大白菜黄化突变基因Bra024218表达模式分析

Expression Pattern Analysis of Etiolated Mutant Gene Bra024218 in Chinese Cabbage

叶色突变不仅能为探究植株叶绿体发育及叶绿素生物合成提供材料,而且在苗期还能作为杂种优势利用的显著标记性状。采用60Co-γ射线照射花蕾与游离小孢子培养相结合的方法,获得了一份稳定遗传的大白菜黄化突变体pem。该突变体全生育期整个植株表现黄化,叶球较小。前期研究已对突变基因进行精细定位,并预测候选突变基因为Bra024218,其启动子区域有一段30bp的片段缺失,拟南芥同源基因AT4G28210注释功能为胚胎缺陷。RT-PCR结果表明:Bra024218在不同组织中均有表达,但在突变体中表达强度相对减弱,尤其在根和花蕾中表达量显著下降。Bra024218在不同发育时期的叶片中均有表达,在突变体中表达强度相对减弱,第1片真叶和第3片真叶的表达量显著下降。启动子核心区域预测及作用元件分析结果表明,将野生型及突变体中Bra024218启动子连接载体后转入拟南芥。T2代拟南芥植株筛选后进行GUS染色分析。与野生型相比,突变体启动子的表达活性在根、茎、叶、花器官和种荚中均较弱。对叶片的GUS酶活性进行测定,突变体GUS酶活性显著下降。利用拟南芥原生质体及烟草叶片进行亚细胞定位,结果证明Bra024218只在叶绿体中表达。可见,突变体pem是研究大白菜叶绿体发育的理想材料,可为今后对于叶色突变机制的研究奠定基础。

Leaf color mutant not only provided the materials for exploring chloroplast development and chlorophyll biosynthesis but also used as marker character in heterosis utilization at seedlings stage.A stable inherited etiolated mutant pem was obtained by radiating the flower buds with60 Co-γray and then combining with free microspore culture in Chinese cabbage.The plant showed etiolated during the whole growth period of the pem,the head was small.The mutant gene had finely been mapped in the previous study and been named as Bra024218.The promoter of mutant gene has a 30 bp deletion region in pem.Its homologous gene AT4 G28210 annotated function was embryo defect in Arabidopsis.RT-PCR results showed that Bra024218 was expressed in different tissues,but the expression was relatively weak in pem compared to the wild type’FT’,especially in roots and flower buds.Bra024218 was expressed in different periods of the leaves.The expression in the pem was relatively weak,especially in first true leaves and third true leaves.According to the promoter core region prediction and functional element analysis,the vector was connected with the Bra024218 promoter and transferred to Arabidopsis thaliana.The GUS analysis was performed after screening the T2 Arabidopsis thaliana.The Gus gene expression activity of the mutant promoter was weak in roots,stems,leaves,floral organs and seed pods compared to the wild type.The GUS enzyme activities of the leaves were measured,and these in the mutant were significantly decreased.Subcellular localization demonstrated that Bra024218 was only expressed in chloroplasts by Arabidopsis protoplasts and tobacco leaves.These results showed that the pem mutant was a useful line for researching chloroplast development in Chinese cabbage and it laid the foundation for studying the mechanism of leaf color mutation in the future.