Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (9): 236-245.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0022
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HAN Hao-zhang(), ZHANG Li-hua, LI Su-hua, ZHAO Rong, WANG Fang, WANG Xiao-li
Received:
2023-01-11
Online:
2023-09-26
Published:
2023-10-24
HAN Hao-zhang, ZHANG Li-hua, LI Su-hua, ZHAO Rong, WANG Fang, WANG Xiao-li. Construction of cDNA Library of Cinnamomun bodinieri Induced by Saline-alkali Stress and Screening of CbP5CS Upstream Regulators[J]. Biotechnology Bulletin, 2023, 39(9): 236-245.
引物名称Primer name | 序列Sequence(5'-3') | 应用Application |
---|---|---|
CDS III/3' PCR primer | AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG | 逆转录 |
SMART IV oligonucleotide | ATTCTAGAGGCCGAGGCGGCCGACATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN | 逆转录 |
P1-F | CATATGGCCATGGAGGCCAGTGAATTCAAGCAGTGGTATCAACGCAGAGTGG | ds cDNA扩增 |
P2-F | CATATGGCCATGGAGGCCAGTGAATTCAAAGCAGTGGTATCAACGCAGAGTGG | ds cDNA扩增 |
P3-F | CATATGGCCATGGAGGCCAGTGAATTCAAAAGCAGTGGTATCAACGCAGAGTGG | ds cDNA扩增 |
P4-R | CATCTGCAGCTCGAGCTCGATGGATCCCTAGAGGCCGAGGCGGCCGACATG | ds cDNA扩增 |
Prime M1 | AAGCAGTGGTATCAACGCAGAGT | 均一化 |
T7-F | taatacgactcactatagggcgag | 菌落PCR |
AD-R | GCACGATGCACAGTTGAAG | 菌落PCR |
Table 1 Oligonucleotide primers used in this study
引物名称Primer name | 序列Sequence(5'-3') | 应用Application |
---|---|---|
CDS III/3' PCR primer | AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG | 逆转录 |
SMART IV oligonucleotide | ATTCTAGAGGCCGAGGCGGCCGACATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN | 逆转录 |
P1-F | CATATGGCCATGGAGGCCAGTGAATTCAAGCAGTGGTATCAACGCAGAGTGG | ds cDNA扩增 |
P2-F | CATATGGCCATGGAGGCCAGTGAATTCAAAGCAGTGGTATCAACGCAGAGTGG | ds cDNA扩增 |
P3-F | CATATGGCCATGGAGGCCAGTGAATTCAAAAGCAGTGGTATCAACGCAGAGTGG | ds cDNA扩增 |
P4-R | CATCTGCAGCTCGAGCTCGATGGATCCCTAGAGGCCGAGGCGGCCGACATG | ds cDNA扩增 |
Prime M1 | AAGCAGTGGTATCAACGCAGAGT | 均一化 |
T7-F | taatacgactcactatagggcgag | 菌落PCR |
AD-R | GCACGATGCACAGTTGAAG | 菌落PCR |
Fig. 2 Agarose gel electrophoresis of ds cDNA M: Marker. 1: ds cDNA amplified with P1-F/P4-R. 2: ds cDNA amplified with P2-F/P4-R. 3: ds cDNA amplified with P3-F/P4-R
Fig. 3 Detection of normalization and removal of small fragments using agarose gel electrophoresis M: Marker. 1: Mixture of ds cDNA amplified with P1-F/P4-R, P2-F/P4-R, and P3-F/P4-R
位点 Site | 位置 Position | 序列 Sequence | 重复次数 Number of repetitions | 功能 Function |
---|---|---|---|---|
ABRE | -1 713 | ACGTG | 1 | 脱落酸响应元件 |
AE-box | +191 | AGAAACAA | 1 | 部分光响应模块 |
ARE | +680、-1 262、-693 | AAACCA | 3 | 厌氧诱导必需元件 |
AT~TATA-box | +551 | TATATA | 1 | 光响应元件 |
Box 4 | -1 846 | ATTAAT | 1 | 光响应中的保守DNA模块 |
CAAT-box | +58、+75、-99、+139、+184、+196、+414、+513、+704、-743、-753、+770、+817、-819、+869、-902、+914、+941、+942、+1 005、-1 008、+1 115、 -1 118、1 140、-1 168、+1 195、+1 198、-1 275、 -1 293、+1 348、-1 351、-1 394、-1 429、+1 479、 +1 480、-1 498、+1 523、+1 540、+1 541、+1 570、+1 591、+1 617、-1 636、-1 813 | CAAAT/CAAT/CCAAT/TGCCAAC | 44 | 启动子和增强子区域普通顺式作用元件 |
CAT-box | +1 976 | GCCACT | 1 | 分生组织表达响应元件 |
CGTCA-motif | -143、-732、+146 | CGTCA | 3 | 茉莉酸甲酯响应的元件 |
ERE | -1 743 | ATTTCATA | 1 | 乙烯响应元件 |
G-Box | +1 713 | CACGTT | 1 | 光响应元件 |
G-box | +80 | CACGAC | 1 | 光响应元件 |
GT1-motif | -610、+1 225、+665 | GGTTAA/GTGTGTGAA | 3 | 光响应元件 |
MYB | +2、-1 258、+1 201、-1 927、+1 001、-1 224 | CAACAG/CAACCA/TAACCA/ | 6 | MYB转录因子结合位点 |
MYB-like sequence | -1 224、-1 927 | TAACCA | 2 | MYB转录因子结合位点 |
MYC | -139、+817、-184 | CATTTG/CAATTG | 3 | bHLH转录因子结合位点 |
Myb | -1 154、+1 485 | TAACTG | 2 | MYB转录因子结合位点 |
Myb-binding site | +2、+1 201 | CAACAG | 2 | MYB转录因子结合位点 |
P-box | -158 | CCTTTTG | 1 | 赤霉素响应元件 |
SARE | -992 | TTCGACCATCTT | 1 | 水杨酸响应元件 |
STRE | -265、+1 080、+1 801、+572、+1 962、-1 402、 -1 834、-281、-712 | AGGGG | 9 | 压力响应元件 |
TATA-box | +202、-550、+267、+551、+552、+553、-1 161、 -1 162、+1 178、-1 179、-1 180、+1 471、-1 472、 -1 473、+1 554、-1 741、+1 850、-1 851、-1 917 | TATA/TATATAA/ccTATAAAaa/TATATA/ATATAA/ATATAT/ATTATA/TATAA/TACAAAA | 19 | -30位置附近核心启动子转录起始位点 |
TATC-box | -258 | TATCCCA | 1 | 赤霉素响应元件 |
TCA-element | -989、+1 408 | CCATCTTTTT | 2 | 水杨酸响应元件 |
TCCC-motif | +1 870 | TCTCCCT | 1 | 光响应元件 |
TGACG-motif | +143、+732、-146 | TGACG | 3 | 茉莉酸甲酯响应元件 |
Unnamed | -234、+1 640、+256、-1 712 | CGTGG | 4 | 未知 |
Unnamed | -628 | TCCACGTAGA | 1 | 未知 |
Unnamed | -9、-1 083、+643、+1 871、-568、1 377、-833、 -1 191、-536、+1 863、+710、-635、-921 | CTCC | 13 | 未知 |
WRE3 | -829、+1 865、-1 098 | CCACCT | 3 | 愈伤调节响应元件 |
WUN-motif | -559 | AAATTACT | 1 | 愈伤调节响应元件 |
Table 2 cis-acting regulatory elements of the CbP5CS promoter in C. bodinieri
位点 Site | 位置 Position | 序列 Sequence | 重复次数 Number of repetitions | 功能 Function |
---|---|---|---|---|
ABRE | -1 713 | ACGTG | 1 | 脱落酸响应元件 |
AE-box | +191 | AGAAACAA | 1 | 部分光响应模块 |
ARE | +680、-1 262、-693 | AAACCA | 3 | 厌氧诱导必需元件 |
AT~TATA-box | +551 | TATATA | 1 | 光响应元件 |
Box 4 | -1 846 | ATTAAT | 1 | 光响应中的保守DNA模块 |
CAAT-box | +58、+75、-99、+139、+184、+196、+414、+513、+704、-743、-753、+770、+817、-819、+869、-902、+914、+941、+942、+1 005、-1 008、+1 115、 -1 118、1 140、-1 168、+1 195、+1 198、-1 275、 -1 293、+1 348、-1 351、-1 394、-1 429、+1 479、 +1 480、-1 498、+1 523、+1 540、+1 541、+1 570、+1 591、+1 617、-1 636、-1 813 | CAAAT/CAAT/CCAAT/TGCCAAC | 44 | 启动子和增强子区域普通顺式作用元件 |
CAT-box | +1 976 | GCCACT | 1 | 分生组织表达响应元件 |
CGTCA-motif | -143、-732、+146 | CGTCA | 3 | 茉莉酸甲酯响应的元件 |
ERE | -1 743 | ATTTCATA | 1 | 乙烯响应元件 |
G-Box | +1 713 | CACGTT | 1 | 光响应元件 |
G-box | +80 | CACGAC | 1 | 光响应元件 |
GT1-motif | -610、+1 225、+665 | GGTTAA/GTGTGTGAA | 3 | 光响应元件 |
MYB | +2、-1 258、+1 201、-1 927、+1 001、-1 224 | CAACAG/CAACCA/TAACCA/ | 6 | MYB转录因子结合位点 |
MYB-like sequence | -1 224、-1 927 | TAACCA | 2 | MYB转录因子结合位点 |
MYC | -139、+817、-184 | CATTTG/CAATTG | 3 | bHLH转录因子结合位点 |
Myb | -1 154、+1 485 | TAACTG | 2 | MYB转录因子结合位点 |
Myb-binding site | +2、+1 201 | CAACAG | 2 | MYB转录因子结合位点 |
P-box | -158 | CCTTTTG | 1 | 赤霉素响应元件 |
SARE | -992 | TTCGACCATCTT | 1 | 水杨酸响应元件 |
STRE | -265、+1 080、+1 801、+572、+1 962、-1 402、 -1 834、-281、-712 | AGGGG | 9 | 压力响应元件 |
TATA-box | +202、-550、+267、+551、+552、+553、-1 161、 -1 162、+1 178、-1 179、-1 180、+1 471、-1 472、 -1 473、+1 554、-1 741、+1 850、-1 851、-1 917 | TATA/TATATAA/ccTATAAAaa/TATATA/ATATAA/ATATAT/ATTATA/TATAA/TACAAAA | 19 | -30位置附近核心启动子转录起始位点 |
TATC-box | -258 | TATCCCA | 1 | 赤霉素响应元件 |
TCA-element | -989、+1 408 | CCATCTTTTT | 2 | 水杨酸响应元件 |
TCCC-motif | +1 870 | TCTCCCT | 1 | 光响应元件 |
TGACG-motif | +143、+732、-146 | TGACG | 3 | 茉莉酸甲酯响应元件 |
Unnamed | -234、+1 640、+256、-1 712 | CGTGG | 4 | 未知 |
Unnamed | -628 | TCCACGTAGA | 1 | 未知 |
Unnamed | -9、-1 083、+643、+1 871、-568、1 377、-833、 -1 191、-536、+1 863、+710、-635、-921 | CTCC | 13 | 未知 |
WRE3 | -829、+1 865、-1 098 | CCACCT | 3 | 愈伤调节响应元件 |
WUN-motif | -559 | AAATTACT | 1 | 愈伤调节响应元件 |
基因ID Gene ID | 读数 Read count | 拟南芥同源序列 Arabidopsis thaliana homolog | E值E-value | 功能注释 Functional annotation |
---|---|---|---|---|
GW020491 | 1 | AT3G47120.1 | 8.00E-76 | 锌指 CCCH结构域蛋白25异构体 x1 |
GW028183 | 15 | AT4G14410.2 | 6.00E-51 | AtbHLH104 |
GW000650 | 3 | AT3G15420.1 | 1.00E-24 | 转录因子 TFIIIC |
GW009089 | 7 | AT4G00755.2 | 6.00E-59 | F-box 家族蛋白 |
GW009287 | 8 | AT1G64230.2 | 3.00E-83 | 泛素连接酶 |
GW012884 | 24 | AT5G15730.2 | 2.00E-137 | 蛋白激酶超家族 |
GW000705 | 24 | AT4G02340.1 | 2.00E-47 | α/β 水解酶超家族蛋白 |
GW003120 | 4 | AT5G04480.1 | 0 | 葡萄糖基转移酶超家族蛋白 |
GW004800 | 7 | AT5G63030.1 | 4.00E-42 | 硫氧还蛋白超家族蛋白 |
GW006791 | 16 | ATMG01360.1 | 0 | 细胞色素氧化酶 |
GW007525 | 13 | AT3G47550.6 | 6.00E-41 | RING/FYVE/PHD锌指超家族蛋白 |
GW008185 | 15 | AT2G20360.1 | 1.00E-161 | NAD(P)结合罗斯曼卷曲超家族蛋白 |
GW008711 | 19 | AT4G14305.1 | 8.00E-79 | 过氧化物酶体膜22 kD(Mpv17/PMP22)家族蛋白 |
GW010514 | 3 | AT2G27030.3 | 2.00E-82 | 钙调素 |
GW011043 | 24 | AT4G35450.1 | 3.00E-115 | 锚蛋白重复序列含有蛋白 |
GW011868 | 16 | AT4G26220.1 | 3.00E-80 | S-腺苷甲硫氨酸依赖的甲基转移酶超家族蛋白 |
GW011935 | 13 | AT1G55160.1 | 2.00E-35 | 功能未知的蛋白质 |
GW012148 | 1 | AT1G54290.1 | 3.00E-48 | 翻译起始因子eIF1 |
GW012186 | 19 | AT5G42850.2 | 4.00E-40 | 硫氧还蛋白超家族蛋白 |
GW014171 | 56 | AT2G44860.2 | 2.00E-62 | 核糖体蛋白质 L24e 家族蛋白 |
GW014289 | 34 | AT2G27020.1 | 1.00E-129 | 20s 蛋白酶体α亚基G1 |
GW014358 | 24 | AT1G68590.1 | 1.00E-43 | 核糖体蛋白质 PSRP-3/Ycf65 |
GW014549 | 10 | AT4G32140.1 | 4.00E-98 | 类EamA转运家族蛋白 |
GW015686 | 1 | AT1G10200.1 | 2.00E-80 | GATA型锌指转录因子家族蛋白 |
GW015796 | 64 | AT1G13950.1 | 4.00E-80 | 真核延伸因子5A-1 |
GW017908 | 15 | AT3G55440.1 | 4.00E-111 | 磷酸丙糖异构酶 |
GW020994 | 23 | AT5G53560.1 | 2.00E-48 | 细胞色素 B5亚型E |
GW022429 | 11 | ATMG01360.1 | 0 | 细胞色素氧化酶 |
GW024339 | 14 | AT4G19420.1 | 2.00E-153 | 果胶乙酰酯酶家族蛋白 |
GW025798 | 12 | AT1G53680.1 | 1.00E-69 | 谷胱甘肽S-转移酶TAU28 |
GW027120 | 11 | AT1G72210.1 | 4.00E-52 | AtbHLH96 |
Table 3 EST blast and annotation results
基因ID Gene ID | 读数 Read count | 拟南芥同源序列 Arabidopsis thaliana homolog | E值E-value | 功能注释 Functional annotation |
---|---|---|---|---|
GW020491 | 1 | AT3G47120.1 | 8.00E-76 | 锌指 CCCH结构域蛋白25异构体 x1 |
GW028183 | 15 | AT4G14410.2 | 6.00E-51 | AtbHLH104 |
GW000650 | 3 | AT3G15420.1 | 1.00E-24 | 转录因子 TFIIIC |
GW009089 | 7 | AT4G00755.2 | 6.00E-59 | F-box 家族蛋白 |
GW009287 | 8 | AT1G64230.2 | 3.00E-83 | 泛素连接酶 |
GW012884 | 24 | AT5G15730.2 | 2.00E-137 | 蛋白激酶超家族 |
GW000705 | 24 | AT4G02340.1 | 2.00E-47 | α/β 水解酶超家族蛋白 |
GW003120 | 4 | AT5G04480.1 | 0 | 葡萄糖基转移酶超家族蛋白 |
GW004800 | 7 | AT5G63030.1 | 4.00E-42 | 硫氧还蛋白超家族蛋白 |
GW006791 | 16 | ATMG01360.1 | 0 | 细胞色素氧化酶 |
GW007525 | 13 | AT3G47550.6 | 6.00E-41 | RING/FYVE/PHD锌指超家族蛋白 |
GW008185 | 15 | AT2G20360.1 | 1.00E-161 | NAD(P)结合罗斯曼卷曲超家族蛋白 |
GW008711 | 19 | AT4G14305.1 | 8.00E-79 | 过氧化物酶体膜22 kD(Mpv17/PMP22)家族蛋白 |
GW010514 | 3 | AT2G27030.3 | 2.00E-82 | 钙调素 |
GW011043 | 24 | AT4G35450.1 | 3.00E-115 | 锚蛋白重复序列含有蛋白 |
GW011868 | 16 | AT4G26220.1 | 3.00E-80 | S-腺苷甲硫氨酸依赖的甲基转移酶超家族蛋白 |
GW011935 | 13 | AT1G55160.1 | 2.00E-35 | 功能未知的蛋白质 |
GW012148 | 1 | AT1G54290.1 | 3.00E-48 | 翻译起始因子eIF1 |
GW012186 | 19 | AT5G42850.2 | 4.00E-40 | 硫氧还蛋白超家族蛋白 |
GW014171 | 56 | AT2G44860.2 | 2.00E-62 | 核糖体蛋白质 L24e 家族蛋白 |
GW014289 | 34 | AT2G27020.1 | 1.00E-129 | 20s 蛋白酶体α亚基G1 |
GW014358 | 24 | AT1G68590.1 | 1.00E-43 | 核糖体蛋白质 PSRP-3/Ycf65 |
GW014549 | 10 | AT4G32140.1 | 4.00E-98 | 类EamA转运家族蛋白 |
GW015686 | 1 | AT1G10200.1 | 2.00E-80 | GATA型锌指转录因子家族蛋白 |
GW015796 | 64 | AT1G13950.1 | 4.00E-80 | 真核延伸因子5A-1 |
GW017908 | 15 | AT3G55440.1 | 4.00E-111 | 磷酸丙糖异构酶 |
GW020994 | 23 | AT5G53560.1 | 2.00E-48 | 细胞色素 B5亚型E |
GW022429 | 11 | ATMG01360.1 | 0 | 细胞色素氧化酶 |
GW024339 | 14 | AT4G19420.1 | 2.00E-153 | 果胶乙酰酯酶家族蛋白 |
GW025798 | 12 | AT1G53680.1 | 1.00E-69 | 谷胱甘肽S-转移酶TAU28 |
GW027120 | 11 | AT1G72210.1 | 4.00E-52 | AtbHLH96 |
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