2021年10月17日 星期日
甘薯查尔酮异构酶基因IbCHI的克隆和表达特性
Cloning and expression characteristics of chalcone isomerase gene IbCHI from Ipomoea batatas
2020年 第29卷 第6期 页码[1-10]    下载全文[12.5MB]  
摘要

根据甘薯〔Ipomoea batatas (Linn.) Lam.〕转录组数据,从甘薯品种‘福菜薯7-6’(‘Fucaishu 7-6’)中克隆获得1个编码查尔酮异构酶的基因,命名为IbCHI;IbCHI基因全长732 bp,GC含量为55.19%,共编码243个氨基酸。生物信息学分析结果表明:在IbCHI基因编码的氨基酸序列第12至第214位包含1个Chalcone结构域;IbCHI蛋白分子式为C1156H1831N291O351S7,理论相对分子质量为25 646.41,理论等电点为pI 5.25,并可能具有1个跨膜螺旋区,定位于细胞核和细胞膜上;其氨基酸序列中包含24个带正电荷和27个带负电荷的氨基酸残基,丙氨酸、丝氨酸、缬氨酸和甘氨酸所占比例较高,半胱氨酸所占比例最低;IbCHI蛋白的不稳定指数和总亲水性平均系数分别为38.49和0.074,表明IbCHI蛋白是一种稳定的亲水性蛋白质;IbCHI蛋白二级结构中,α螺旋、随机卷曲、延伸链和β转角所占比例分别为39.51%、31.69%、20.16%和8.64%。多重比对结果显示:IbCHI基因与同属种类圆叶牵牛(I. purpurea Lam.)和牵牛〔I. nil (Linn.) Roth〕CHI基因编码的氨基酸序列高度相似(相似度90%以上),且在系统发育树上聚为同一支。IbCHI基因启动子区包含启动子基本元件TATA-box和CAAT-box,以及胁迫响应元件、茉莉酸甲酯响应元件和光响应元件等26个顺式作用元件。qRT-PCR分析结果表明:IbCHI基因在供试4个甘薯品种‘EC15’、‘EC16’、‘福菜薯18’(‘Fucaishu 18’)和‘福菜薯7-6’的根、茎、茎尖和叶中均有表达,但其在不同品种和器官间的相对表达量存在明显差异,其中,‘福菜薯7-6’茎和叶中IbCHI基因的相对表达量显著高于其他3个品种。经干旱(质量体积分数30%PEG6000)和盐(0.2 mol·L-1 NaCl)处理后,IbCHI基因的相对表达量随处理时间的延长均呈先下降后上升的趋势,并在处理24 h后该基因的相对表达量显著(P<0.05)升高。综合分析结果表明:甘薯IbCHI蛋白属于Chalcone超级家族,具有较高的保守性;IbCHI基因的表达具有组织和品种特异性,且在调控甘薯对非生物胁迫的响应过程中起重要作用。

Abstract

 According to transcriptome data of Ipomoea batatas (Linn.) Lam., a gene named IbCHI encoding chalcone isomerase was cloned from I. batatas ‘fucaishu 7-6’; the full length of IbCHI gene is 732 bp, the GC content is 55.19%, and a total of 243 amino acids are encoded. Bioinformatics analysis result shows that there is a Chalcone domain at the position from 12th to 214th of amino acid sequence encoded by IbCHI gene; the molecular formula of IbCHI protein is C1156H1831N291O351S7, the theoretical relative molecular mass is 25 646.41, the theoretical isoelectric point is pI 5.25, and there may be a transmembrane helix region located on the nucleus and cell membrane; there are 24 positively charged and 27 negatively charged amino acid residues in its amino acid sequence, the proportion of alanine, serine, valine and glycine is higher, while that of cysteine is the lowest; the instability index and average coefficient of total hydrophilicity of IbCHI protein are 38.49 and 0.074, respectively, indicating that IbCHI protein is a stable hydrophilic protein; in the secondary structure of IbCHI protein, the proportion of α-helix, random coil, extended chain and β-turn is 39.51%, 31.69%, 20.16% and 8.64%, respectively. Multiple alignment result shows that the amino acid sequence encoded by IbCHI gene are highly similar (similarity more than 90%) to those encoded by CHI gene from the same genus of I. purpurea Lam. and I. nil (Linn.) Roth, and cluster into the same branch in phylogenetic tree. The promoter region of IbCHI gene contains 26 cis-acting elements including promoter common element of TATA-box and CAAT-box, and stress-responsive element, MeJA-responsive element and lightresponsive element, etc. The qRT-PCR analysis result shows that IbCHI gene is expressed in root, stem, stem tip and leaf of four test I. batatas cultivars of ‘EC15’, ‘EC16’, ‘Fucaishu 18’ and ‘Fucaishu 7-6’, but there are obvious differences in its relative expression among different cultivars and organs, in which, the relative expression of IbCHI gene in stem and leaf of ‘Fucaishu 7-6’ is significantly higher than that of other three cultivars. After drought (mass volume fraction of 30% PEG6000) and salt (0.2 mol·L-1NaCl) treatments, the relative expression of IbCHI gene shows a tendency to first decrease and then increase with the elongation of treatment time, and increases significantly (P<0.05) after treatment for 24 h. The comprehensive analysis results show that IbCHI protein belongs to Chalcone superfamily and has relatively high conservation; IbCHI gene expression has tissue and cultivar specificity, and plays an important role in regulating I. batatas response to abiotic stress.

关键词甘薯; IbCHI基因; qRT-PCR; 亚细胞定位; 表达特性; 非生物胁迫
Key wordsIpomoea batatas (Linn.) Lam.; IbCHI gene; qRT-PCR; subcellular location; expression characteristics; abiotic stress
作者饶莉萍1,2, 苏文瑾1,2, 刘意1,3, 宋天晓1,2, SOVIGUIDI Deka Reine Judess1, 张文英2, 杨新笋
所在单位1. 湖北省农业科学院粮食作物研究所, 湖北 武汉 430064; 2. 长江大学农学院, 湖北 荆州 434025; 3. 海南大学园艺学院, 海南 海口 570228
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基金项目国家重点研发计划(2018YFD1000700; 2018YFD1000705); 湖北省技术创新专项(对外科技合作类)(2018AHB012); 国家现代甘薯产业技术体系建设项目(CARS-11-C-15)