Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (7): 146-155.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0362
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SHAN Cao-mei(), YE Lei, ZHANG Lian-hu, KUANG Wei-gang, SUN Xiao-tang, MA Jian, CUI Ru-qiang()
Received:
2021-03-22
Online:
2021-07-26
Published:
2021-08-13
Contact:
CUI Ru-qiang
E-mail:StrawM@126.com;cuiruqiang@jxau.edu.cn
SHAN Cao-mei, YE Lei, ZHANG Lian-hu, KUANG Wei-gang, SUN Xiao-tang, MA Jian, CUI Ru-qiang. Cloning,and Functional Analysis of Gene OsRAI1 Resistant to Hirschmanniella mucronate in Rice[J]. Biotechnology Bulletin, 2021, 37(7): 146-155.
名称Name | 序列 Sequences(5'-3') | 作用 Function |
---|---|---|
OsRAI1-F | ATGGAGCTTGACGAG | 基因克隆 Gene cloning |
OsRAI1-R | CAGACACCTTCCGCC | |
T7 | CCTATAGTGAGTCGTATTA | 原核表达载体鉴定 Identification of prokaryotic expression vectors |
T7ter | CTAGCATAACCCCTTGGGGC | |
pET28a-OsRAI1-F | CAGCAAATGGGTCGCGGATCCATGGAGCTTGACGAG | 添加酶切位点 Adding enzyme cutting site |
pET28a-OsRAI1-R | ACTCGAGCACCACCAGCGGCCGCCAGACACCTTCCGCC | |
1302-OsRAI1-F | ACCATGGTAGATCTGACTAGTATGGAGCTTGACGAGGAGTCC | 添加酶切位点 Adding enzyme cutting site |
1302-OsRAI1-R | AAGTTCTTCTCCTTTACTAGTCAGACACCTTCCGCCATAGC | |
pCA-F | TGAGACTTTTCAACAAAGGGTAATATCCGG | 过表达载体鉴定 Identification of overexpression vectors |
pCA-R | GATCTAGTAACATAGATGACACCGC |
Table 1 Information of PCR primers
名称Name | 序列 Sequences(5'-3') | 作用 Function |
---|---|---|
OsRAI1-F | ATGGAGCTTGACGAG | 基因克隆 Gene cloning |
OsRAI1-R | CAGACACCTTCCGCC | |
T7 | CCTATAGTGAGTCGTATTA | 原核表达载体鉴定 Identification of prokaryotic expression vectors |
T7ter | CTAGCATAACCCCTTGGGGC | |
pET28a-OsRAI1-F | CAGCAAATGGGTCGCGGATCCATGGAGCTTGACGAG | 添加酶切位点 Adding enzyme cutting site |
pET28a-OsRAI1-R | ACTCGAGCACCACCAGCGGCCGCCAGACACCTTCCGCC | |
1302-OsRAI1-F | ACCATGGTAGATCTGACTAGTATGGAGCTTGACGAGGAGTCC | 添加酶切位点 Adding enzyme cutting site |
1302-OsRAI1-R | AAGTTCTTCTCCTTTACTAGTCAGACACCTTCCGCCATAGC | |
pCA-F | TGAGACTTTTCAACAAAGGGTAATATCCGG | 过表达载体鉴定 Identification of overexpression vectors |
pCA-R | GATCTAGTAACATAGATGACACCGC |
Fig. 5 Development and evolution of OsRAI1 protein system A: Phylogenetic tree of OsRAI1. B: Conserved motif element prediction of OsRAI1 protein sequences
Fig. 6 Electropherogram of PCR product for OsRAI1 gene A:Amplified product of gene OsRAI1 via prokaryotic expression; B:Amplified product of gene OsRAI1 via overexpression. M:2 000 marker; 1-3:Amplified product
Fig. 7 Identification of positive clones of OsRAI1 gene via vectors A:Identification of positive clones of gene OsRAI1 via prokaryotic expression vector; M:2000 marker. 1-10:PCR product of single colony. B:Identification of positive clones of gene OsRAI1 via overexpression vector; M:5000 marker. 1-10:PCR product of single colony
Fig. 8 Subcellular localization of gene OsRAI1 in the leaves of Nicotiana benthamiana A:Target protein fluorescence channel. B:Chloroplast fluorescence channel. C:Brightfield. D:Overlay chart
Fig. 9 WB analysis of recombinant protein expression products induced by different concentrations of IPTG M:Protein marker. 1:Whole bacteria induced by 1 mmol/L. 2:Supernatant induced by 0.8 mmol/L. 3:Whole bacteria induced by 0.8 mmol/L. 4:Supernatant induced by 0.6 mmol/L. 5:Whole bacteria induced by 0.6 mmol/L. 6:Supernatant induced by 0.2 mmol/L. 7:Whole bacteria induced by 0.2 mmol/L
Fig. 10 SDS-PAGE analysis of recombinant protein expression products induced by different inducing time and temperature M:Protein Marker. 1:Whole bacteria induced by 37℃ for 3 h. 2:Whole bacteria induced by 37℃ for 6 h. 3:Whole bacteria induced by 37℃ for 18 h. 4:Whole bacteria induced by 28℃ for 3 h. 5:Whole bacteria induced by 28℃ for 6 h. 6:Whole bacteria induced by 28℃ for 18 h. 7:Whole bacteria induced by 16℃ for 3 h. 8:Whole bacteria induced by 16℃ for 6 h. 9:Whole bacteria induced by 16℃ for 18 h. 10:Supernatant induced by 37℃ for 3 h. 11:Supernatant induced by 37℃ for 6 h. 12:Supernatant induced by 37℃ for 18 h. 13:Supernatant induced by 28℃ for 3 h. 14:Supernatant induced by 28℃ for 6 h. 15:Supernatant induced by 28℃ for 18 h. 16:Supernatant induced by 16℃ for 3 h. 17:Supernatant induced by 16℃ for 6 h. 18:Supernatant induced by 16℃ for 18 h
基因名称 Gene name | 蛋白描述 Protein description | 功能结构域 Functional domain | 氨基酸数/个 Number of amino acids | 互作系数 Interaction coefficient |
---|---|---|---|---|
Os05T0586300-00 | Expressed protein;Os03g0137400 protein | HLH | 387 | 0.644 |
Os04T0486400-01 | OJ000223_09.13 protein;Os04g0486400 protein | SANT | 385 | 0.612 |
Os03T0137400-01 | Expressed protein;Os03g0137400 protein | transmembrane helix region | 792 | 0.578 |
Os01T0752500-00 | Putative ethylene responsive protein Os01g0752500 protein | AP2 | 212 | 0.570 |
Os06T0164400-01 | annotation not available;Os06g0164400 protein | coiled coil region;HLH | 197 | 0.503 |
Table 2 Functional interaction protein prediction of OsRAI1
基因名称 Gene name | 蛋白描述 Protein description | 功能结构域 Functional domain | 氨基酸数/个 Number of amino acids | 互作系数 Interaction coefficient |
---|---|---|---|---|
Os05T0586300-00 | Expressed protein;Os03g0137400 protein | HLH | 387 | 0.644 |
Os04T0486400-01 | OJ000223_09.13 protein;Os04g0486400 protein | SANT | 385 | 0.612 |
Os03T0137400-01 | Expressed protein;Os03g0137400 protein | transmembrane helix region | 792 | 0.578 |
Os01T0752500-00 | Putative ethylene responsive protein Os01g0752500 protein | AP2 | 212 | 0.570 |
Os06T0164400-01 | annotation not available;Os06g0164400 protein | coiled coil region;HLH | 197 | 0.503 |
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