闽侯野生蕉LDOX基因的克隆及表达特性

Cloning and expression characteristics of the LDOX gene in Musa itinerans

为探讨闽侯野生蕉无色花青素双加氧酶基因(MiLDOX)的序列和表达特性,利用反转录PCR(RT-PCR)克隆MiLDOX基因cDNA序列,并对其进行系列生物信息学和不同组织器官中的表达特性分析.结果显示,MiLDOX基因包含1 083 bp完整的开放阅读框,可编码分子量(Mr)为40.73×10~3、包含360个氨基酸的蛋白质. MiLDOX基因属于α-酮戊二酸依赖性双加酶基因家族,其编码蛋白为不含信号肽和跨膜结构的亲水性蛋白,理论等电点为5.71.MiLDOX二级结构中不规则卷曲和α-螺旋占比较大.亚细胞定位预测结果显示MiLDOX可能定位于细胞质中.系统进化树显示MiLDOX与小果野蕉MaLDOX和阿比西尼亚红脉蕉EvLDOX亲缘关系最近.实时荧光定量PCR结果显示MiLDOX在闽侯野生蕉不同组织部位都有表达,在雄花中的表达量最高,且与类黄酮和花色素苷含量呈显著正相关.此外,通过研究MiLDOX基因在不同转色阶段果皮中的表达情况,发现它的表达随着果皮色素积累逐步升高.本研究表明MiLDOX基因可能分别参与闽侯野生蕉类黄酮和花色素苷的积累,并在果皮转色等过程发挥着重要作用.(图9表1参37)

To investigate the sequence and expression of the leucocyanidin dioxygenase gene(MiLDOX)in Minhou wild banana(Musa itinerans),full-length MiLDOX cDNA was cloned from Minhou wild banana using reverse transcription-polymerase chain reaction(RT-PCR),and expression pattern and bioinformatics analysis were performed.Sequence analysis showed that MiLDOX CDS contained 1083 base pairs and encoded a protein with a molecular mass of 40.73×103 that consisted of 360 amino acids.The MiLDOX gene belongs to the family ofα-ketoglutarate-dependent dioxygenase enzyme genes.Its encoded protein was hydrophilic with a theoretical pI of 5.71 and did not contain signal peptides nor transmembrane structure.Protein secondary structure analysis showed that MiLDOX was rich in irregular curling andα-helices and that it may be located in the cytoplasm.The phylogenetic analysis showed that MiLDOX was closely related to Musa acuminata subsp.malaccensis LDOX(MaLDOX)and Ensete ventricosum LDOX(EvLDOX).Quantitative real time PCR indicated that MiLDOX was expressed in different tissues and organs of Musa itinerans with the highest expression in staminate flowers.Correlation analysis revealed positive correlations between MiLDOX expression and flavonoid content and between its expression and anthocyanin content.Moreover,by determining the gene expression pattern in pericarps during the different pigment accumulation stages,we found that its expression increased as the pericarp color deepened.This study indicated that the MiLDOX gene may participate in flavonoid and anthocyanin accumulation and that it contributed greatly to pigmentation processes,including pericarp pigmentation.