荔枝LcAsr基因的生物信息学分析与载体构建

Bioinformatic Analysis and Construction of Plant Expression Vector of LcAsr Gene from Litchi

根据荔枝果皮cDNA微阵列信号分析结果,对荔枝cDNA文库的BAO5-H4克隆测序。NCBI(http://www.ncbi.nlm.nih.gov/)ORF分析发现其含有1个完整的开放读码框,编码152个氨基酸残基的多肽,为一全长cDNA序列。Blastx比对分析表明该基因编码的蛋白与其它植物来源的ASR(abscisic acid,stress and ripening inducible)蛋白高度同源,该基因被命名为LcAsr,通过PCR的方法获得其DNA全长序列。生物信息学分析结果表明,LcAsr基因的DNA序列全长为1177bp,读码框中间包含1个488bp的内含子。Protparam预测分析结果表明LcAsr基因编码蛋白的分子式为C756H1130N228O238S1,相对分子质量为17252.7,等电点pI6.10,富含Glu、His、Lys、Ala。Predictprotein预测该蛋白的二级结构主要是α螺旋,有1个酪氨酸激酶磷酸化位点、2个酪蛋白激酶II磷酸盐化位点、3个豆蔻酰化位点等,并具有很高的亲水性,预测该蛋白位于细胞核。构建了植物荧光表达载体pCAMBIA-LcAsr,并通过基因枪转化法转化洋葱表皮细胞进行瞬时表达,对LcASR蛋白进行亚细胞定位,结果表明LcASR蛋白定位于细胞核。

The BAO5-H4 from Litchi cDNA library was sequenced based on the results of cDNA microarry, and a full-length eDNA was obtained and designated as LcAsr. Sequence analysis shows that the eDNA of LcAsr is 799 bp, containing an open reading frame encoding 152 amino acids. The encoding protein is homologous with the ASR （abscises acid, stress and ripening inducible） protein from other plants. The DNA sequence of the LcAsr was obtained by using PCR method. Sequence analysis shows the fulllength LcAsr DNA is 1 177 bp, containing a 488 bp intron. Protparam predicted the LcASR protein was unstable with Mr=17 252.7, pI=6.10 and strong hydrophilicity, located to nucleus. The deduced amino acid sequence was rich in Glu, His, Lys and Ala. Protein predict PROSITE motif search revealed that the LcA SR had several motifs, such as Casein kinase II phosphorylation site, N-myristoylation site, Tyrosine kinase phosphorylation site and Amidation site. The secondary structure of the LcA SR protein was primarily composed of alpha helices with strong hydrophilicity, and hence the LcA SR may be located in nucleus. A plant expression vector pCAMBIA-LcAsr with a Gfp （green fluorescent protein） gene was constructed and transformed into onion skin cells by the gene gun method. Transient expression showed that the LcA SR protein was located in the nucleus which was in line with the characteristics of transcription factors.