绣球‘杜丽'AP3基因克隆与基因编辑载体构建

AP3 gene cloning and gene-editing vector construction of Hydrangea macrophylla ‘Dooley'

关键词:  绣球, MADS-box家族, AP3, CRISPR/Cas9, 载体构建

Key words:  Hydrangea macrophylla, MADS-box family, AP3, CRISPR/Cas9, vector construction

摘要:

绣球(Hydrangea macrophylla)是以花序为主要观赏部位的园林植物,多用作切花装饰和景观营造,在亚洲、美洲、欧洲广泛栽培。为探究AP3基因在绣球花萼形成过程中的功能,加快重瓣绣球新品种培育进程,该研究以绣球‘杜丽'为材料,克隆其MADS-box B类基因HmAP3,并结合生物信息学方法预测基因功能; 根据HmAP3序列信息,筛选出高特异性编辑靶点并构建CRISPR/Cas9基因编辑载体,通过农杆菌转化法将载体整合到绣球基因组中。结果表明:(1)克隆到1段HmAP3基因的cDNA序列,其序列全长546 bp,共编码181个氨基酸,测序结果表明其氨基酸序列与参考序列一致性为100%,与拟南芥AtAP3相似度为58.8%。(2)不同属植物AP3氨基酸序列差异较大,在同属不同物种中,AP3蛋白主要结构较为保守,仅在少数基序上存在差异。(3)在HmAP3中共鉴定到2个高特异性靶点,并成功构建2个单靶点CRISPR/Cas9基因编辑载体。(4)该研究共获得5株基因组内含有Cas9序列的抗性芽,但其靶点均未突变,在抗性芽中没有检测到Cas9表达。该研究探讨了AP3基因在重瓣绣球育种中的价值,对绣球的CRISPR/Cas9基因编辑技术进行了初探,为绣球优良品种繁育工作奠定了基础。

Abstract:

Hydrangea macrophylla is a garden plant widely cultivated in Asia, America, and Europe with its inflorescence as main ornamental feature. It is commonly used in interior decoration and landscape creation. To investigate the role of AP3 gene in hydrangea during calyx formation, H. macrophylla ‘Dooley' was used as the material. The MADS-box class B gene HmAP3 was cloned, and its gene function was predicted by bioinformatics analysis. To explore methods for quicker breeding new varieties, highly-specific editing targets were screened and CRISPR/Cas9 gene-editing vectors were constructed. The vector sequence was integrated into the H. macrophylla genome by agrobacterium-mediated transformation. The results were as follows:(1)The cDNA sequence full length of HmAP3 was 546 bp, encoding 181 amino acids. Its amino acid sequence was 100% similar to the reference sequence and 58.8% similar to Arabidopsis thaliana.(2)AP3 differed greatly in different genera. Within the same genus, the main structure of AP3 protein was conserved and differed only in a few motifs.(3)There were two highly specific targets in HmAP3. Sequencing results indicated that two single-target CRISPR/Cas9 gene-editing vectors were constructed successfully.(4)There were five resistant buds with Cas9 sequences in their genomes. However, their target sequences did not change due to the absence of Cas9 expression. In this study, the potential of AP3 gene in the breeding work of double flower phenotype was investigated, and a preliminary exploration of CRISPR/Cas9 gene-editing technology for Hydrangea macrophylla was conducted. These results provide a basis for the breeding of H. macrophylla.