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绣球‘杜丽’AP3 基因克隆与基因编辑载体构建

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) HmAP3 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 蛋白主要结构则较为保守,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)本研究共获得 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。本研究探讨了 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基础。

英文摘要: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 quicker methods for 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 full length of HmAP3 was 546 bp, encoding 181 amino acids. Its protein 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 was conserved and differed only in a few motifs. (3) There were 2 highly specific targets in HmAP3. Sequencing results indicated that both single-target CRISPR/Cas9 gene-editing vectors were constructed successfully. (4) There were 5 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 was conducted. These results provide a basis for the breeding of H. macrophylla

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[V1] 2022-07-05 17:05:49 chinaXiv:202207.00014V1 下载全文
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