结缕草叶肉细胞原生质体瞬时基因表达系统的构建

doi:10.11686/cyxb2022348

摘要/Abstract

摘要：

为了结合转基因方法探索结缕草基因功能，建立高效、快速的结缕草原生质体制备及瞬时基因表达系统，利用正交试验对影响原生质体制备的主要因素进行优化，同时利用原生质体进行亚细胞定位和蛋白互作研究。结果表明，当酶配比为2% （w/v） cellulase R10和1.5% （w/v） macerozyme R10，酶解时间为7 h时，原生质体产量和活性均达到最高，分别为1.23×10⁷个·mL^-1和98%以上，满足后续转化所需。多次重复试验表明，加入5~10 μg质粒，原生质体转化率可达75%以上。利用原生质体转化体系进行了亚细胞定位检测，结果发现结缕草ZjNOL和ZjNYC1定位在叶绿体中，ZjZFN定位在细胞核中。双分子荧光互补分析证明ZjNOL和ZjNYC1在叶绿体中相互作用。原生质体制备及转化方法与遗传学和组学技术相结合，能够为结缕草基因功能研究和基因编辑提供支持。

关键词: 结缕草, 原生质体, 瞬时基因表达, 亚细胞定位, 蛋白互作

Abstract:

An efficient leaf mesophyll protoplast isolation and transient gene expression system in Zoysia japonica is highly desirable for in vivo gene functional studies in its homologous system. The main factors influencing the isolation of protoplasts from Z. japonica were optimized by orthogonal design. The transformation protocol was used for protein subcellular localization and protein-protein interaction. In this study， a highly efficient protoplast （1.23×10⁷ protoplasts·mL^-1） was generated using 2% （w/v） cellulase R10 and 1.5% （w/v） macerozyme R10 for digestion over 7 h. FDA staining results showed that approximately 99% of protoplasts exhibited high activity. Repeated experiments showed that by adding 5-10 μg plasmid， the transformation efficiency reached more than 75%. Using this protocol to identify subcellular locations， ZjNOL and ZjNYC1 were proved to be localized in chloroplasts， and ZjZFN was visualized in nuclei. The bimolecular fluorescence complementation assay showed that ZjNOL and ZjNYC1 interacted with each other in the chloroplast. The protoplast isolation and transformation protocol， when combined with genetics and omics techniques， will allow further study of gene functions in Z. japonica.

Key words: Zoysia japonica, protoplast, transient gene expression, subcellular localization, protein-protein interaction

管瑾, 郭一荻, 刘凌云, 尹淑霞, 滕珂. 结缕草叶肉细胞原生质体瞬时基因表达系统的构建[J]. 草业学报, 2023, 32(7): 61-71.

Jin GUAN, Yi-di GUO, Ling-yun LIU, Shu-xia YIN, Ke TENG. An efficient protocol for Zoysia japonica mesophyll protoplast isolation and transformation， and its application in subcellular localization and protein interaction analysis[J]. Acta Prataculturae Sinica, 2023, 32(7): 61-71.

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引物名称Primer name	序列Sequence （5'-3'）
ZjNYC1-F	CTCGTCCCGACCTTATCCGC
ZjNYC1-R	GGGCACGGGGTCATCCAG
ZjNOL-F	CTCCACACAAGACTCCATTCG
ZjNOL-R	GAAAAAGGAATGGTTCAAAACAGAT
ZjZFN-F	ATGTCGTCCGCCATGGAATT
ZjZFN-R	TCACGCGGTCATGAGGAGGC
3302Y3-ZjNYC1-F	cctactagtcctagggacgtcaATGGCCGCCGCCGTCGCGCA
3302Y3-ZjNYC1-R	tgctcaccatacgcgttacagaTGTGCCTGGAAGAGGACCAC
3302Y3-ZjNOL-F	cctactagtcctagggacgtcaATGGCTGCCAGCGTCAGCATCG
3302Y3-ZjNOL-R	tgctcaccatacgcgttacagaATCTTCAACAACATACTTAT
3302Y3-ZjZFN-F	cacgggggactcttgaccatggtaATGTCGTCCGCCATGGAATT
3302Y3-ZjZFN-R	ggtacacgcgtactagtcagatcCGCGGTCATGAGGAGGCGGG
35S-pSPY-ZjNYC1-F	caggcctggcgcgccactagtgATGGCCGCCGCCGTCGCGCA
35S-pSPY-ZjNYC1-R	ggtcgacagtactatcgatggaTGTGCCTGGAAGAGGACCAC
35S-pSPY-ZjNOL-F	caggcctggcgcgccactagtgATGGCTGCCAGCGTCAGCATCG
35S-pSPY-ZjNOL-R	ggtcgacagtactatcgatggaATCTTCAACAACATACTTAT

引物名称Primer name	序列Sequence （5'-3'）
ZjNYC1-F	CTCGTCCCGACCTTATCCGC
ZjNYC1-R	GGGCACGGGGTCATCCAG
ZjNOL-F	CTCCACACAAGACTCCATTCG
ZjNOL-R	GAAAAAGGAATGGTTCAAAACAGAT
ZjZFN-F	ATGTCGTCCGCCATGGAATT
ZjZFN-R	TCACGCGGTCATGAGGAGGC
3302Y3-ZjNYC1-F	cctactagtcctagggacgtcaATGGCCGCCGCCGTCGCGCA
3302Y3-ZjNYC1-R	tgctcaccatacgcgttacagaTGTGCCTGGAAGAGGACCAC
3302Y3-ZjNOL-F	cctactagtcctagggacgtcaATGGCTGCCAGCGTCAGCATCG
3302Y3-ZjNOL-R	tgctcaccatacgcgttacagaATCTTCAACAACATACTTAT
3302Y3-ZjZFN-F	cacgggggactcttgaccatggtaATGTCGTCCGCCATGGAATT
3302Y3-ZjZFN-R	ggtacacgcgtactagtcagatcCGCGGTCATGAGGAGGCGGG
35S-pSPY-ZjNYC1-F	caggcctggcgcgccactagtgATGGCCGCCGCCGTCGCGCA
35S-pSPY-ZjNYC1-R	ggtcgacagtactatcgatggaTGTGCCTGGAAGAGGACCAC
35S-pSPY-ZjNOL-F	caggcctggcgcgccactagtgATGGCTGCCAGCGTCAGCATCG
35S-pSPY-ZjNOL-R	ggtcgacagtactatcgatggaATCTTCAACAACATACTTAT

因素 Factors	Cellulase R10 （%， w/v）	Macerozyme R10 （%， w/v）	酶解时间 Enzymolysis time （h）
1	1	0.5	4
2	2	1.0	5
3	3	1.5	6
4	4	2.0	7

因素 Factors	Cellulase R10 （%， w/v）	Macerozyme R10 （%， w/v）	酶解时间 Enzymolysis time （h）
1	1	0.5	4
2	2	1.0	5
3	3	1.5	6
4	4	2.0	7

溶液名称 Solution name	溶液配方 Solution composition	储存条件 Storage	用途 Usage
酶解液Enzyme solution	cellulase R10，macerozyme R10，甘露醇Mannitol，10 mmol·L^-1 MES，pH 5.7，10 mmol·L^-1 CaCl₂，0.1% （w/v） BSA	现用现配，室温Freshly prepared， room temperature	制备原生质体Preparation of protoplasts
W5溶液 W5 solution	154 mmol·L^-1 NaCl，125 mmol·L^-1 CaCl₂，5 mmol·L^-1KCl，2 mmol·L^-1 MES，pH 5.7	4 ℃	释放和清洗原生质体Release and wash protoplasts
MMG溶液 MMG solution	0.4 mol·L^-1 甘露醇Mannitol， 15 mmol·L^-1 MgCl₂， 4 mmol·L^-1 MES，pH 5.7	4 ℃	重悬原生质体Resuspend protoplasts
PEG溶液 PEG solution	40% （w/v） PEG 4000， 0.2 mol·L^-1 甘露醇Mannitol，0.1 mol·L^-1 CaCl₂	现用现配，室温Freshly prepared， room temperature	介导原生质体转化Transform plasmids into protoplasts
WI溶液 WI solution	0.5 mol·L^-1 甘露醇Mannitol，20 mmol·L^-1 KCl，4 mmol·L^-1 MES，pH 5.7	4 ℃	重悬转化体系Resuspend transformation system