苦瓜 McPDS基因克隆及CRISPR/Cas9基因编辑载体构建

Cloning and CRISPR/Cas9 gene editing vector construction of McPDS in bitter gourd (Momordica charantia L.)

摘要

以苦瓜八氢番茄红素脱氢酶(phytoene dehydrogenase, PDS)基因为靶标，构建其特异gRNA的CRISPR/Cas9基因编辑载体，以期为建立苦瓜CRISPR/Cas9基因编辑技术体系奠定基础。以苦瓜自交系B07叶片cDNA为模板，同源克隆苦瓜 McPDS基因CDS序列。结果表明， McPDS基因CDS序列全长1731 bp, 编码576个氨基酸，蛋白质相对分子质量为64.44 kD，理论等电点(PI)为7.09。跨膜结构分析结果表明，该蛋白为亲水性非跨膜蛋白。系统进化树分析结果表明，McPDS与黄瓜、甜瓜等葫芦科植物中的PDS蛋白同源性较高。此外，以 McPDS为靶标基因，在5’端筛选2个高特异性靶点，经设计引物，成功构建1个双靶点CRISPR/Cas9基因编辑载体，为苦瓜基因编辑体系的建立奠定了技术基础。

Abstract

A gRNA-specific CRISPR/Cas9 gene editing vector targeting phytoene dehydrogenase (PDS) gene of bitter gourd was constructed, hoping to lay a foundation for the establishment of CRISPR/Cas9 gene editing technology system of bitter gourd. In this study, the CDS region of McPDS gene was cloned from the leaf cDNA of bitter gourd inbred line B07. The results showed that the coding region length of McPDS was 1731 bp, encoded 576 amino acids, the theoretical relative molecular weight was 64.44 kD, and the theoretical isoelectric point (PI) was 7.09. The transmembrane structure analysis showed that the protein was a hydrophilic non-transmembrane protein. Phylogenetic analysis showed that McPDS had high homology with PDS proteins in cucurbit plants such as cucumber and melon. In addition, using McPDS as the target gene, two highly specific targets were screened at the 5' end, primers were designed, and a double-target CRISPR/Cas9 gene editing vector was successfully constructed, which laid a technical foundation for the establishment of bitter gourd gene editing system.

关键词

苦瓜 / McPDS / 基因克隆 / 载体构建 / 基因编辑

Key words

Bitter gourd / McPDS / Gene cloning / Vector construction / Gene editing

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基金资助

广东省农业科学院协同创新中心项目(XTXM202203)

广州市科技计划项目(202206010170)

广州市科技计划项目(202201010493)

广东省自然科学基金项目(2022A1515010343)

广东省农业农村厅种业振兴行动专项(2022-NPY-00-027)

广东省农业科学院创新基金项目(202301)

广东省农业科学院学科团队建设项目(202130TD)