利用CRISPR/Cas9系统研究REVOLUTA参与烟草叶芽发育的调控

doi:10.13560/j.cnki.biotech.bull.1985.2023-0364

摘要/Abstract

摘要：

作物叶芽受到分生组织调控，调控叶芽是作物增产的有效措施之一。目前关于烟草分生组织调控的分子机理研究偏少，可用于株型改良的种质资源缺乏。本研究通过CRISPR/Cas9编辑系统靶向突变烟草REVOLUTA（REV）基因，分别构建两个不同REV单靶点序列C15NtREV和C16NtREV，通过农杆菌介导的叶盘转化方法获得再生苗，利用PCR测序鉴定转基因阳性单株，测序结果表明Ko-C15Ntrev突变体在NtREV氨基酸第26位置之后发生移码突变，而Ko-C16Ntrev突变体在NtREV氨基酸第60位置之后发生移码突变。此外，借助扫描电镜分别观测两个单靶点纯合突变体顶芽表型，结果表明烟草Ko-C15Ntrev双拷贝同源突变体出现顶芽缺失和叶片畸形的表型，而Ko-C16Ntrev单拷贝同源突变体未表现出顶芽缺失，但顶芽发育迟缓。相较于野生型烟草，Ko-C16Ntrev突变体自然株高较野生型增加3.76%，Ko-C16Ntrev突变体叶片数和腋芽鲜重分别较野生型减少21.47%和23.41%，且均达到极显著差异，说明NtREV参与烟草顶端分生组织发育，进而调节叶和腋芽发育。这些突变体为后续研究烟草的叶芽发育分子机理提供了重要研究材料。

关键词: CRISPR/Cas9, REVOLUTA, 叶芽, 株型, 侧生分生组织

Abstract:

The leaf buds of crop are regulated by the meristem, which is one of the effective measures to increase crop yield. At present, there are few studies on the molecular mechanism of meristem regulation in tobacco, and there is a lack of germplasm resources that can be used for plant type improvement. In this study, two different REVOLUTA（REV）single-target sequences, C15NtREV and C16NtREV, were constructed respectively by targeting mutation of REV gene through CRISPR/Cas9 editing system, and regenerated seedlings were obtained by Agrobacterium-mediated leaf disc transformation method, and the transgenic plants were identified by PCR sequencing. The sequencing results showed that the Ko-C15Ntrev mutant had a shift mutation after amino acid position 26 of NtREV, while the Ko-C16Ntrev mutant had a shift mutation after amino acid position 60 of NtREV. In addition, the apical bud phenotypes of two single-target homozygous mutants were observed separately by scanning electron microscope, the results showed that the double-copy homologous mutants of Ko-C15Ntrev had apical bud deletion and leaf deformities, while the single-copy homologous mutants of Ko-C16Ntrev did not have the absence of apical buds, but the apical buds development delayed. Compared with wild type, the natural plant height of the Ko-C16Ntrev mutant increased by 3.76%, while the number of leaf blades and fresh axillary bud weight of the Ko-C16Ntrev mutant decreased by 21.47% and 23.41%, respectively, and both reached extremely significant differences, indicating that NtREV is involved in the development of shoot apical meristem in tobacco, which in turn regulates the development of leaf and axillary bud. These mutants provided important research materials for the subsequent study of the molecular mechanism of bud development in tobacco.

Key words: CRISPR/Cas9, REVOLUTA, leaf bud, plant architecture, axillary meristem

王兵, 赵会纳, 余婧, 陈杰, 骆梅, 雷波. 利用CRISPR/Cas9系统研究REVOLUTA参与烟草叶芽发育的调控[J]. 生物技术通报, 2023, 39(10): 197-208.

WANG Bing, ZHAO Hui-na, YU Jing, CHEN Jie, LUO Mei, LEI Bo. Regulation of Leaf Bud by REVOLUTA in Tobacco Based on CRISPR/Cas9 System[J]. Biotechnology Bulletin, 2023, 39(10): 197-208.

图/表 10

表1 本研究引物序列及用途

Table 1 Sequences and purpose of primers in this study

引物Primer	序列Sequence（5'-3'）	作用Purpose
C16NtREV-T1F	ATTGGCTCGATATTCGACAGAATAGGG	靶点接头引物 NtREV target construction
C16NtREV-T1R	AAACCCCTATTCTGTCGAATATCGAGC
C15NtREV-T1F	ATTGACAGTAGTGGAAAGTATGTCCGG
C15NtREV-T1R	AAACCCGGACATACTTTCCACTACTGT
1300-gRNA-F	CCAGTCACGACGTTGTAAAAC	SgRNA表达盒检测 Detection of SgRNA expression kit
1300-gRNA-R	CAATGAATTTCCCATCGTCGAG	SgRNA表达盒检测 Detection of SgRNA expression kit
Hyg-F	CGATTGCGTCGCATCGACC	潮霉素抗性基因检测 Detection of hygromycin resistance gene
Hyg-R	TTCTACAACCGGTCGCGGAG	潮霉素抗性基因检测 Detection of hygromycin resistance gene
C16NtREV-CRISPR test-F	TTTTGGTTTGGGATTTTGAGG	检测转基因突变 Detection of transgenic mutations
C16NtREV-CRISPR test-R	CATTGTTCAATTTGGATTACTCC
C16NtREV-CRISPR test-R	GGAGCAATCAAAATTGAACAGCG
C15NtREV-CRISPR test-F	CTATGGTTGCACAGCAGCACA
C15NtREV-CRISPR test-R	ACTTCATCCATCACTGATCTAAC
C15NtREV-CRISPR test-R	ACTAGAAGCACAGTGATATATTC

图1 NtREV1（Nitab4.5_0004624g0050.1）和NtREV2（Nitab4.5_0003131g0030.1）氨基酸序列的同源性比较

Fig. 1 Homology comparison of NtREV1 and NtREV2 amino acid sequences

图2 烟草REV结构示意图及靶位点选择与载体构建 A：REV基因外显子和内含子示意图以及靶点编辑序列所在位置；B ：REV基因靶点编辑序列的双酶切核酸电泳

Fig. 2 Schematic structure and target site selection of NtREV and vector construction A: Schematic diagram of REV gene exons and introns, as well as the locations of the target editing sequences. B: Nucleic acid electrophoresis of double enzyme digestion of REV gene the target editing sequences

图3 植物双元表达载体pCAMBIA1300-pYAO-Cas9-NtREV的构建 A：pCAMBIA1300-pYAO-Cas9-NtREV的PCR 扩增的核酸电泳； B ：pCAMB-IA1300-pYAO-Cas9-NtREV双酶切的核酸电泳

Fig. 3 Construction of the plant binary expression vector pCAMBIA1300-pYAO-Cas9-NtREV A: Nucleic acid electrophoresis of PCR amplification of pCAMBIA1300-pYAO-Cas9-NtREV. B: Nucleic acid electrophoresis of double enzyme digestion of pCAMBIA1300-pYAO-Cas9-NtREV

图4 转基因植株检测的核酸电泳 A ：REV基因靶点C15编辑植株检测的核酸电泳；B ：REV基因靶点C16编辑植株检测的核酸电泳

Fig. 4 Nucleic acid electrophoresis for the detection of transgenic plants A: Nucleic acid electrophoresis for detecting plant editing of the REV gene at target C15. B: Nucleic acid electrophoresis for detecting plant editing of the REV gene at target C16

图5 CRISPR/Cas9编辑NtREV基因突变体形式分析 A ：REV基因靶点C16序列的突变情况；B ：REV基因靶点C15序列的突变情况。下划线表示PAM序列，蓝色字表示替换碱基，红色字表示插入碱基，-表示缺失的碱基

Fig. 5 Mutation form analysis of NtREV edited by the CRISPR/Cas9 A: Mutation of REV target C16 sequence; B: mutation of REV target C15 sequence. Underscores indicate PAM sequences, blue words indicate replacement bases, red words indicate insertion bases, and - indicates missing bases

图6 突变体材料和野生型材料的叶形态表型A-F：双拷贝同源突变体材料叶发育形态，包括漏斗形叶、荷叶形叶等形态；G-H：单拷贝同源突变体材料叶发育形态； I：对应时期野生型生长状态；标尺：1 cm

Fig. 6 Leaf morphological phenotypes of mutant and wild-type materials A-F: Leaf development morphology of the double-copy homologous mutants, including funnel-shaped leaves, lotus-shaped leaves and other morphologies; G-H: leaf development morphology of the single-copy homologous mutants; I: wild type growth state in the corresponding period; bar=1 cm

图7 烟草Ko-C15Ntrev和Ko-C16Ntrev突变体编辑形式Ko-C15Ntrev编辑形式为双拷贝同源突变体；Ko-C16Ntrev编辑形式为单拷贝同源突变体

Fig. 7 Editing forms of Ko-C15Ntrev and Ko-C16Ntrev mutants in tobacco The double-copy homologous mutants of Ko-C15NtREV; Ko-C16NtREV editing form is the single-copy homologous mutants

图8 突变体材料和野生型材料的顶芽观测 A，B，C分别为体式显微镜下烟草苗期野生型WT、Ko-C16Ntrev和Ko-C15Ntrev突变体顶芽表型；D，E，F分别为扫描电子显微镜下烟草苗期WT、Ko-C16Ntrev和Ko-C15Ntrev突变体顶端分生组织；G，H，I分别为上述表型对应的烟草苗期WT、Ko-C16Ntrev和Ko-C15Ntrev突变体材料

Fig. 8 Observations of apical buds of mutant and wild-type ones A, B, C: Apical bud phenotypes of tobacco seedling wild-type, Ko-C16Ntrev and Ko-C15Ntrev mutant under stereomicroscope, respectively; D, E, F: apical meristem phenotypes of tobacco seedling wild-type,Ko-C16Ntrev and Ko-C15Ntrev mutant under scanning electron microscopy, respectively; G, H, I: tobacco seedling wild-type, Ko-C16Ntrev and Ko-C15Ntrev mutant corresponding to the above phenotypes, respectively

图9 Ko-C16Ntrev突变体的自然株高、叶数、腋芽鲜重表型 A，B，C分别为WT和Ko-C16Ntrev突变体的自然株高、叶数、腋芽鲜重的统计；D，E分别为WT和Ko-C16Ntrev突变体侧芽表型图。数据表示平均值 ± SD，其中 n ≥ 10 ；**表示显著性 P<0.01；标尺：5 cm

Fig. 9 Phenotypes of plant height, leaf numbers and fresh weight of C16Ntrev mutants A, B, C: Statistical analysis of plant height, leaf numbers and fresh weight content of axillary buds of WT and Ko-C16Ntrev mutants. D and E: The phenotype of lateral buds of WT and Ko-C16Ntrev mutants, respectively. Bar= 5 cm. The data represent mean ± SD, n ≥ 10; ** indicates significance P<0.01

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