Zhang Xuan, Zhou Huiyue, Zang Xiaonan, Gong Le, Sun Hengyi, Zhang Xuecheng
Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao, 266003, Shandong province, China.
Mar Biotechnol (NY). 2014 Aug;16(4):475-83. doi: 10.1007/s10126-014-9565-0. Epub 2014 Feb 18.
To study the Ca(2+)/Calmodulin (CaM) signal transduction pathway of Gracilaria lemaneiformis under heat stress, myo-inositol-1-phosphate synthase (MIPS), a calmodulin-binding protein, was isolated using the yeast two-hybrid system. cDNA and DNA sequences of mips were cloned from G. lemaneiformis by using 5'RACE and genome walking procedures. The MIPS DNA sequence was 2,067 nucleotides long, containing an open reading frame (ORF) of 1,623 nucleotides with no intron. The mips ORF was predicted to encode 540 amino acids, which included the conserved MIPS domain and was 61-67 % similar to that of other species. After analyzing the amino acid sequence of MIPS, the CaM-Binding Domain (CaMBD) was inferred to be at a site spanning from amino acid 212 to amino acid 236. The yeast two-hybrid results proved that MIPS can interact with CaM and that MIPS is a type of calmodulin-binding protein. Next, the expression of CaM and MIPS in wild-type G. lemaneiformis and a heat-tolerant G. lemaneiformis cultivar, "981," were analyzed using real-time PCR under a heat shock of 32 °C. The expression level displayed a cyclical upward trend. Compared with wild type, the CaM expression levels of cultivar 981 were higher, which might directly relate to its resistance to high temperatures. This paper indicates that MIPS and CaM may play important roles in the high-temperature resistance of G. lemaneiformis.
为研究龙须菜在热胁迫下的Ca(2+)/钙调蛋白（CaM）信号转导途径，利用酵母双杂交系统分离了一种钙调蛋白结合蛋白——肌醇-1-磷酸合酶（MIPS）。通过5'RACE和基因组步移技术从龙须菜中克隆了mips的cDNA和DNA序列。MIPS的DNA序列长2067个核苷酸，包含一个1623个核苷酸的开放阅读框（ORF），无内含子。mips的ORF预计编码540个氨基酸，其中包括保守的MIPS结构域，与其他物种的相似性为61%-67%。分析MIPS的氨基酸序列后，推断钙调蛋白结合结构域（CaMBD）位于氨基酸212至236的位点。酵母双杂交结果证明MIPS能与CaM相互作用，且MIPS是一种钙调蛋白结合蛋白。接下来，在32℃热激条件下，利用实时荧光定量PCR分析了野生型龙须菜和耐高温龙须菜品种“981”中CaM和MIPS的表达情况。表达水平呈周期性上升趋势。与野生型相比，品种981的CaM表达水平更高，这可能与其耐高温性直接相关。本文表明MIPS和CaM可能在龙须菜的耐高温性中发挥重要作用。
