萱草3种蔗糖转化酶基因的分离及对低温和渗透胁迫响应的分析

doi:10.16420/j.issn.0513-353x.2020-0346

摘要/Abstract

摘要：

从‘Athlone’萱草（Hemerocallis fulva）中克隆了细胞质、细胞壁和液泡蔗糖转化酶基因各1个,命名为HfCIN3、HfCWIN1和HfVIN1,其ORF分别为1 941、1 701、1 920 bp,编码646、566和639个氨基酸,氨基酸序列相似性为18.81% ~ 41.86%。HfCIN3与石斛、野香蕉等的氨基酸序列相似性为75.16% ~ 82.79%,HfCWIN1与芦笋、油棕榈等的相似性为58.95% ~ 68.88%,HfVIN1与龙舌兰相似性为73.4%。HfCIN3具有信号肽和典型的β-fructofuranosidase domain、glycoside hydrolase family 100结构域,而HfCWIN1和HfVIN1有glycosyl hydrolases family 32特征结构域,HfCWIN1还含有72个氨基酸与细胞壁糖分子识别有关的Malectin domain,HfVIN1含有可能的液泡识别motif“ILPD”,并在N端有典型跨膜结构。HfCWIN1和HfVIN1同时存在N-糖基化位点和O-β-GlcNAc糖基化位点,HfCIN3仅有O-β-GlcNAc糖基化位点。系统进化分析显示,在不同类型转化酶分支里,可以较清楚地区分单子叶和双子叶植物,CWIN和VIN之间的亲缘关系较近,HfCIN3、HfCWIN1、HfVIN1聚于单子叶植物分支。瞬时表达分析显示,35S:HfCIN3-GFP在叶绿体中表达,35S:HfVIN1-GFP在液泡中表达,35S:HfCWIN1-YFP在细胞壁、保卫细胞中均有明显表达。HfCIN3、HfCWIN1、HfVIN1在萱草叶片中的表达水平显著高于根和花组织,在叶片中表达水平HfVIN1 > HfCWIN1 > HfCIN3。0 ℃处理24 h后,HfVIN1的表达水平较5 ℃上调2.2倍,HfCWIN1的表达也显著上调,HfCIN3的表达水平峰值出现在5 ℃,其他两个基因表达峰值出现在0 ℃。5%或10% PEG处理24 h后,HfCIN3和HfVIN1的表达水平与对照差异不显著,HfCWIN1显著低于对照。在不同组织中CIN酶活性与基因表达结果基本一致,而其他两种酶与基因表达不一致;随着温度降低,叶片中CIN酶活性整体呈下降趋势,CWIN先升高后降低,VIN先下降后升高再下降;PEG可以诱导VIN活性上升。低温和渗透胁迫处理后酶活性与基因表达存在不同步现象,推测基因转录后修饰和翻译后修饰对转化酶活性起着重要作用。本结果证实萱草3种转化酶的亚细胞定位明显不同,不同组织间基因表达有较大差异,HfVIN1响应低温胁迫,HfCWIN1响应渗透胁迫。

关键词: 萱草, 蔗糖, 转化酶, 非生物胁迫, 基因表达

Abstract:

In this study,three invertase genes were cloned from Hemerocallis fulva‘Athlone’,named HfCIN3,HfCWIN1 and HfVIN1. The ORFs of the three invertase genes were 1 941,1 701,1 920 bp,encoding 646,566 and 639 amino acids,respectively,with the similarity of 18.81%-41.86%. The amino acid sequence of HfCIN3 was 75.16%-82.79% similar to those of dendrobium and wild banana,HfCWIN1 was 58.95%-68.88% similar to asparagus and oil palm,and HfVIN1 was 73.4% similar to agave. HfCIN3 had a signal peptide and typical glycoside hydrolases family 100 domain,HfCWIN1 and HfVIN1 had glycosyl hydrolases family 32 domain. HfCWIN1 also contained a 72 amino acid Malectin domain related to cell wall sugar recognition. HfVIN1 contained a possible vacuolar motif“ILPD”and had a typical transmembrane domain at the N terminus. Both HfCWIN1 and HfVIN1 possessed the N-glycosylation and O-β-GlcNAc sites,whereas HfCIN3 only had the O-β-GlcNAc sites. Phylogenetic analysis showed that monocotyledons and dicotyledons could clearly be distinguished regarding different types of invertase;CWIN and VIN were closely related,and HfCIN3,HfCWIN1 and HfVIN1 were clustered in monocots. Transient expression analysis showed 35S:HfCIN3-GFP was expressed in chloroplasts;35S:HfVIN1-GFP was expressed in vacuoles;and 35S:HfCWIN1-YFP was detected in cell walls and guard cells. The expression levels of HfCIN3,HfCWIN1 and HfVIN1 were significantly higher in leaves than that of the root and flower tissues;the expression levels in leaves were HfVIN1 > HfCWIN1 > HfCIN3. After 24 h treatment at 0 ℃,the expression of HfVIN1 was increased by 2.2 times compared with 5 ℃,and the expression level of HfCWIN1 was increased significantly at 0 ℃ compared with the other temperatures. The highest expression of HfVIN3 was recorded at 0 ℃. After 24 h treatment with 5% or 10% PEG,the expression levels of HfCIN3 and HfVIN1 were not significantly different from the control,but that of HfCWIN1 was significantly lower than the control. The CIN activity and gene expression were similar in different tissues,but CWIN and VIN were not consistent with the gene expression. As temperature decreased,CIN declined,CWIN increased at first and then decreased,and VIN decreased at first then increased,and then decreased;PEG induced VIN activity. The enzyme activity and gene expression were not synchronized after the low temperature and osmotic stress treatment,possibly because the gene post-transcriptional modifications played an important role in invertase activity. The results of this study established the differences in subcellular localizations,gene expression patterns and the responses to abiotic stresses among the three various types of Hemerocallis fulva invertase,HfVIN1 responded to low temperature,whereas HfCWIN1 responded to osmotic stress.

Key words: Hemerocallis fulva, sucrose, invertase, abiotic stress, gene expression

中图分类号:

S682.1 ⁺9

白露, 张志国, 张世杰, 黄东梅, 秦巧平. 萱草3种蔗糖转化酶基因的分离及对低温和渗透胁迫响应的分析[J]. 园艺学报, 2021, 48(2): 300-312.

BAI Lu, ZHANG Zhiguo, ZHANG Shijie, HUANG Dongmei, QIN Qiaoping. Isolation of Three Types of Invertase Genes from Hemerocallis fulva and Their Responses to Low Temperature and Osmotic Stress[J]. Acta Horticulturae Sinica, 2021, 48(2): 300-312.

图/表 8

表1 本研究中所用的PCR引物序列

Table 1 PCR primer sequences used in this study

用途 Application	引物名称 Primer	引物序列（5′-3′） Sequence
cDNA克隆	HfVIN1-F1/R1	ATGGTACCGGACCAATGGTA/CAAGTTGGGGAAGTGTAAGGTGT
cDNA clone	HfCWIN1-F1/ R1	ATGGCTGCTGGTTCATTGCT/TTAGCTAATTATCGCTTTTGCCA
	HfCIN3-F1/ R1	ATGGGAGTTTGTGATTTATC/TCATACGATATATGTCTTCTTCA
qPCR	HfVIN1-F2/R2	CTCCTCCTCCCTTCCGATTC/CGGGCTTCATTAGTGTTGGG
	HfCWIN1-F2/R2	TGGTTCCGTGACCCTTCTAC/AAGTCCGGACATTCCCACAT
	HfCIN3-F2/R2	TGGCAACTACACAGCAATCG/AGGCCAAGAACCTCCATTGT
pBI221-GFP载体构建补英文。 Construction of PBI221-GFP vector	HfCIN3-F3/R3	gaggatctcgagcggtctagaATGGGAGTTTGTGATTTATC/agcggccgctgtacaggtaccTACGATATATGTCTTCTTCA
	HfVIN1-F3/R3	gaggatctcgagcggtctagaATGGGGTCACGTGACTTGGA/agcggccgctgtacaggtaccCAAGTTGGGGAAGTGTAAGG
	HfCWIN1-F3/R3	gaggatctcgagcggtctagaATGGCTGCTGGTTCATTGCT/agcggccgctgtacaggtaccGCTAATTATCGCTTTTGCCA
pC131-YFP载体构建 Construction of pC131-YFP vector	HfCWIN1-F4/R4	gaggatctcgagcgggaattcATGGCTGCTGGTTCATTGCT/agcggccgctgtacaactagtGCTAATTATCGCTTTTGCCA
qPCR内参补英文 qRT-PCR reference gene	HfUBQ-F/R	AACGTGAAGGCCAAGATAC/AGACGGAGCACCAGGTGGA

图1 萱草‘Athlone’转化酶基因的克隆

Fig. 1 Cloning of invertase genes from‘Athlone’daylily

表2 萱草3种转化酶基因编码蛋白质的理化性质

Table 2 Predicted physical and chemical characteristics of three types of invertase from daylily

理化性质 Characteristics	分子量/kD Molecular weight	pI	不稳定性指数 Instability index	亲水性平均系数 Grand average of hydropathicity	磷酸化位点Phosphoryla- tion sites	糖基化位点Glycosylation site
理化性质 Characteristics	分子量/kD Molecular weight	pI	不稳定性指数 Instability index	亲水性平均系数 Grand average of hydropathicity	磷酸化位点Phosphoryla- tion sites	N-Glycosyl- ation	O-β-GlcNAc
HfCIN3	74.840	6.35	46.98（不稳定Unstable）	-0.275	5	-	√
HfCWIN1	63.496	5.38	38.66（稳定Stable）	-0.326	0	√	√
HfVIN1	71.340	5.33	37.42（稳定Stable）	-0.312	6	√	√

图2 萱草3种转化酶基因编码的氨基酸序列比对 A：Clustal序列比对,TMHMM和NCBI domain预测;B：MEME motif预测。

Fig. 2 Alignment of amino acid sequences encoded by three types of invertase from daylily A：Clustal alignment,domain prediction by TMHMM and NCBI;B：Motif Prediction by MEME. Glyco_hydro_100：Glycosyl hydrolases family 100;Glyco_hydro_32N：Glycosyl hydrolases family 32 N terminal domain; Glyco_hydro_32C：Glycosyl hydrolases family 32 C terminal domain.

图3 植物不同类型转化酶的系统进化树

Fig. 3 Phylogenetic tree of three types of invertase from different plants

图4 萱草转化酶GFP/YFP融合蛋白的表达

Fig. 4 Expression of fusion proteins of daylily invertases with GFP/YFP

图5 萱草转化酶在不同组织和低温及渗透胁迫下的基因表达变化不同小写字母表示相同基因不同处理间有显著差异（P < 0.05）。

Fig. 5 Expression of daylily invertase genes in different tissues and under low temperature or osmotic stress Different lowercase letters indicate significant differences among different treatments for the same gene（P < 0.05）.

图6 萱草转化酶在不同组织和低温及渗透胁迫下的酶活性变化不同小写字母表示相同酶不同处理间有显著差异（P < 0.05）。

Fig. 6 Enzyme activities in different tissues and under low temperature or osmotic stress Different lowercase letters indicate significant differences among different treatments for the same enzyme（P < 0.05）.

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