甘蓝型油菜BnDHAR基因的克隆及表达分析

Molecular Cloning and Expression Analysis of BnDHAR in Brassica napus L.

摘要:

在甘蓝型油菜矮化突变体与其高秆野生型亲本构建的消减杂交文库中得到一长约230bp与编码脱氢抗坏血酸还原酶的DHAR基因的一段核苷酸序列高度相似的DNA片段。采用同源克隆技术，在甘蓝型油菜中克隆得到了该基因的全长cDNA序列，命名为BnDHAR. BnDHAR与已公布的甘蓝型油菜基因组中的该基因核苷酸序列完全一致。矮秆突变体与其野生型亲本DHAR全长cDNA核苷酸序列并无差异，但对BnDHAR基因进行二叶一心期、四叶一心期、抽薹期、花期不同组织的表达分析的结果显示，BnDHAR基因在野生型甘蓝型油菜苗期的叶中表达量是最高的，接近矮化突变体的5倍，在根、茎中表达极低，矮化突变体与高秆野生型差异不大。高温胁迫条件下，BnDHAR表达量具有升高的趋势；盐胁迫条件下，BnDHAR表达量在处理9h时达最高，然后呈现下降的趋势；干旱胁迫条件下，表达量则是在处理12h时最高，也呈现出先上升再下降的趋势。综上所述，在甘蓝型油菜中BnDHAR基因的表达具有组织特异性，与生长发育及非生物胁迫响应有关。

Abstract:

About 230bp of DHAR partial DNA segments was obtained from Subtractive hybridization library, which is highly similar with the nucleotide sequence of DNA encoding DHAR gene, the Subtractive hybridization library was constructed by dwarf mutant and wild-type parent. A full-length cDNA sequence of the gene named BnDHAR was cloned by homologous cloning from Brassica napus. BnDHAR was fully consistent with nucleotide sequence of the gene in the published Brassica napus genome. There were no difference in the DHAR full-length cDNA sequence between dwarf mutant and its wild-type parent. We used qRT-PCR technology analysised the BnDHAR gene expression in different organizations at various periods of Brassica napus , the results showed that: BnDHAR gene expression in the wild-type Brassica napus leaves is the highest approximately 5 times in the dwarf mutant at the seeding stage, and extremely low expression in roots and stems, dwarf mutant and tall wild-type didn’t have any significant difference. Under the high temperature stress, the expression of the BnDHAR gene increased; under the salt stress, BnDHAR reached the highest expression after dealing with 9h, then showed a downward trend; under the drought stress, the expression reached the highest after 12 hours, also showed a downward trend after the first rise. In summary, the BnDHAR gene exhibited a tissue specific expression in Brassica napus and also related with growth and abiotic stress response.