草业学报 ›› 2023, Vol. 32 ›› Issue (4): 129-141.DOI: 10.11686/cyxb2022180
• 研究论文 • 上一篇
收稿日期:
2022-04-20
修回日期:
2022-06-27
出版日期:
2023-04-20
发布日期:
2023-01-29
通讯作者:
徐彬
作者简介:
E-mail: binxu@njau.edu.cn基金资助:
Jia-ming YAO(), Huan-huan HAO, Jing ZHANG, Bin XU()
Received:
2022-04-20
Revised:
2022-06-27
Online:
2023-04-20
Published:
2023-01-29
Contact:
Bin XU
摘要:
tRNA可以将多个sgRNAs连接起来合并成一个多顺反子基因,再与CRISPR/Cas9表达载体结合形成多顺反子tRNA-sgRNA/Cas9(PTG/Cas9)系统来对多靶点进行基因编辑。该系统已经在水稻中验证其可促进sgRNAs的转录和提高多靶点的编辑效率。因此,为了在多年生黑麦草中实现高效的基因编辑,本研究中,构建了2个带有tRNA的CRISPR中间载体,并提供了一种快速、灵活的PTG/Cas9载体构建方法。为了快速验证PTG/Cas9系统是否可以在多年生黑麦草基因组中发挥功能,用聚乙二醇4000(PEG 4000)介导PTG/Cas9质粒转化多年生黑麦草原生质体,后提取原生质体DNA扩增目的序列检测是否有目标基因被编辑的细胞。试验结果显示,PTG/Cas9系统可用于多年生黑麦草基因编辑,且基因编辑效率约为6.7%。试验结果为多年生黑麦草遗传研究和育种提供了重要的基础。
姚佳明, 郝欢欢, 张敬, 徐彬. tRNA-sgRNA/Cas9系统介导多年生黑麦草原生质体的基因编辑[J]. 草业学报, 2023, 32(4): 129-141.
Jia-ming YAO, Huan-huan HAO, Jing ZHANG, Bin XU. The use of the tRNA-sgRNA/Cas9 system for gene editing in perennial ryegrass protoplasts[J]. Acta Prataculturae Sinica, 2023, 32(4): 129-141.
引物Primer | 引物序列Primer sequence (5′-3′) |
---|---|
S1F | TTGCAGTATGGGCCGGCCCATTACG |
S1R | ACGTTGTAAAACGACGGCCAGTGCC |
S2F | CCATGATTACGAATTCGAGCTCGGTACCCGG |
S2R | CTGCACATCTGATTCCTCCAAGATCCAT |
F1 | CTCCGTTTTACCTGTGGAATC |
R1 | |
F2 | |
R2 | CGGAGGAAAATTCCATCCAC |
F3 | GGCTCGTATGTTGTGTGG |
R3 | |
F4 | |
F5 | TTCAGAGGTCTCTACCGTGGAATCGGCAGCAAA |
R5 | TTCAGAGGTCTCA |
F6 | GAGGTCTCG |
R6 | AGCGTGGGTCTCGCTCGTCCATCCACTCCAAGC |
F7 | GCGAGCCTTATAAGCAG |
R7 | GCTGAGATAACGAGCCAA |
表1 试验所用引物
Table 1 Primers used in this study
引物Primer | 引物序列Primer sequence (5′-3′) |
---|---|
S1F | TTGCAGTATGGGCCGGCCCATTACG |
S1R | ACGTTGTAAAACGACGGCCAGTGCC |
S2F | CCATGATTACGAATTCGAGCTCGGTACCCGG |
S2R | CTGCACATCTGATTCCTCCAAGATCCAT |
F1 | CTCCGTTTTACCTGTGGAATC |
R1 | |
F2 | |
R2 | CGGAGGAAAATTCCATCCAC |
F3 | GGCTCGTATGTTGTGTGG |
R3 | |
F4 | |
F5 | TTCAGAGGTCTCTACCGTGGAATCGGCAGCAAA |
R5 | TTCAGAGGTCTCA |
F6 | GAGGTCTCG |
R6 | AGCGTGGGTCTCGCTCGTCCATCCACTCCAAGC |
F7 | GCGAGCCTTATAAGCAG |
R7 | GCTGAGATAACGAGCCAA |
图1 sgRNA在多年生黑麦草基因组中的位置(a)及tRNA-sgRNA多顺反子(PTG)表达盒的结构(b)黄色字体碱基序列代表间隔序列1,紫色代表间隔序列2,红棕色代表间隔序列3。下同。Yellow colored nucleotides represented the spacer 1, purple ones represented the spacer 2, reddish-brown ones represented the spacer 3. The same below.
Fig.1 Location of sgRNAs in the genome of perennial ryegrass and the structure of the polycistronic tRNA-sgRNA (PTG) cassette
步骤Steps | 反应组分Reaction components | 组分名称Component name | 体积Volume (μL) |
---|---|---|---|
PCR组分 Components of the PCR | PCR缓冲液PCR buffer | 2×Phanta Max buffer | 15.0 |
DRT | dNTP Mix (10 mmol·L-1) | 0.6 | |
模板Template | POT/PT (100 ng·L-1) | 1.0 | |
上游引物Forward primer | F1/F2/F3/F4 (10 μmol·L-1) | 1.2 | |
下游引物Reverse primer | R1/R2/R3/R2 (10 μmol·L-1) | 1.2 | |
DNA聚合酶DNA polymerase | Phanta Max Super-Fidelity DNA polymerase | 0.6 | |
双蒸水Double distilled water | ddH2O | 10.4 | |
共计Total | 30.0 | ||
重叠式PCR Overlapping PCR | PCR缓冲液PCR buffer | 2×Phanta Max buffer | 15.0 |
DRT | dNTP Mix (10 mmol·L-1) | 0.6 | |
模板Template | 产物1、产物2/产物3、产物4 Product 1, product 2/product 3, product 4 | 1.0+1.0 | |
上游引物Forward primer | F5/F6 (10 μmol·L-1) | 1.2 | |
下游引物Reverse primer | R5/R6 (10 μmol·L-1) | 1.2 | |
DNA聚合酶DNA polymerase | Phanta Max Super-Fidelity DNA polymerase | 0.6 | |
双蒸水Double distilled water | ddH2O | 9.4 | |
共计Total | 30.0 | ||
金门组装反应 Golden gate assembly | 缓冲液Buffer | 10×CutSmart | 1.0 |
10×T4 DNA ligase buffer | 1.0 | ||
连接产物Products for connection | 产物5 Product 5 | 2.5 | |
产物6 Product 6 | 2.5 | ||
表达载体Expression vector | pYLCRISPR Cas9Pubi-H | 2.0 | |
限制性内切酶Restriction enzyme | Bsa I | 0.5 | |
连接酶Ligase | T4 DNA ligase | 0.5 | |
共计Total | 10.0 |
表2 PCR反应体系
Table 2 PCR reaction mix
步骤Steps | 反应组分Reaction components | 组分名称Component name | 体积Volume (μL) |
---|---|---|---|
PCR组分 Components of the PCR | PCR缓冲液PCR buffer | 2×Phanta Max buffer | 15.0 |
DRT | dNTP Mix (10 mmol·L-1) | 0.6 | |
模板Template | POT/PT (100 ng·L-1) | 1.0 | |
上游引物Forward primer | F1/F2/F3/F4 (10 μmol·L-1) | 1.2 | |
下游引物Reverse primer | R1/R2/R3/R2 (10 μmol·L-1) | 1.2 | |
DNA聚合酶DNA polymerase | Phanta Max Super-Fidelity DNA polymerase | 0.6 | |
双蒸水Double distilled water | ddH2O | 10.4 | |
共计Total | 30.0 | ||
重叠式PCR Overlapping PCR | PCR缓冲液PCR buffer | 2×Phanta Max buffer | 15.0 |
DRT | dNTP Mix (10 mmol·L-1) | 0.6 | |
模板Template | 产物1、产物2/产物3、产物4 Product 1, product 2/product 3, product 4 | 1.0+1.0 | |
上游引物Forward primer | F5/F6 (10 μmol·L-1) | 1.2 | |
下游引物Reverse primer | R5/R6 (10 μmol·L-1) | 1.2 | |
DNA聚合酶DNA polymerase | Phanta Max Super-Fidelity DNA polymerase | 0.6 | |
双蒸水Double distilled water | ddH2O | 9.4 | |
共计Total | 30.0 | ||
金门组装反应 Golden gate assembly | 缓冲液Buffer | 10×CutSmart | 1.0 |
10×T4 DNA ligase buffer | 1.0 | ||
连接产物Products for connection | 产物5 Product 5 | 2.5 | |
产物6 Product 6 | 2.5 | ||
表达载体Expression vector | pYLCRISPR Cas9Pubi-H | 2.0 | |
限制性内切酶Restriction enzyme | Bsa I | 0.5 | |
连接酶Ligase | T4 DNA ligase | 0.5 | |
共计Total | 10.0 |
常规PCR Regular PCR | 重叠式PCR Overlapping PCR | 金门组装Golden gate assembly | ||||||
---|---|---|---|---|---|---|---|---|
步骤Step | 温度Temperature (℃) | 时间Time (s) | 步骤Step | 温度Temperature (℃) | 时间Time (s) | 步骤Step | 温度Temperature (℃) | 时间Time (s) |
1 | 95 | 180 | 1 | 95 | 180 | 1 | 37 | 300 |
2 | 95 | 15 | 2 | 95 | 15 | 2 | 16 | 600 |
3 | 60 | 15 | 3 | 60 | 15 | 3 | - | 7200 |
4 | 72 | 30 | 4 | 72 | 30 | |||
5 | - | 2100 | 5 | - | 2100 | |||
6 | 72 | 300 | 6 | 72 | 3000 |
表3 PCR反应条件
Table 3 PCR reaction condition
常规PCR Regular PCR | 重叠式PCR Overlapping PCR | 金门组装Golden gate assembly | ||||||
---|---|---|---|---|---|---|---|---|
步骤Step | 温度Temperature (℃) | 时间Time (s) | 步骤Step | 温度Temperature (℃) | 时间Time (s) | 步骤Step | 温度Temperature (℃) | 时间Time (s) |
1 | 95 | 180 | 1 | 95 | 180 | 1 | 37 | 300 |
2 | 95 | 15 | 2 | 95 | 15 | 2 | 16 | 600 |
3 | 60 | 15 | 3 | 60 | 15 | 3 | - | 7200 |
4 | 72 | 30 | 4 | 72 | 30 | |||
5 | - | 2100 | 5 | - | 2100 | |||
6 | 72 | 300 | 6 | 72 | 3000 |
溶液Solution | 成分Ingredient |
---|---|
酶解液Enzyme solution | 10 mmol·L-1 MES,1.5%纤维素酶R10 Cellulase R10,0.75%离析酶R10 Macerozyme R10,20 mmol·L-1 KCl,10 mmol·L-1 CaCl2,0.1% BSA,0.6 mol·L-1甘露醇Mannitol |
W5 | 154 mmol·L-1 NaCl,125 mmol·L-1 CaCl2,5 mmol·L-1 KCl,2 mmol·L-1 MES |
MMg | 0.4 mol·L-1甘露醇Mannitol,15 mmol·L-1 MgCl2,4 mmol·L-1 MES |
PEG 4000 | 20% PEG 4000,10 mmol·L-1 CaCl2,0.3 mol·L-1甘露醇Mannitol |
表4 多年生黑麦草原生质体提取及转化所用溶液
Table 4 Solutions used to extract and transform perennial ryegrass protoplasts
溶液Solution | 成分Ingredient |
---|---|
酶解液Enzyme solution | 10 mmol·L-1 MES,1.5%纤维素酶R10 Cellulase R10,0.75%离析酶R10 Macerozyme R10,20 mmol·L-1 KCl,10 mmol·L-1 CaCl2,0.1% BSA,0.6 mol·L-1甘露醇Mannitol |
W5 | 154 mmol·L-1 NaCl,125 mmol·L-1 CaCl2,5 mmol·L-1 KCl,2 mmol·L-1 MES |
MMg | 0.4 mol·L-1甘露醇Mannitol,15 mmol·L-1 MgCl2,4 mmol·L-1 MES |
PEG 4000 | 20% PEG 4000,10 mmol·L-1 CaCl2,0.3 mol·L-1甘露醇Mannitol |
图2 构建tRNA-sgRNA多顺反子(PTG)表达盒的CRISPR/Cas9载体a: pYLsgRNA-OsU3原始载体pYLsgRNA-OsU3 original vector; b: pYLsgRNA-OsU3-tRNA(POT)载体pYLsgRNA-OsU3-tRNA (POT) vector; c: pYLsgRNA-tRNA(PT)载体pYLsgRNA-tRNA (PT) vector; d: pYLCRISPRCas9Pubi-H载体(Cas9双元载体) pYLCRISPRCas9Pubi-H vector (Cas9 binary vector).
Fig.2 CRISPR/Cas9 vectors for constructing the polycistronic tRNA-sgRNA (PTG) cassette
图4 Bsa I连接位点的选择及连接过程黑色加粗部分为Bsa Ⅰ识别序列,虚线方框为Bsa Ⅰ连接位点。The bold black parts are the Bsa Ⅰ recognition sequences, and the dotted boxs are the Bsa Ⅰ connection sites.
Fig.4 Selection of Bsa Ⅰ connection site and the connection process
图5 多年生黑麦草原生质体制备a: 制备的原生质体Isolated protoplasts; b~c: 原生质体的FDA染色观察Observation of FDA stained protoplasts; 原生质体经FDA染色后,b为一个视野中明场下的细胞总数量,c为相同视野中绿色荧光通路下的活细胞数量After the protoplasts were stained by FDA, Figure b shows the total number of cells under the bright light, and Figure c shows the number of viable cells under the green fluorescence light in the same field.
Fig.5 Perennial ryegrass protoplasts preparation
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