非洲菊GjMnSOD基因的克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0077

摘要/Abstract

摘要：

以非洲菊品种‘玲珑’为材料,利用RT-PCR技术克隆获得一条CDS长为519 bp的SOD基因（GenBank ID:MH708534.1）。利用生物信息学手段分析了该基因及其编码蛋白的序列特征,并利用实时荧光定量PCR（qRT-PCR）技术分析了该基因在3种激素（100 µmol/L IAA、SA和GA₃）和3种非生物胁迫（200 mmol/L NaCl盐胁迫、30% PEG模拟干旱和4℃低温处理）下的表达模式。结果显示:该基因可编码分子量为19 203.85、等电点（pI）为7.93、无信号肽及跨膜结构的稳定亲水蛋白。该蛋白含有典型Sod-Fe-C保守结构域,在141-148位氨基酸之间含有一个MnSOD特征序列（DVWEHAYY）,因此命名为GjMnSOD。Wolf Psort预测结果显示GjMnSOD定位于线粒体。GjMnSOD与刺苞菜蓟MnSOD相似性最高（为93.86%）且在进化上亲缘关系最近。qRT-PCR结果显示:GjMnSOD的表达受IAA、GA₃、SA、盐胁迫和低温显著抑制;在干旱胁迫下,GjMnSOD的表达呈“先降后升”的表达趋势,于处理4 h后显著高于对照。研究结果表明GjMnSOD参与非洲菊对多种逆境胁迫的响应,尤其在非洲菊应答干旱胁迫过程中发挥重要作用。

关键词: 非洲菊, 锰超氧化物歧化酶, 基因克隆, 表达分析, 逆境胁迫响应

Abstract:

Having the ‘Linglong’ Gerbera jamesonni as experimental material,RT-PCR technology was used to successfully clone a SOD gene（GenBank accession number:MH708534.1）with CDS length of 519 bp. Then,bioinformatics analysis was to analyze the sequences of the gene and its encoded protein,and quantitative real time PCR（qRT-PCR）was adapted to investigate the gene expression patterns under treatments of 3 phytohormones（100 µmol/L IAA,GA₃ and SA）and 3 abiotic stresses（salt stress using 200 mmol/L NaCl,mimicked drought stress using 30% PEG,and cold stress using 4℃）. Results showed that the SOD gene encoded a stable hydrophilic protein with no signal peptide and transmembrane structure and its molecular weight was 19 203.85 and pI was 7.93. The protein contained typical Sod-Fe-C conserved domain and a MnSOD characteristic sequence（DVWEHAYY）ranging from 141 ^th to 148^th amino acids,thus it was named as GjMnSOD. Wolf Psort prediction revealed that GjMnSOD was located in mitochondria. GjMnSOD shared the highest similarity（93.86%）with Cynara cardunculus MnSOD,and they were in the closest genetic relationship. qRT-PCR result showed that the expression of GjMnSOD was significantly inhibited by IAA,SA and GA₃treatments,salt and 4℃ low temperature stresses. Under drought treatment,it showed ‘fall-rise’ expression pattern and its expression level was found to be significantly higher than control from 4 h post-treatment. The above results indicate that GjMnSOD is widely involved in the stress responses of G. jamesonni,especially in drought response.

Key words: Gembera jamesonni, manganese superoxide dismutase, gene cloning, expression analysis, stress response

武欢, 卢珍红, 郝向阳, 王斌, 焦元辰, 杨春梅, 程春振. 非洲菊GjMnSOD基因的克隆及表达分析[J]. 生物技术通报, 2021, 37(10): 17-25.

WU Huan, LU Zhen-hong, HAO Xiang-yang, WANG Bin, JIAO Yuan-chen, YANG Chun-mei, CHENG Chun-zhen. Cloning and Expression Analysis of GjMnSOD Gene in Gerbera jamesonni[J]. Biotechnology Bulletin, 2021, 37(10): 17-25.

图/表 8

图1 cDNA扩增产物电泳检测结果 M:DL2000 marker;1:目的条带

Fig. 1 Electrophoresis detection result of cDNA amplifica-tion products M: DL2000 marker; 1: target band

图2 GjMnSOD蛋白磷酸化位点及保守结构域预测结果 A:磷酸化位点预测;B:保守结构域预测

Fig.2 Prediction of GjMnSOD protein phosphorylation site and conserved domain A:Phosphorylation site prediction; B:conserved domain prediction

图3 GjMnSOD二级结构预测结果

Fig. 3 Prediction of secondary structure of GjMnSOD

表1 GjMnSOD蛋白保守基序

Table 1 GjMnSOD protein conserved motif

基序Motif	E-value	宽度Width	位点Location
RAIDQNFG SAIKFNGG	1.8e+001	8	76-84 40-47
FWKNLTP QYKNVRP	2.3e+001	7	55-61 150-156
GSGWVW VGIDVW	2.8e+002	6	101-106 138-143
LTAKMN YITNYN	3.4e+002	6	88-93 12-17
HQKHHQ HVNHST	3.3e+002	6	5-10 49-54
KYMESD KGDAST	7.9e+002	6	161-166 29-34
ETTANQ EGAAVQ	1.0e+003	6	119-124 95-100
PPHGSL PTLVPL	2.0e+003	6	69-74 132-137
KELKPL KALEQL	4.9e+003	6	111-116 18-23
TREGGG KLERPK	7.0e+003	6	62-69 167-172
EHAYYL DPLVTK	1.0e+004	6	144-149 125-130

图4 GjMnSOD三级结构 A:蛋白三级结构图;B:活性位点示意图

Fig. 4 Predicted tertiary structure of GjMnSOD A: Tertiary structure diagram of protein; B: Schematic diagram of active site

图5 不同植物MnSOD蛋白序列多重比对结果红框内为MnSOD特征序列

Fig. 5 Multiple alignment results of MnSOD proteins from different plants In the red box: MnSOD characteristic sequence

图6 非洲菊GjMnSOD与其他植物MnSOD系统进化树

Fig. 6 Phylogenetic tree of GjMnSOD in G. jamesonni and MnSOD proteins from other plants

图7 激素与非生物胁迫处理对GjMnSOD相对表达量的影响 A:IAA处理;B:GA3处理;C:SA处理;D:盐胁迫处理;E:PEG处理;F:低温（4℃）胁迫;不同字母表示0.01水平上有显著差异

Fig. 7 Effects of hormone and abiotic stresses on the relative expression of GjMnSOD A: IAA treatment; B: GA3 treatment; C: SA treatment; D: salt stress treatment; E: PEG treatment; F: low temperature (4℃) stress. Different letters indicate significant differences at the 0.01 level

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