柠条锦鸡儿F3H基因克隆及功能分析  

Cloning and expression analysis of F3H gene in Caragana korshinskii

作　　者：郑晟[1] 毛玉珊 张腾国[1] 聂亭亭[1] 

机构地区：[1]西北师范大学生命科学学院,兰州730070

出　　处：《广西植物》2017年第6期723-733,共11页Guihaia

基　　金：国家自然科学基金(31460099);西北师范大学青年教师科研能力提升计划项目(NWNU-LKQN-14-23)~~

摘　　要：柠条锦鸡儿为豆科灌木,对各种环境胁迫具有较强的适应能力,类黄酮是天然的抗氧化剂,花青素属类黄酮化合物,逆境胁迫会影响植物体内花青素的合成,而黄烷酮3-羟化酶(F3H)是花青素生物合成所必需的一种关键酶。该研究成功分离克隆了柠条锦鸡儿的F3H基因,命名为CkF3H。CkF3H基因的开放阅读框(ORF)为1095 bp,编码364个氨基酸,推测的蛋白质分子量为41.3 kDa,理论等电点为5.9。生物信息学分析发现,CkF3H基因序列与其它植物F3H有较高的一致性,推测CkF3H蛋白与其它植物F3H蛋白具有相似的功能。利用染色体步移法克隆得到CkF3H起始密码子ATG上游468 bp的启动子序列,PlantCARE软件分析表明,该序列具有启动子的基本元件CAAT-box和TATA-box以及多种与逆境胁迫相关的顺式调控元件。实时荧光定量PCR分析表明,CkF3H在柠条的根、茎和叶中均有表达,没有组织特异性;CkF3H的表达受低温、高盐、干旱和高温胁迫的诱导,并且在低温胁迫下,CkF3H的表达还受到光周期的影响。综上所述,研究结果表明CkF3H基因在柠条锦鸡儿适应低温、高盐、干旱和高温胁迫的过程中发挥作用。Caragana korshinskii is a kind of shrub belonging to the leguminosae category and it has characteristic of tolerance to stressful conditions. Flavonoids are natural antioxidants that include the groups of notable pigments such as anthocyanins. The adversity stress can influence the biosynthesis of anthocyanins in plants. Flavanone 3-Hydroxylase（F3H） is a key enzyme needed for the biosynthesis of anthocyanins. In this study,we successfully isolated,cloned F3H gene from C. korshinskii. The full length c DNA of C. korshinskii F3H gene（ designated as CkF3H） had an open reading frame（ORF） of 1095 bp encoding 364 amino acids with a calculated molecular mass of 41.3 k Da as well as an isoelectric point of 5.9. Comparative and bioinformatical analyses revealed that the cloned CkF3H from C. korshinskii has high identity with F3H from other plant species. The deduced CkF3H protein showed similarities with other available plant F3H proteins. The promoter of CkF3H gene was isolated by chromosomal walking and 468 bp sequence was obtained by sequencing. Plant CARE analysis of this sequence showed that the peomoter contained some typical elements CAAT-box and TATA-box and kinds of Cis-acting elements involved in defence and stress responsiveness. RT-PCR analysis revealed that CkF3H was expressed in roots,stems,and leaves with almost no tissue specificity. The transcript level of CkF3H was increased in response to cold,high salt,drought and high temperature stress. The gene expression of CkF3H was influenced by photoperiod in response to cold stresses. The results suggested that CkF3H played an important role during cold,high salt,drought and high temperature stress in C. korshinskii.

关 键 词：柠条锦鸡儿 CkF3H基因 基因克隆 表达分析 非生物胁迫 

分 类 号：Q943.2[生物学—植物学]