青花菜萝卜硫素合成相关基因BoCYP83A1的克隆与表达分析

Cloning and Expression Analysis of Sulforaphane Synthesis-related Gene BoCYP83A1 in Broccoli

CYP83A1基因是萝卜硫素合成代谢中的关键基因,该试验以青花菜品种CDBY-10为材料,利用RACE和RT-PCR方法,获得BoCYP83A1基因的全长序列。该基因全长1 509bp,编码502个氨基酸,包含保守的P450结构域。通过实时荧光定量PCR分析了BoCYP83A1基因在不同品种、不同组织以及不同激素处理下的表达水平。系统进化树分析表明,BoCYP83A1与结球甘蓝亲缘关系最近。BoCYP83A1基因在不同品种间的组织特异性不同:在青花菜品种CDBY-10的根、茎、叶3个组织间表达水平差异较小,在品种CDBY-12中表现为茎>叶>根,在CDBY-14中则表现为根>茎>叶。MeJA及SA处理均能够引起BoCYP83A1基因表达量的变化:经MeJA处理后,BoCYP83A1基因表达量升高至对照的1.9倍,而后有少量下降;经SA处理后,BoCYP83A1基因表达水平迅速降低,在6h时降低至对照的0.1倍,而后逐渐回升至对照的表达水平。研究表明,BoCYP83A1基因在不同青花菜品种中的表达特性不同,其表达能够被MeJA和SA所调控。青花菜BoCYP83A1的克隆及鉴定为培育高萝卜硫素含量的青花菜新品种奠定了理论基础。

CY P83A1 is a key gene in sulforaphane biosynthesis and metabolism .The full length of BoC-Y P83A1 was obtained from broccoli CDBY-10 by the methods of RACE and RT-PCR .BoCY P83A1 was 1 509 bp in length ,encoded a deduced protein of 502 amino acids ,and contained the conserved P450 domain . The expression level of BoCY P83A1 in different cultivars ,tissues ,or seedlings treated with different hor-mones was analyzed by real-time fluorescence quantitative PCR .Phylogenetic tree analysis showed that BoCYP83A1 had the closest relationship with Brassica oleracea var .oleracea BoCYP83A1 .Tissue-specific expression of BoCY P83A1 in cultivars was different ：expression difference in CDBY-10 was small .It showed stem〉leaf〉 root in CDBY-12 ,while root 〉 stem 〉 leaf in CDBY-14 .The treatments of MeJA＆amp;nbsp;and SA led to the changes of BoCY P83A1 expression ：after the MeJA treatment ,the expression of BoC-Y P83A1 was raised to 1 .9 times of the control ,and then decreased a little ;After the SA treatment ,the ex-pression of BoCY P83 A1 reduced quickly ,and upto 0 .1 time of the control at 6 h ,then restored gradually to the expression level of control .The results indicated that the expression characteristics of BoCY P83A1 in broccoli cultivars were different ,and BoCY P83A1 expression can be regulated by MeJA and SA .Clo-ning and characterization of BoCY P83 A1 gene provide the theoretical basis for breeding new broccoli culti-vars with high sulforaphane content .