Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (7): 195-205.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1304
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YU Hui(), WANG Jing, LIANG Xin-xin, XIN Ya-ping, ZHOU Jun, ZHAO Hui-jun()
Received:
2022-10-24
Online:
2023-07-26
Published:
2023-08-17
Contact:
ZHAO Hui-jun
E-mail:1632570918@qq.com;zhaohuijun1022@163.com
YU Hui, WANG Jing, LIANG Xin-xin, XIN Ya-ping, ZHOU Jun, ZHAO Hui-jun. Isolation and Functional Verification of Genes Responding to Iron and Cadmium Stresses in Lycium barbarum[J]. Biotechnology Bulletin, 2023, 39(7): 195-205.
引物Primer | 序列Sequence(5'-3') | 用途Purpose |
---|---|---|
LbIRT2 | F: ATGGCTGCTCAAACCCAAGT R: TCACAACTCCATGCCAGTAGTAGA | 基因克隆 Gene clone |
LbFRO | F: ATGGGTGAACTCTCAGGGCAA R: CTAGAGGTCAAAATTGTGGCAGTTG | 基因克隆 Gene clone |
LbNRAMP3 | F: ATGCCTCCACACGATGAAGAACA R: TCAACTCTCTATGCTCGTGATACTCTT | 基因克隆 Gene clone |
LbActin | F: TCTACGAGGGTTACGCTTTG R: TCCCGTTCAGCAGTGGTT | 枸杞内参引物 Internal primer of Lycium barbarum L. |
qRT-LbFRO | F: TCAAGCCACCCAGGGAACT R: CATGAAAATTGGTAAACTGAACATCA | 荧光定量引物 Fluorescent quantitative PCR primer |
qRT-LbNRAMP3 | F: GCAGAGAGCGTTAATAACAAGAAGTTG R: TTAAGCCACTCGTTGAGAACATCT | 荧光定量引物 Fluorescent quantitative PCR primer |
qRT-LbIRT1 | F: AGGAGCAATTCATCAAGATGCA R: TGCTACATGCTCCGGCTACTAG | 荧光定量引物 Fluorescent quantitative PCR primer |
qRT-LbIRT2 | F: TCCAAATTACCATTCCTAAAGACCAT R: CGAACCCACTGTGATCTTTGAG | 荧光定量引物 Fluorescent quantitative PCR primer |
qRT-LbYSL | F: TGCAGTGTTCATGAGCGGTATAA R: TCACATCCTCCGAATTCTTCTTC | 荧光定量引物 Fluorescent quantitative PCR primer |
Table 1 Primers for gene cloning and specific expressions
引物Primer | 序列Sequence(5'-3') | 用途Purpose |
---|---|---|
LbIRT2 | F: ATGGCTGCTCAAACCCAAGT R: TCACAACTCCATGCCAGTAGTAGA | 基因克隆 Gene clone |
LbFRO | F: ATGGGTGAACTCTCAGGGCAA R: CTAGAGGTCAAAATTGTGGCAGTTG | 基因克隆 Gene clone |
LbNRAMP3 | F: ATGCCTCCACACGATGAAGAACA R: TCAACTCTCTATGCTCGTGATACTCTT | 基因克隆 Gene clone |
LbActin | F: TCTACGAGGGTTACGCTTTG R: TCCCGTTCAGCAGTGGTT | 枸杞内参引物 Internal primer of Lycium barbarum L. |
qRT-LbFRO | F: TCAAGCCACCCAGGGAACT R: CATGAAAATTGGTAAACTGAACATCA | 荧光定量引物 Fluorescent quantitative PCR primer |
qRT-LbNRAMP3 | F: GCAGAGAGCGTTAATAACAAGAAGTTG R: TTAAGCCACTCGTTGAGAACATCT | 荧光定量引物 Fluorescent quantitative PCR primer |
qRT-LbIRT1 | F: AGGAGCAATTCATCAAGATGCA R: TGCTACATGCTCCGGCTACTAG | 荧光定量引物 Fluorescent quantitative PCR primer |
qRT-LbIRT2 | F: TCCAAATTACCATTCCTAAAGACCAT R: CGAACCCACTGTGATCTTTGAG | 荧光定量引物 Fluorescent quantitative PCR primer |
qRT-LbYSL | F: TGCAGTGTTCATGAGCGGTATAA R: TCACATCCTCCGAATTCTTCTTC | 荧光定量引物 Fluorescent quantitative PCR primer |
Fig. 1 Screening and hot map of ion transporter genes in the leaf tissue and root tissue of L. barbarum T12hL/CKL: The genes changed in the leaves at the 12th hour of Cd stress with the transcriptome data of the control; T48hL/CKL: the genes changed in the leaves at the 48th hour of Cd stress with the transcriptome data of the control; T12hR/CKR: the genes changed in the roots at the 12th hour of Cd stress with the transcriptome data of the control; T48hR/CKR: the genes changed in the roots at the 48th hour of Cd stress, the transcriptome data of the control
基因ID Gene ID | 基因名称 Gene name | log2 (T12hR/CKR) | log2 (T48hR/CKR) | 长度 Length/bp | 相似度 Similarity/% | 筛选部位 Isolation location |
---|---|---|---|---|---|---|
CL7788.Contig3 | 离子转运蛋白基因 Ion transport protein | 6.67 | 6.82 | 1 948 | 89.94 | 根系Root |
Unigene39380 | OPT寡肽转运蛋白基因 OPT oligopeptide transporter | 8.372 403 | 10.0 693 | 1 739 | 99.47 | 根系Root |
Unigene61286 | 锌转运体5 Zinc transporter 5-like | 4.88 | 5.86 | 1 431 | 82.17 | 根系Root |
CL11868.Contig2 | ZIP家族锌转运蛋白基因 ZIP Zinc transporter | 7.28 | 7.23 | 1 167 | 98.77 | 根系Root |
CL4044 | Nramp3金属离子转运蛋白基因 Metal transporter Nramp3-like | 5.3 | 4.23 493 | 3 830 | 92.62 | 根系Root |
CL1976.Contig4_ | 机械敏感通道蛋白基因8 Mechanosensitive ion channel protein 8-like | 7.852 761 | 7.92 | 3 088 | 72.81 | 根系Root |
Unigene60599 | 黄色条纹蛋白基因YSL2 Metal-nicotianamine transporter YSL2-like | 2.178 219 | 2.525 766 | 2 161 | 86.78 | 根系Root |
CL1155.Contig | 黄色条纹蛋白基因YSL3 Metal-nicotianamine transporter YSL3-like | 9.070 304 | 6.348 448 | 3 045 | 91.95 | 根系Root |
CL3488.Contig3 | 铁氧还原酶基因6 Ferric reduction oxidase 6-like | 1.875 425 | 2.782 025 | 3 193 | 89.01 | 叶片Leaf |
Unigene44038 | 铁氧还原酶基因8 Ferric reduction oxidase 8 | 2.515 402 | 2.019 701 | 1 935 | 89.08 | 叶片Leaf |
CL9188.Contig6 | 液泡膜铁离子转运蛋白基因4 Vacuolar iron transporter homolog 4-like | 3.63 367 | 2.881 373 | 588 | 84.66 | 叶片Leaf |
CL12735.Contig1 | 铁氧还原酶基因2 Ferric reduction oxidase 2-like | 3.527 493 | 4.06 572 | 2 158 | 91.53 | 叶片Leaf |
Unigene21912 | 寡肽转运蛋白基因4 Oligopeptide transporter 4 | 2.264 557 | 0.763 512 | 1 713 | 98.03 | 叶片Leaf |
CL1352.Contig23 | 多向耐药性PDR跨膜转运蛋白基因1 Pleiotropic drug resistance protein 1-like | 9.955 166 | 10.28 298 | 5 054 | 90.33 | 叶片Leaf |
CL17567.Contig5 | Nramp6金属离子转运蛋白基因 Metal transporter Nramp6 | 6.758 793 | 6.640 834 | 1 159 | 95.06 | 叶片Leaf |
Unigene36942 | Nramp5金属离子转运蛋白基因 Metal transporter Nramp5 | 8.152 608 | 7.285 907 | 415 | 95.48 | 叶片Leaf |
Table 2 Genes related to ion transporter selected from the root and leaf
基因ID Gene ID | 基因名称 Gene name | log2 (T12hR/CKR) | log2 (T48hR/CKR) | 长度 Length/bp | 相似度 Similarity/% | 筛选部位 Isolation location |
---|---|---|---|---|---|---|
CL7788.Contig3 | 离子转运蛋白基因 Ion transport protein | 6.67 | 6.82 | 1 948 | 89.94 | 根系Root |
Unigene39380 | OPT寡肽转运蛋白基因 OPT oligopeptide transporter | 8.372 403 | 10.0 693 | 1 739 | 99.47 | 根系Root |
Unigene61286 | 锌转运体5 Zinc transporter 5-like | 4.88 | 5.86 | 1 431 | 82.17 | 根系Root |
CL11868.Contig2 | ZIP家族锌转运蛋白基因 ZIP Zinc transporter | 7.28 | 7.23 | 1 167 | 98.77 | 根系Root |
CL4044 | Nramp3金属离子转运蛋白基因 Metal transporter Nramp3-like | 5.3 | 4.23 493 | 3 830 | 92.62 | 根系Root |
CL1976.Contig4_ | 机械敏感通道蛋白基因8 Mechanosensitive ion channel protein 8-like | 7.852 761 | 7.92 | 3 088 | 72.81 | 根系Root |
Unigene60599 | 黄色条纹蛋白基因YSL2 Metal-nicotianamine transporter YSL2-like | 2.178 219 | 2.525 766 | 2 161 | 86.78 | 根系Root |
CL1155.Contig | 黄色条纹蛋白基因YSL3 Metal-nicotianamine transporter YSL3-like | 9.070 304 | 6.348 448 | 3 045 | 91.95 | 根系Root |
CL3488.Contig3 | 铁氧还原酶基因6 Ferric reduction oxidase 6-like | 1.875 425 | 2.782 025 | 3 193 | 89.01 | 叶片Leaf |
Unigene44038 | 铁氧还原酶基因8 Ferric reduction oxidase 8 | 2.515 402 | 2.019 701 | 1 935 | 89.08 | 叶片Leaf |
CL9188.Contig6 | 液泡膜铁离子转运蛋白基因4 Vacuolar iron transporter homolog 4-like | 3.63 367 | 2.881 373 | 588 | 84.66 | 叶片Leaf |
CL12735.Contig1 | 铁氧还原酶基因2 Ferric reduction oxidase 2-like | 3.527 493 | 4.06 572 | 2 158 | 91.53 | 叶片Leaf |
Unigene21912 | 寡肽转运蛋白基因4 Oligopeptide transporter 4 | 2.264 557 | 0.763 512 | 1 713 | 98.03 | 叶片Leaf |
CL1352.Contig23 | 多向耐药性PDR跨膜转运蛋白基因1 Pleiotropic drug resistance protein 1-like | 9.955 166 | 10.28 298 | 5 054 | 90.33 | 叶片Leaf |
CL17567.Contig5 | Nramp6金属离子转运蛋白基因 Metal transporter Nramp6 | 6.758 793 | 6.640 834 | 1 159 | 95.06 | 叶片Leaf |
Unigene36942 | Nramp5金属离子转运蛋白基因 Metal transporter Nramp5 | 8.152 608 | 7.285 907 | 415 | 95.48 | 叶片Leaf |
Fig. 2 Expression pattern of target gene under 200 μmol/L Fe-EDTA stress *, ** and *** denote P<0.05, P<0.01 and P<0.001, compared with the control(CK). The same below
基因名称 Gene name | 氨基酸长度 Amino acid length/aa | 分子量 Molecular weight/kD | 等电点 pI | 脂肪指数 Aliphatic index | 不稳定指数 Instability index | 亲疏水性 GRAVY |
---|---|---|---|---|---|---|
LbIRT2 | 215 | 22 607.41 | 8.81 | 97.26 | 40.50 | 0.340 |
LbFRO | 740 | 83 469.16 | 8.48 | 112.19 | 36.60 | 0.371 |
LbNRAMP3 | 511 | 56 347.21 | 5.83 | 119.10 | 37.17 | 0.556 |
Table 3 Physicochemical properties of functional genes responsible to iron and cadmium stress in L. barbarum
基因名称 Gene name | 氨基酸长度 Amino acid length/aa | 分子量 Molecular weight/kD | 等电点 pI | 脂肪指数 Aliphatic index | 不稳定指数 Instability index | 亲疏水性 GRAVY |
---|---|---|---|---|---|---|
LbIRT2 | 215 | 22 607.41 | 8.81 | 97.26 | 40.50 | 0.340 |
LbFRO | 740 | 83 469.16 | 8.48 | 112.19 | 36.60 | 0.371 |
LbNRAMP3 | 511 | 56 347.21 | 5.83 | 119.10 | 37.17 | 0.556 |
基因名称 Gene name | α-螺旋 α-helix/% | 延伸链 Extended strand/% | β-转角 β-turn/% | 无规则卷曲 Random coil/% |
---|---|---|---|---|
LbIRT2 | 55.35 | 12.09 | 8.37 | 24.19 |
LbFRO | 37.03 | 21.35 | 3.11 | 38.51 |
LbNRAMP3 | 55.58 | 12.33 | 3.91 | 28.18 |
Table 4 Prediction of secondary structure of functional genes in L. barbarum response to iron and cadmium stress
基因名称 Gene name | α-螺旋 α-helix/% | 延伸链 Extended strand/% | β-转角 β-turn/% | 无规则卷曲 Random coil/% |
---|---|---|---|---|
LbIRT2 | 55.35 | 12.09 | 8.37 | 24.19 |
LbFRO | 37.03 | 21.35 | 3.11 | 38.51 |
LbNRAMP3 | 55.58 | 12.33 | 3.91 | 28.18 |
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