2022,46(5):49-57.
[13] 曾益春,刘刚,代洁,等.桑树花青素合成相关基因MaLAR和MaUGAT的克隆与表达分析[J].蚕业科学,2023,49(5):385-394.
[14] SUN S H,QI X J,ZHANG Z Z,et al.A structural variation in the promoter of the leucoanthocyanidin reductase gene AaLAR1 enhances freezing tolerance by modulating proanthocyanidin accumulation in kiwifruit (Actinidia arguta)[J].Plant, cell & environment,2024,47(10):4048-4066.
[15] PAOLOCCI F,ROBBINS M P,MADEO L,et al.Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. structure, expression analysis, and genetic control of Leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus[J].Plant physiology,2007,143(1):504-516.
[16] WANG L J,JIANG Y Z,YUAN L,et al.Isolation and characterization of cDNAs encoding leucoanthocyanidin reductase and anthocyanidin reductase from Populus trichocarpa[J].PLoS one,2013,8(5): e64664.
[17] WANG P Q,ZHANG L J,JIANG X L,et al.Evolutionary and functional characterization of leucoanthocyanidin reductases from Camellia sinensis[J].Planta,2018,247(1):139-154.
[18] LIU Y,SHI Z,MAXIMOVA S,et al.Proanthocyanidin synthesis in Theobroma cacao: Genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase[J].BMC plant biology,2013, 13:202.
[19] TAO X,FANG Y,XIAO Y,et al.Comparative transcriptome analysis to investigate the high starch accumulation of duckweed (Landoltia punctata) under nutrient starvation[J].Biotechnology for biofuels,2013,6(72):1-15.
[20] 唐娅丽,于昌江,刘宇,等.浮萍合成生物学研究进展[J].生命科学,2020,32(2):100-109.
[21] LIU Y,WANG Y,XU S Q,et al.Efficient genetic transformation and CRISPR/Cas9-mediated genome editing in Lemna aequinoctialis[J].Plant biotechnology journal,2019,17(11):2143-2152.
[22] 李志丹,方扬,靳艳玲,等.少根紫萍转录因子及其营养胁迫下的表达[J].应用与环境生物学报,2018,24(1):97-101.
[23] ZHU Y R,LI X X,GAO X,et al.Molecular mechanism underlying the effect of maleic hydrazide treatment on starch accumulation in S. Polyrrhiza 7498 fronds[J]. Biotechnology for biofuels,2021,14:99.
[24] 胡月,赵舒媛,张亚美,等.川西獐牙菜SmC4H基因的生物信息学分析和表达分析[J].分子植物育种,2024,22(8):2528-2536.
[25] TANNER G J,FRANCKI K T,ABRAHAMS S,et al.Proanthocyanidin biosynthesis in plants. Purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA[J].Journal of biological chemistry,2003, 278(34):31647-31656.
[26] CHEMLER J A,FOWLER Z L,MCHUGH K P,et al.Improving NADPH availability for natural product biosynthesis in Escherichia coli by metabolic engineering[J].Metabolic engineering,2010,12(2):96-104.
[27] BOGS J,DOWNEY M O,HARVEY J S,et al.Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves[J]. Plant physiology,2005,139(2):652-663.
[28] WANG L,LI S L,SUN L,et al.Over-expression of phosphoserine aminotransferase-encoding gene (AtPSAT1) prompts starch accumulation in L. turionifera under nitrogen starvation[J].International journal of molecular sciences,2022,23(19):11563.
[29] TAO X,FANG Y,HUANG M J,et al.High flavonoid accompanied with high starch accumulation triggered by nutrient starvation in bioenergy crop duckweed (Landoltia punctata)[J].BMC genomics,2017,18(1):166.
[30] MA B,SONG Y,FENG X H,et al.Integrated metabolome and transcriptome analyses reveal the mechanisms regulating flavonoid biosynthesis in blueberry leaves under salt stress[J].Horticulturae,2024,10(10):1084.
[31] RANKENBERG T,GELDHOF B,VAN VEEN H,et al.Age-dependent abiotic stress resilience in plants[J]. Trends in plant science,2021,26(7):692-705.