枇杷试管苗2个CAT基因成员的克隆及表达分析

Cloning, Characterization and Expression Analysis of Two CAT Genes from Eriobotrya japonica

以早钟6号枇杷试管苗叶片为试验材料,采用同源克隆方法从叶片mRNA中分离得到CAT基因的2个成员Ej-CAT1和Ej-CAT2,Ej-CAT1 cDNA序列全长1 738 bp,包括1 479 bp ORF和257 bp 3′-UTR;Ej-CAT2序列全长1 742 bp,包含1 479 bp ORF及261 bp 3′-UTR。2个CAT基因成员的核苷酸序列相似性为98.04%,其开放阅读框推导的氨基酸序列具有97.32%的相似性。生物信息学分析结果表明:2个蛋白均编码492个氨基酸,是亲水蛋白,不含信号肽,不存在螺旋卷曲结构,是非跨膜蛋白,亚细胞定位于过氧化物酶体;Ej-CAT1及Ej-CAT2蛋白分别有18、17个功能位点,22、19个磷酸化位点。CAT基因在枇杷试管苗离体保存过程中不同时期具有不同的表达量,整体趋势呈"W"形状,在离体保存1个月时表达量最高,保存7个月时表达量最低。CAT表达量变化可能跟枇杷试管苗的生长发育模式有关。

Two CA T genes, Ej-CA T1 and Ej-CA T2 were cloned by homologous cloning, from the leaves of the in vitro plantlets in Eriobotrya japonica cv. Zaozhong No.6. The full length of Ej-CATI was 1 738 bp, including an open reading frame of 1 479bp and a 3＇untranslated region of 257 bp; the length of Ej-CAT2 was 1742 bp, and it consisted of an open reading frame of 1 479 bp and a 3＇untranslated region of 261 bp. The homology of their nucleotide sequence and translation of amino acids was 98.04% and 97.32% respectively. Bioinformation analysis showed that both the two CAT protein encoded 492 amino acids, and they were a kind of hydrophilic protein without signal peptide, did not contain coiled coil domain, and belonged to the non-transmembrane protein. The Ej-CAT1 protein contained 18 functional sites and 22 phosphorylation sites, while Ej-CAT2 protein contained 17 functional sites and 19 phosphorylation sites. With Actin gene as the reference gene, the expressions of the CAT gene during in vitro conservation were investigated by real-time quantitative PCR technology in loquat. The results showed that the CA T gene expressed at all six stages during in vitro conservation.The overall trend of CA T gene expression level showed the shape of the letter ＂W＂ -shaped, the expression level of CA T reached the highest on the first month and the lowest on the seventh month. The change of CAT expression might be associated with the pattern of growth and development of in vitro plantlets in loquat.