基于CRISPR/Cas9的辣椒(Capsicum annuum L.)中植物烯去饱和酶(PDS)基因的基因组编辑

IF 3.5 Q3 Biochemistry, Genetics and Molecular Biology
Mallesham Bulle , Ajay Kumar Venkatapuram , Sadanandam Abbagani , P.B. Kirti
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引用次数: 0

摘要

在一种具有重要商业价值的作物--辣椒--中开发出了一种有效的 CRISPR/Cas9 试剂递送系统,该系统使用了一种构建体,该构建体含有两个不同的 gRNA,分别针对植物烯去饱和酶(CaPDS)基因的第 14 和 15 号外显子,该基因的功能缺失突变会导致光漂白表型,并损害类胡萝卜素的生物合成。在含有 80 毫克/升卡那霉素的培养基上再生的子叶和通过生物媒介将 CRISPR/Cas9 试剂转移到辣椒细胞后再生的植株中,观察到携带两个 sgRNA 的构建体产生了明显的白化表型。对 CRISPR/Cas9 基因组编辑事件的分析,包括卡那霉素筛选突变体和使用 T7 内切酶测定法(T7E1)评估同质性,结果显示 62.5%的转化植株成功编辑了目标区域,并表现出白化和马赛克两种表型。有趣的是,序列分析表明,所有株系的目标 CaPDS 区域都存在插入和替换。检测到的突变主要是破坏外显子-内含子交界处的 12 至 24 个 bp 的缺失,以及碱基置换和在目标位点的原间隔邻接基序(PAM)区域插入 1 个 bp。基因敲除辣椒品系中基本光合色素(叶绿素 a、叶绿素 b 和类胡萝卜素)的减少进一步证明 CaPDS 基因的功能已被破坏。在本研究中,我们报告了将 CRISPR/Cas9 试剂生物递送到辣椒中的方法非常有效,并能在短时间内产生多种突变事件。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
CRISPR/Cas9 based genome editing of Phytoene desaturase (PDS) gene in chilli pepper (Capsicum annuum L.)

An effective CRISPR/Cas9 reagent delivery system has been developed in a commercially significant crop, the chilli pepper using a construct harboring two distinct gRNAs targeting exons 14 and 15 of the Phytoene desaturase (CaPDS) gene, whose loss-of-function mutation causes a photo-bleaching phenotype and impairs the biosynthesis of carotenoids. The construct carrying two sgRNAs was observed to create visible albino phenotypes in cotyledons regenerating on a medium containing 80 mg/L kanamycin, and plants regenerated therefrom after biolistic-mediated transfer of CRISPR/Cas9 reagents into chilli pepper cells. Analysis of CRISPR/Cas9 genome-editing events, including kanamycin screening of mutants and assessing homozygosity using the T7 endonuclease assay (T7E1), revealed 62.5 % of transformed plants exhibited successful editing at the target region and displayed both albino and mosaic phenotypes. Interestingly, the sequence analysis showed that insertions and substitutions were present in all the plant lines in the targeted CaPDS region. The detected mutations were mostly 12- to 24-bp deletions that disrupted the exon–intron junction, along with base substitutions and the insertion of 1-bp at the protospacer adjacent motif (PAM) region of the target site. The reduction in essential photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoid) in knockout chilli pepper lines provided further evidence that the CaPDS gene had been functionally disrupted. In this present study, we report that the biolistic delivery of CRISPR/Cas9 reagents into chilli peppers is very effective and produces multiple mutation events in a short span of time.

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来源期刊
Journal of Genetic Engineering and Biotechnology
Journal of Genetic Engineering and Biotechnology Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
5.70
自引率
5.70%
发文量
159
审稿时长
16 weeks
期刊介绍: Journal of genetic engineering and biotechnology is devoted to rapid publication of full-length research papers that leads to significant contribution in advancing knowledge in genetic engineering and biotechnology and provide novel perspectives in this research area. JGEB includes all major themes related to genetic engineering and recombinant DNA. The area of interest of JGEB includes but not restricted to: •Plant genetics •Animal genetics •Bacterial enzymes •Agricultural Biotechnology, •Biochemistry, •Biophysics, •Bioinformatics, •Environmental Biotechnology, •Industrial Biotechnology, •Microbial biotechnology, •Medical Biotechnology, •Bioenergy, Biosafety, •Biosecurity, •Bioethics, •GMOS, •Genomic, •Proteomic JGEB accepts
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