不结球白菜花青苷合成负调控基因BrcLBD39的克隆和表达分析及其对外源6-BA的响应

[目的]本文旨在探究LBD39基因在不结球白菜紫色和绿色材料中的特性及其对外源6-BA的响应，为花青苷调控机制奠定理论基础。[方法]以不结球白菜紫色自交系NJZX1-3及其绿色突变体NJZX1-0为材料，同源克隆LBD39基因的全长，应用生物信息学方法分析其核酸和蛋白序列及其进化关系，并在线预测蛋白质二级和三级结构。采用外源6-BA处理后测定叶片中花青苷含量，并通过实时荧光定量PCR技术测定了LBD39基因在外源6-BA处理后的表达水平。[结果]在2个不同材料中克隆获得的LBD39基因完全相同，序列长为876 bp，包含编码区（长为699 bp）及非编码区（长为177 bp）。其编码区序列含有1个长为696 bp的开放阅读框（ORF），编码232个氨基酸，将该基因命名为BrcLBD39。亚细胞定位预测该蛋白分布在细胞核中，蛋白相对分子质量为25.30×103，等电点为8.85。进化树分析表明，BrcLBD39蛋白序列与大白菜BraLBD39相似性为100%，其次与油菜和甘蓝关系最近，相似性分别为99%和93%。外源6-BA处理24 h以后紫色材料NJZX1-3叶片中花青苷含量均高于对照，而绿色突变体叶片基本不含花青苷；BrcLBD39基因在2个材料中具有相似的表达模式，即先下降后上升而后又下降，其中在处理后48 h时BrcLBD39基因表达量急剧增加，在紫色和绿色材料中增量分别为74.84%和49.87%。此外，BrcLBD39表达量在绿色材料中明显高于紫色材料。[结论]不结球白菜中花青苷负调控基因BrcLBD39既响应外源6-BA，又与叶片中花青苷的积累密切相关，在花青苷的生物合成过程中起着重要的调控作用。
[Objectives]The aim of the study is to investigate the characteristic of LBD39 gene responsive to 6-BA in non-heading Chinese cabbage,providing the theoretical basis of the regulation of anthocyanins. [Methods]In this experiment,the full length of LBD39 gene was cloned from two materials of non-heading Chinese cabbage,that was the purple self-line NJZX1-3 and its green mutant line NJZX1-0. The gene sequences and protein sequences were analyzed by bioinformatics,and the econdary structure and tertiary structure of BrcLBD39 protein were predicted online. The materials were treated by 6-BA,and the expression level of LBD39 was determined by quantitative real-time PCR and the total anthocyanins content was also measured. [Results]The study indicated that the sequences of the two cDNA clones were coincident completely,with a full-length of 876 bp,including the code area(699 bp) and the noncode area(177 bp). The open reading frame(ORF) of the code area of LBD39 was 696 bp in length and encoded 232 predicted amino acids,designated as BrcLBD39. Subcellular localization predicted that the protein was distributed in cell nucleus. The molecular weight of BrcLBD 39 was 25.30×103 and pI value was 8.85. The BrcLBD39 protein had 100% identities to BraLBD39,and shared the most close relationship with Brassica rapa,then Brassica napus and Brassica oleracea,with the identities of 99% and 93%,respectively. With the treatment of 6-BA,total anthocyanins content in the purple self-line NJZX1-3 increased and were higher than that of control after 24 h,however,anthocyanins were absent in the green mutant line NJZX1-0. At the same time,BrcLBD39 expressed in the two different self-lines with similar expression patterns,which declined first and then rose significantly and then fell again. The expression of BrcLBD39 increased to the maximum at 48 h,with the increment