2018年12月16日 星期日
茶树鸟苷酸交换因子CsRopGEF1和CsRopGEF3 基因的克隆及表达特性
Cloning and expression properties of CsRopGEF1 and CsRopGEF3 genes of guanine nucleotide exchange factor in Camellia sinensis
2018年 第27卷 第4期 页码[1-10]
摘要

从茶树〔Camellia sinensis (Linn.) O. Ktze.〕品种‘龙井长叶’(‘Longjingchangye’)叶片中克隆获得鸟苷酸交换因子RopGEF的编码基因CsRopGEF1和CsRopGEF3;用生物信息学方法分析了这2个基因编码的氨基酸序列的同源性及理化性质;并用qRT-PCR研究了CsRopGEF1和CsRopGEF3基因在茶树不同组织中的表达模式及其对外源ABA处理的响应模式。结果显示:茶树CsRopGEF1和CsRopGEF3基因全长分别为1 844和1 509 bp,分别包含长度1 731和1 434 bp的开放阅读框,各编码576和477个氨基酸。茶树CsRopGEF1和CsRopGEF3基因编码的氨基酸序列均含有保守的PRONE结构域,其中均包含保守基序Motif 1至Motif 7;分子式分别为C2797H4402N762O856S32和C2356H3696N648O752S29,理论相对分子质量分别为63 420和54 060,理论等电点分别为pI 6.69和pI 5.10,不稳定系数均大于40,亲水性平均系数分别为-0.368和-0.552,并存在多个磷酸化位点,以丝氨酸磷酸化位点最多,表明这2个基因编码的氨基酸序列均属于不稳定的酸性、亲水性蛋白,且其功能可能受磷酸化调控。茶树CsRopGEF1和CsRopGEF3基因编码的氨基酸序列与水稻(Oryza sativa Linn.)等植物的RopGEF1和RopGEF3氨基酸序列的相似度均在75%以上,且与双子叶植物同类氨基酸序列的同源性较高,而与单子叶植物同类氨基酸序列的同源性较低。qRT-PCR分析结果显示:茶树CsRopGEF1基因的相对表达量在茎中最高、叶片中最低,且差异显著(P<0.05);而CsRopGEF3基因的相对表达量在根和茎中均较高,且均显著高于叶片,表明CsRopGEF1和CsRopGEF3基因表达存在组织特异性。经100 μmol·L-1 外源ABA处理后,茶树CsRopGEF1基因的相对表达量整体显著下调,而CsRopGEF3基因的相对表达量则整体上调,说明这2个基因对外源ABA的响应模式存在差异。根据研究结果,推测茶树CsRopGEF1和CsRopGEF3基因在ABA信号转导途径中受不同的调控作用,且可能在茶树根和茎的生长发育中发挥重要作用。

 

Abstract

 The coding gene CsRopGEF1 and CsRopGEF3 of guanine nucleotide exchange factor RopGEF were cloned from leaves of cultivar ‘Longjingchangye’ of Camellia sinensis (Linn.) O. Ktze.; the homology and physicochemical properties of amino acid sequences encoded by these two genes were analyzed by bioinformatics method; the expression patterns of CsRopGEF1 and CsRopGEF3 genes in different tissues of C. sinensis and their response patterns to exogenous ABA treatment were studied by qRT-PCR. The results show that the full lengths of CsRopGEF1 and CsRopGEF3 genes from C. sinensis are 1 844 and 1 509 bp, which contain open reading frames with length of 1 731 and 1 434 bp, and encode 576 and 477 amino acids, respectively. Both amino acid sequences encoded by CsRopGEF1 and CsRopGEF3 genes from C. sinensis contain conserved PRONE domain with conserved motif Motif 1 to Motif 7; the molecular formulas are C2797H4402N762O856S32 and C2356H3696N648O752S29, the theoretical relative molecular masses are 63 420 and 54 060, and the theoretical isoelectric points are pI 6.69 and pI 5.10, the instability coefficients are all greater than 40, the average hydrophilicity coefficient are -0.368 and -0.552, respectively, and there are multiple phosphorylation sites, and phosphorylation sites of serine are the most, it is indicating that the amino acid sequences encoded by these two genes belong to unstable acidic and hydrophilic proteins, and their functions may be regulated by phosphorylation. The similarity degree between amino acid sequences encoded by CsRopGEF1 and CsRopGEF3 genes from C. sinensis and those of RopGEF1 and RopGEF3 from Oryza sativa Linn., etc. is higher than 75%, and the homology with the same kind of amino acid sequence from dicotyledons is higher, but that with the same kind of amino acid sequence from monocotyledons is lower. The result of qRT-PCR analysis shows that the relative expression level of CsRopGEF1 gene from C. sinensis is the highest in stem and the lowest in leaf, and the difference is significant (P<0.05); the relative expression level of CsRopGEF3 gene is higher both in root and stem, and significantly higher than that in leaf, it is indicating that the expressions of CsRopGEF1 and CsRopGEF3 genes show tissue specificity. After 100 μmol·L-1 exogenous ABA treatment, the relative expression level of CsRopGEF1 gene from C. sinensis is generally significantly downregulated, while that of CsRopGEF3 gene is generally upregulated, it is indicating that these two genes have different response patterns to exogenous ABA. According to the results, it is speculated that CsRopGEF1 and CsRopGEF3 genes from C. sinensis are regulated differently in ABA signal transduction pathway, and may play important roles in growth and development of root and stem of C. sinensis.
 

关键词茶树; RopGEF基因; 理化特性; 同源性; 表达特性
Key wordsCamellia sinensis (Linn.) O. Ktze.; RopGEF gene; physicochemical property; homology; expression property
作者赵悦1, 申加枝1, 马媛春1, 文博1, 周琳1,2, 朱旭君1, 王玉花1, 房婉萍1
所在单位1. 南京农业大学茶叶科学研究所, 江苏 南京 210095; 2. 上海市农业科学院林木果树研究所, 上海 201106
点击量75
基金项目国家自然科学基金资助项目(31370688; 31770733); 江苏省科技品种后补助项目(BE2016417); 国家茶叶产业技术体系农业部财政专项(CARS-19); 江苏省现代农业(茶叶)产业技术体系良种繁育(SXGC[2017]279)