沙田柚无机焦磷酸酶基因的cDNA克隆及序列分析

Cloning and sequence analysis of inorganic pyrophosphatase gene from Citrus grandis var. shatianyu

关键词:  沙田柚  无机焦磷酸酶  全长cDNA  基因克隆  序列分析

Key words:  Citrus grandis var. shatianyu  inorganic pyrophosphatase  full-length cDNA  gene cloning  sequence analysis

摘要:

植物自交不亲和性是植物生殖过程中普遍存在的一种现象,是植物特异性识别并拒绝自身花粉或亲缘关系很相近的花粉的一种遗传机制。无机焦磷酸酶(inorganic pyrophosphatase,IPPase)在植物生长发育方面起重要作用。该研究根据沙田柚花柱消减文库中EST序列(无机焦磷酸酶基因内部片段),设计了2对特异引物5'-GSP1,5'-nGSP1,3'-GSP2 and 3'-nGSP2,通过SMART-RACE PCR技术从所构建的沙田柚花柱抑制性消减文库中克隆了沙田柚无机焦磷酸酶基因的cDNA全长序列,利用Blastn、DNAman和Expasy软件对所克隆的基因进行同源性分析,以及基因编码的氨基酸的分子量、等电点、疏水性等理化性质分析。结果表明:IPPase基因cDNA全长为1 136 bp(GenBank登录号为KF990474),开放阅读框(ORF)全长为654 bp,共编码217个氨基酸,包括170 bp 5'UTR和312 bp的3'UTR; 编码的蛋白质的分子量为24.4 kDa,等电点为5.96; 蛋白结构域分析显示沙田柚IPPase与焦磷酸酶具有相同的保守结构域; 对沙田柚IPPase蛋白质序列进行疏水性分析,结果表明沙田柚IPPase基因编码的肽链中疏水性最大值约为3.21,最小值约为-2.98,属于亲水性蛋白,无跨膜区域; Blastn搜索的结果显示,沙田柚IPPase基因序列与多种植物的IPP基因高度同源; 序列分析表明,沙田柚IPPase基因核苷酸的同源性与毛果杨(Populus trichocarpa)和橡胶树(Hevea brasiliensis)IPPase基因均为87%; 氨基酸序列与克莱门柚(Citrus clementina)无机焦磷酸酶完全一致。该研究结果可为深入研究无机焦磷酸酶在沙田柚自交不亲和中的作用机理提供基础。

Abstract:

Self-incompatibility(SI)is the prevalence of phenomenon in the process of plant reproduction,it is an intraspecific reproductive barrier adopted by angiosperms that allows the pistil to distinguish between self(genetically related)and non-self(genetically unrelated)pollen. Inorganic pyrophosphatase(IPPase)play important roles in regulating the growth and development in plants. In order to better understand the mechanism of IPP gene in the self-incompatibility of Citrus grandis var. shatianyu,the inorganic pyrophosphatase gene of C. grandis var. shatianyu was cloned and physicochemical properties of pyrophosphatase were analyzed. The total RNA was isolated from style of C. grandis var. shatianyu used the total RNA Purification System(Invitrogen)and following the manufacture's protocols. According to the EST sequence(internal fragment of inorganic pyrophosphatase gene)in suppression subtractive hybridization libraries of C. grandis var. shatianyu style,4 specific primers 5'-GSP1,5'-nGSP1,3'-GSP2 and 3'-nGSP2 were designed for amplifying 3-RACE and 5-RACE of the gene. The full-length sequences of cDNA of inorganic pyrophosphatase gene were obtained from suppression subtractive hybridization libraries of C. grandis var. shatianyu style by the SMART-RACE PCR method. A comparison of the similarity of the full-length cDNA sequence of the inorganic pyrophosphatase gene was performed in the GenBank database used the BLAST program. DNAman software was used for mino acid sequence and homology analysis. Prediction of molecular weight,isoeletric point(pI)and hydrophobicity were performed by using on line software ExpASy and DNAman. A 150 bp band in 3-RACE,and a 900 bp band in 5-RACE were cloned by nested and non nested PCR method. For the full-length cDNA sequences,the middle sequence,3-RACE and 5-RACE sequence of the inorganic pyrophosphatase gene were spliced and formed the full-length cDNA sequences. As a result,the cDNA of inorganic pyrophosphatase was 1 136 bp in length containing an 654 bp open reading frame(ORF),which encoded a protein of 217 amino acids with a 170 bp 5 untranslated region(5 UTR)and a 321 bp 3 UTR. The sequence of the cloned cDNA of the inorganic pyrophosphatase from C. grandis var. shatianyu was registered in GenBank under the accession No. KF990474. In addition,through the DNAman software analysis,the deduced molecular weight of encoded protein of the full-length cDNA sequence was 24.4 kDa and theoretical pI value of 5.96. Protein domain analysis showed that IPPase of C. grandis var. shatianyu and pyrophosphatase have the same conserved domain. Bioinformatics analysis showed that the IPPase gene was a hydrophilic protein without any signal peptide,has no winded helix structure and transmembrane domain. The full length inorganic pyrophosphatase cDNA from C. grandis var. shatianyu was cloned and characterized. Blastn search results showed that the sequence of IPP gene of C. grandis var. shatianyu was highly homologous with IPP gene of variety plants. The homology analysis indicated that C. grandis var. Shatianyu IPPase gene shared 87% nucleotide sequence homology with Populus trichocarpa and Hevea brasiliensis IPPase,and 100% amino acid sequence homology with Citrus clementina IPPase. The research results would provide the basis for further exploration of the function of inorganic pyrophosphatase in self-incompatibility of C. grandis var. shatianyu.