甘蔗可溶性酸性转化酶(SoSAI1)基因的克隆及表达分析

Cloning and Expression Analysis of A Soluble Acid Invertase Gene(SoSAI1) of Sugarcane

【目的】克隆甘蔗可溶性酸性转化酶基因(SoSAI1)全长cDNA序列和5侧翼启动子序列,并分析其序列特征和基因表达模式。【方法】利用RACE技术克隆SoSAI1的全长cDNA序列,应用生物信息学软件分析SoSAI1预期编码蛋白特征;采用Genome Walking技术克隆SoSAI1的启动子序列;采用实时荧光定量PCR分析不同生长期SoSAI1在甘蔗叶和茎中的表达,以及PEG 6000、100 mmol·L-1NaCl和6℃胁迫下,SoSAI1在甘蔗苗期根和叶中表达模式。【结果】SoSAI1的cDNA序列全长为2 387 bp,ORF长2 058 bp,编码685个氨基酸,预测其分子量和等电点分别为74.44 kD和5.6,GenBank登录号为JQ406875。5侧翼启动子序列长417 bp,含有胚乳特异表达顺式作用元件和参与干旱诱导的MYB结合位点,GenBank登录号为KC862314。SoSAI1表达在生理成熟期的花序和花序轴中较高,而在成熟茎和老茎中较低。15%PEG和6℃能诱导叶中SoSAI1表达,而15%PEG和NaCl能诱导根中SoSAI1表达。【结论】获得SoSAI1全长cDNA序列和部分启动子序列,SoSAI1在甘蔗生长发育和蔗糖积累,并在应对环境胁迫中发挥作用。

[Objective] The objective of this study is to clone the soluble acid invertase gene （SoSAH） and 5＇ promoter sequence from sugarcane, to analyze its sequence and expression patterns. [Method] The full-length eDNA sequence of SoSAI1 gene was amplified from the leaves of sugarcane by the technology of rapid amplification of cDNA ends （RACE）. The cDNA sequence and the deduced amino acid sequence were analyzed by bioinformatics method. The promoter sequence of SoSAI1 gene was cloned by genome walking. The spatial-specific expressions of SoSAH were determined by real-time fluorescent quantitative PCR （qRT-PCR） in leaves and stems at different growth stages of sugarcane, and in roots and leaves under treatment of PEG6000 100 mmol-L1 NaC1 or 6℃ stress conditions. [Result] The cloned full length of SoSAH cDNA sequence was 2 387 bp, containing a 2 055 bp open reading frame （ORF） which encodes 685 amino acids with a theoretical molecular mass of 74.44 kD. The 5＇ promoter sequence length DNA sequence was 417 bp, which contained endosperm specific expression of eis-acting elements and participate in the drought induced MYB binding sites. The result of real time PCR exhibited that SoSA11 was the highest abundance in inflorescence and the lowest in old and matured internodes of stems. The conditions of 15% PEG or 6~C could induce the expression of SOS.411 gene in leaves, while 15% PEG or NaC1 could induce the expression of SoSAI1 gene in roots. [ Conclusion] The full length of SoSAI1 and part of the promoter were cloned from sugarcane. The analysis by qRT-PCR suggested that SoSAI1 played an important role in sugarcane for growth and development, sucrose accumulation and response to environmental stress.