荸荠淀粉分支酶CwSBEII基因的克隆及其表达分析

Cloning and Expression of SBEII in Eleocharis tuberosa

【目的】克隆荸荠(Eleocharis tuberosa)淀粉分支酶CwSBEII基因,分析该基因的蛋白功能特性及其在荸荠不同组织以及不同膨大时期的表达模式,为进一步深入研究CwSBEII基因的功能提供理论参考。【方法】采用RTPCR的方法克隆得到CwSBEII基因的cDNA序列,通过生物信息学分析其蛋白功能特性,利用荧光定量PCR法检测该基因在荸荠球茎中的时空表达特征。【结果】成功克隆得到CwSBEII基因,该基因的开放阅读框(ORF)长度为2 547 bp,编码848个氨基酸。CwSBEII蛋白分子量为96 100.52 Da;理论等电点(pI)值为5.24,不稳定指数为41.07,GRAVY为-0.473,属于不稳定亲水蛋白。系统进化分析表明,荸荠与禾本科的小麦(Triticum aestivum)、水稻(Oryza sativa)、玉米(Zea mays)的SBEII蛋白聚为一支,亲缘关系最近。荧光定量PCR结果表明,CwSBEII基因在根、叶片、荸荠皮以及荸荠肉中均有表达,但在叶片中的表达量最高。在荸荠球茎膨大初期CwSBEII基因表达出现明显上调。【结论】CwSBEII基因在荸荠中特异性表达,在叶片中的表达量最高,膨大初期表达明显上调,推测该基因在荸荠淀粉合成中发挥着重要作用。

【Objective】 CwSBEII in Eleocharis tuberosa was cloned to understand the functions and expressions of the gene in various tissues and corms at different expansion stages. 【Methods】 The cDNA sequence of CwSBEII of Chinese water chestnut was cloned by RT-PCR, functions deciphered by bioinformatics, and spatiotemporal expressions in tissues and corms determined by fluorescence quantitative PCR. 【Results】 CwSBEII had an ORF length of 2 547 bp encoding 848 amino acids. The molecular weight of this unstable hydrophilic protein was 96 100.52 Da with a theoretical pI of 5.24, an instability index of 41.07, and a GRAVY reading at-0.473. It was closely related to and clustered with the SBEIIs of wheat(Triticum aestivum), rice(Oryza sativa), and maize(Zea Mays) of the grass family. Although found in the roots, leaves, peels as well as corm, CwSBEII was most highly expressed in the leaves and significantly upregulated in the early stage of corm expansion of Chinese water chestnut. 【Conclusion】 CwSBEII was specifically expressed in various tissues of Chinese water chestnut and most highly in the leaves. It was significantly upregulated during the early stage of corm expansion and might play an important role in the starch synthesis of E. tuberosa.