Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (4): 202-216.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0830
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HAO Qing-qing(), YAO Sheng, LIU Jia-he, CHEN Pei-zhen, ZHANG Meng-yang, JI Kong-shu()
Received:
2021-06-30
Online:
2022-04-26
Published:
2022-05-06
Contact:
JI Kong-shu
E-mail:hqq@njfu.edu.cn;ksji@njfu.edu.cn
HAO Qing-qing, YAO Sheng, LIU Jia-he, CHEN Pei-zhen, ZHANG Meng-yang, JI Kong-shu. Cloning and Expression Analysis of NAC Transcription Factor PmNAC8 in Pinus massoniana[J]. Biotechnology Bulletin, 2022, 38(4): 202-216.
引物名称 Primer name | 引物序列 Primer sequence (5'-3') | 用途 Purpose |
---|---|---|
PmNAC8-Mid F | GAAGGTATGTGAAAATGGGG | 中间片段扩增前引物 Forward primer of middle fragment amplification |
PmNAC8-Mid R | GGGAATCTCAGTGTTTTGAAG | 中间片段扩增后引物 Reverse primer of middle fragment amplification reverse primer |
3' RACE-PmNAC8 F | GGCAGGGGAATCTCAGTGTTTTGAAG | 3'-巢氏PCR前引物 Forward primer of 3'-nested PCR |
3' RACE-PmNAC8 R | TACCGTCGTTCCACTAGTGATTT | 3'-巢氏PCR后引物 Reverse primer of 3'-nested PCR |
5' RACE-PmNAC8 F | CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT | 5'-巢氏PCR前引物 Forward primer of 5'-nested PCR |
5' RACE-PmNAC8 R | CAATACCATCTTGGGACTGAGGACGA | 5'-巢氏PCR后引物 Reverse primer of 5'-nested PCR |
PmNAC8-F | CAAAGTGGAAACTCTGAGCAGAGC | 全长扩增前引物 Forward primer of total length of amplification |
PmNAC8-R | TTCCGAAGACGGCAAAAATAGTTG | 全长扩增后引物 Reverse primer of total length of amplification |
PmNAC8-ORF F | ATGGCGAGACCATGGATTACTG | 编码阅读框前引物 Forward primer of code reading frame forward primer |
PmNAC8-ORF R | TTGGCTGTTACCAAAGTTCTTTAACC | 编码阅读框后引物 Reverse primer of code reading frame forward primer |
Tubulin alpha F | CACAGAAAGCTGCTCATGGTAA | 内参基因前引物 Forward primer of internal genes |
Tubulin alpha R | CTGCGACTATGAGTGGAGTGA | 内参基因后引物 Reverse primer of internal genes |
Q-PmNAC8 F | TCTGATTGTGGTCGGATTGGAGG | 定量及半定量前引物 Forward primer of the quantitative and semi-quantitative |
Q-PmNAC8 R | AGGCTTTCTTGGGAACTGAACTG | 定量及半定量后引物 Reverse primer of the quantitative and semi-quantitative |
PmNAC8-GFP F | gagaacacgggggactctagaATGGCGAGACCATGGATTACTG | 重组表达载体前引物 Forward primer of recombinant expression vector |
PmNAC8-GFP R | gcccttgctcaccatggatccTTGGCTGTTACCAAAGTTCTTTAACC | 重组表达载体后引物 Reverse primer of recombinant expression vector |
GSP1 | TTGCGATTCTCCTACCATCAGTAA | 启动子扩增特异性引物1 The promoter amplification specificity primer 1 |
GSP2 | ACATCATTTCTCCCGTTCTCGTTT | 启动子扩增特异性引物2 The promoter amplification specificity primer 2 |
PmNAC8-Pro F | TTTGGTTAGCGTAGAGGGTTGTCA | 启动子全长克隆前引物 Forward primer of total length of the promoter cloning |
PmNAC8-Pro R | CCGTGTCGCCTTACATCATTTCTC | 启动子全长克隆后引物 Reverse primer of total length of the promoter cloning |
Table 1 Sequences of primers used in this study
引物名称 Primer name | 引物序列 Primer sequence (5'-3') | 用途 Purpose |
---|---|---|
PmNAC8-Mid F | GAAGGTATGTGAAAATGGGG | 中间片段扩增前引物 Forward primer of middle fragment amplification |
PmNAC8-Mid R | GGGAATCTCAGTGTTTTGAAG | 中间片段扩增后引物 Reverse primer of middle fragment amplification reverse primer |
3' RACE-PmNAC8 F | GGCAGGGGAATCTCAGTGTTTTGAAG | 3'-巢氏PCR前引物 Forward primer of 3'-nested PCR |
3' RACE-PmNAC8 R | TACCGTCGTTCCACTAGTGATTT | 3'-巢氏PCR后引物 Reverse primer of 3'-nested PCR |
5' RACE-PmNAC8 F | CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT | 5'-巢氏PCR前引物 Forward primer of 5'-nested PCR |
5' RACE-PmNAC8 R | CAATACCATCTTGGGACTGAGGACGA | 5'-巢氏PCR后引物 Reverse primer of 5'-nested PCR |
PmNAC8-F | CAAAGTGGAAACTCTGAGCAGAGC | 全长扩增前引物 Forward primer of total length of amplification |
PmNAC8-R | TTCCGAAGACGGCAAAAATAGTTG | 全长扩增后引物 Reverse primer of total length of amplification |
PmNAC8-ORF F | ATGGCGAGACCATGGATTACTG | 编码阅读框前引物 Forward primer of code reading frame forward primer |
PmNAC8-ORF R | TTGGCTGTTACCAAAGTTCTTTAACC | 编码阅读框后引物 Reverse primer of code reading frame forward primer |
Tubulin alpha F | CACAGAAAGCTGCTCATGGTAA | 内参基因前引物 Forward primer of internal genes |
Tubulin alpha R | CTGCGACTATGAGTGGAGTGA | 内参基因后引物 Reverse primer of internal genes |
Q-PmNAC8 F | TCTGATTGTGGTCGGATTGGAGG | 定量及半定量前引物 Forward primer of the quantitative and semi-quantitative |
Q-PmNAC8 R | AGGCTTTCTTGGGAACTGAACTG | 定量及半定量后引物 Reverse primer of the quantitative and semi-quantitative |
PmNAC8-GFP F | gagaacacgggggactctagaATGGCGAGACCATGGATTACTG | 重组表达载体前引物 Forward primer of recombinant expression vector |
PmNAC8-GFP R | gcccttgctcaccatggatccTTGGCTGTTACCAAAGTTCTTTAACC | 重组表达载体后引物 Reverse primer of recombinant expression vector |
GSP1 | TTGCGATTCTCCTACCATCAGTAA | 启动子扩增特异性引物1 The promoter amplification specificity primer 1 |
GSP2 | ACATCATTTCTCCCGTTCTCGTTT | 启动子扩增特异性引物2 The promoter amplification specificity primer 2 |
PmNAC8-Pro F | TTTGGTTAGCGTAGAGGGTTGTCA | 启动子全长克隆前引物 Forward primer of total length of the promoter cloning |
PmNAC8-Pro R | CCGTGTCGCCTTACATCATTTCTC | 启动子全长克隆后引物 Reverse primer of total length of the promoter cloning |
Fig. 1 Full-length and ORF amplification of PmNAC8 gene in P. massoniana A:Electropherogram of full-length PmNAC8 cDNA of P. massoniana. B:Electropherogram of ORF of PmNAC8 of P. massoniana. M:DL2502 DNA marker
Fig.2 Bioinformatics analysis of PmNAC8 protein A:Conserved domain prediction of PmNAC8 protein. B:Hydrophobicity prediction of PmNAC8 protein. C:Signal peptide prediction of PmNAC8 protein
物种 Species | 登录号 GenBank accession number | A3s | C3s | G3s | T3s | ENc | CAI | GC3s/% | GC/% |
---|---|---|---|---|---|---|---|---|---|
马尾松 P. massoniana | MZ291447 | 0.443 | 0.182 | 0.241 | 0.451 | 50.19 | 0.187 | 31.10 | 42.50 |
Table 2 Codon selection preference parameters of PmNAC8 gene
物种 Species | 登录号 GenBank accession number | A3s | C3s | G3s | T3s | ENc | CAI | GC3s/% | GC/% |
---|---|---|---|---|---|---|---|---|---|
马尾松 P. massoniana | MZ291447 | 0.443 | 0.182 | 0.241 | 0.451 | 50.19 | 0.187 | 31.10 | 42.50 |
氨基酸 Amino acid | 密码子 Codon | 相对密码子 使用度RSCU | 个数 Number | 频率 Frequency | 比例 Fraction | 氨基酸 Amino acid | 密码子 Codon | 相对密码子 使用度RSCU | 个数 Number | 频率 Frequency | 比例 Fraction | |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Phe | UUU | 1.54 | 10 | 24.814 | 0.769 | Tyr | UAU | 1.71 | 6 | 14.888 | 0.857 | |
UUC | 0.46 | 3 | 7.444 | 0.231 | UAC | 0.29 | 1 | 2.481 | 0.143 | |||
Leu | UUA | 1.50 | 6 | 14.888 | 0.250 | TER | UAA | 0.00 | 0 | 0.000 | 0.000 | |
UUG | 1.25 | 5 | 12.407 | 0.208 | UAG | 0.00 | 0 | 0.000 | 0.000 | |||
CUU | 1.75 | 7 | 17.370 | 0.92 | His | CAU | 1.47 | 11 | 27.295 | 0.733 | ||
CUC | 1.00 | 4 | 9.926 | 0.167 | CAC | 0.53 | 4 | 9.926 | 0.267 | |||
CUA | 0.50 | 2 | 4.963 | 0.083 | Gln | CAA | 1.10 | 11 | 27.295 | 0.550 | ||
CUG | 0.00 | 0 | 0.000 | 0.000 | CAG | 0.90 | 9 | 22.333 | 0.450 | |||
Ile | AUU | 1.43 | 10 | 24.814 | 0.476 | Asn | AAU | 1.09 | 12 | 29.777 | 0.545 | |
AUC | 0.57 | 4 | 9.926 | 0.191 | AAC | 0.91 | 10 | 24.814 | 0.455 | |||
AUA | 1.00 | 7 | 17.370 | 0.333 | Lys | AAA | 1.04 | 11 | 27.295 | 0.478 | ||
Met | AUG | 1.00 | 7 | 17.370 | 1.000 | AAG | 0.96 | 12 | 29.777 | 0.522 | ||
Val | GUU | 1.25 | 5 | 12.407 | 0.313 | Asp | GAU | 1.57 | 22 | 54.591 | 0.786 | |
GUC | 0.75 | 3 | 7.444 | 0.187 | GAC | 0.43 | 6 | 14.888 | 0.214 | |||
GUA | 0.75 | 3 | 7.444 | 0.187 | Glu | GAA | 1.22 | 22 | 54.591 | 0.611 | ||
GUG | 1.25 | 5 | 12.407 | 0.313 | GAG | 0.78 | 14 | 34.739 | 0.389 | |||
Ser | UCU | 1.31 | 7 | 17.370 | 0.318 | Cys | UGU | 1.54 | 10 | 24.814 | 0.769 | |
UCC | 0.38 | 2 | 4.963 | 0.091 | UGC | 0.46 | 3 | 7.444 | 0.231 | |||
UCA | 2.44 | 13 | 32.258 | 0.591 | TER | UGA | 3.00 | 1 | 2.481 | 1.000 | ||
UCG | 0.00 | 0 | 0.000 | 0.000 | Trp | UGG | 1.00 | 6 | 14.888 | 1.000 | ||
Pro | CCU | 1.57 | 11 | 27.295 | 0.393 | Arg | CGU | 1.67 | 5 | 12.407 | 0.625 | |
CCC | 0.57 | 4 | 9.926 | 0.143 | CGC | 0.00 | 0 | 0.000 | 0.000 | |||
CCA | 1.86 | 13 | 32.258 | 0.464 | CGA | 0.67 | 2 | 4.963 | 0.250 | |||
CCG | 0.00 | 0 | 0.000 | 0.00 | CGG | 0.33 | 1 | 2.481 | 0.125 | |||
Thr | ACU | 1.60 | 10 | 24.814 | 0.400 | Ser | AGU | 0.56 | 3 | 7.444 | 0.300 | |
ACC | 0.16 | 1 | 2.481 | 0.040 | AGC | 1.31 | 7 | 13.370 | 0.700 | |||
ACA | 1.60 | 10 | 24.814 | 0.400 | Arg | AGA | 1.67 | 5 | 12.407 | 0.500 | ||
ACG | 0.64 | 4 | 9.926 | 0.160 | AGG | 1.67 | 5 | 12.407 | 0.500 | |||
Ala | GCU | 0.86 | 3 | 7.444 | 0.214 | Gly | GGU | 0.88 | 7 | 13.370 | 0.700 | |
GCC | 0.29 | 1 | 2.481 | 0.071 | GGC | 0.38 | 3 | 7.444 | 0.300 | |||
GCA | 2.57 | 9 | 22.333 | 0.643 | GGA | 1.63 | 13 | 32.258 | 0.591 | |||
GCG | 0.29 | 1 | 2.481 | 0.072 | GGG | 1.13 | 9 | 22.333 | 0.409 |
Table 3 RSCU of PmNAC8 gene
氨基酸 Amino acid | 密码子 Codon | 相对密码子 使用度RSCU | 个数 Number | 频率 Frequency | 比例 Fraction | 氨基酸 Amino acid | 密码子 Codon | 相对密码子 使用度RSCU | 个数 Number | 频率 Frequency | 比例 Fraction | |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Phe | UUU | 1.54 | 10 | 24.814 | 0.769 | Tyr | UAU | 1.71 | 6 | 14.888 | 0.857 | |
UUC | 0.46 | 3 | 7.444 | 0.231 | UAC | 0.29 | 1 | 2.481 | 0.143 | |||
Leu | UUA | 1.50 | 6 | 14.888 | 0.250 | TER | UAA | 0.00 | 0 | 0.000 | 0.000 | |
UUG | 1.25 | 5 | 12.407 | 0.208 | UAG | 0.00 | 0 | 0.000 | 0.000 | |||
CUU | 1.75 | 7 | 17.370 | 0.92 | His | CAU | 1.47 | 11 | 27.295 | 0.733 | ||
CUC | 1.00 | 4 | 9.926 | 0.167 | CAC | 0.53 | 4 | 9.926 | 0.267 | |||
CUA | 0.50 | 2 | 4.963 | 0.083 | Gln | CAA | 1.10 | 11 | 27.295 | 0.550 | ||
CUG | 0.00 | 0 | 0.000 | 0.000 | CAG | 0.90 | 9 | 22.333 | 0.450 | |||
Ile | AUU | 1.43 | 10 | 24.814 | 0.476 | Asn | AAU | 1.09 | 12 | 29.777 | 0.545 | |
AUC | 0.57 | 4 | 9.926 | 0.191 | AAC | 0.91 | 10 | 24.814 | 0.455 | |||
AUA | 1.00 | 7 | 17.370 | 0.333 | Lys | AAA | 1.04 | 11 | 27.295 | 0.478 | ||
Met | AUG | 1.00 | 7 | 17.370 | 1.000 | AAG | 0.96 | 12 | 29.777 | 0.522 | ||
Val | GUU | 1.25 | 5 | 12.407 | 0.313 | Asp | GAU | 1.57 | 22 | 54.591 | 0.786 | |
GUC | 0.75 | 3 | 7.444 | 0.187 | GAC | 0.43 | 6 | 14.888 | 0.214 | |||
GUA | 0.75 | 3 | 7.444 | 0.187 | Glu | GAA | 1.22 | 22 | 54.591 | 0.611 | ||
GUG | 1.25 | 5 | 12.407 | 0.313 | GAG | 0.78 | 14 | 34.739 | 0.389 | |||
Ser | UCU | 1.31 | 7 | 17.370 | 0.318 | Cys | UGU | 1.54 | 10 | 24.814 | 0.769 | |
UCC | 0.38 | 2 | 4.963 | 0.091 | UGC | 0.46 | 3 | 7.444 | 0.231 | |||
UCA | 2.44 | 13 | 32.258 | 0.591 | TER | UGA | 3.00 | 1 | 2.481 | 1.000 | ||
UCG | 0.00 | 0 | 0.000 | 0.000 | Trp | UGG | 1.00 | 6 | 14.888 | 1.000 | ||
Pro | CCU | 1.57 | 11 | 27.295 | 0.393 | Arg | CGU | 1.67 | 5 | 12.407 | 0.625 | |
CCC | 0.57 | 4 | 9.926 | 0.143 | CGC | 0.00 | 0 | 0.000 | 0.000 | |||
CCA | 1.86 | 13 | 32.258 | 0.464 | CGA | 0.67 | 2 | 4.963 | 0.250 | |||
CCG | 0.00 | 0 | 0.000 | 0.00 | CGG | 0.33 | 1 | 2.481 | 0.125 | |||
Thr | ACU | 1.60 | 10 | 24.814 | 0.400 | Ser | AGU | 0.56 | 3 | 7.444 | 0.300 | |
ACC | 0.16 | 1 | 2.481 | 0.040 | AGC | 1.31 | 7 | 13.370 | 0.700 | |||
ACA | 1.60 | 10 | 24.814 | 0.400 | Arg | AGA | 1.67 | 5 | 12.407 | 0.500 | ||
ACG | 0.64 | 4 | 9.926 | 0.160 | AGG | 1.67 | 5 | 12.407 | 0.500 | |||
Ala | GCU | 0.86 | 3 | 7.444 | 0.214 | Gly | GGU | 0.88 | 7 | 13.370 | 0.700 | |
GCC | 0.29 | 1 | 2.481 | 0.071 | GGC | 0.38 | 3 | 7.444 | 0.300 | |||
GCA | 2.57 | 9 | 22.333 | 0.643 | GGA | 1.63 | 13 | 32.258 | 0.591 | |||
GCG | 0.29 | 1 | 2.481 | 0.072 | GGG | 1.13 | 9 | 22.333 | 0.409 |
氨基酸 Amino acid | 密码子 Codon | 马尾松PmNAC8 | 拟南芥基因组At | 烟草基因组Nt | 酵母基因组Sc | 大肠杆菌基因组Ec | 欧洲山杨基因组Pt | PmNAC8/At | PmNAC8/Nt | PmNAC8/Sc | PmNAC8/Ec | PmNAC8/Pt |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Phe | UUU | 24.814 | 21.8 | 25.1 | 26.1 | 22.2 | 28.2 | 1.14 | 0.99 | 0.95 | 1.12 | 0.10 |
UUC | 7.444 | 20.7 | 18.0 | 18.4 | 15.9 | 17.3 | 0.36 | 0.36 | 0.40 | 0.47 | 0.43 | |
Leu | UUA | 14.888 | 12.7 | 13.4 | 26.2 | 13.8 | 14.0 | 1.17 | 1.11 | 0.57 | 1.08 | 1.06 |
UUG | 12.407 | 20.9 | 22.3 | 27.2 | 13.0 | 26.3 | 0.59 | 0.56 | 0.55 | 0.95 | 0.47 | |
CUU | 17.370 | 24.1 | 24.0 | 12.3 | 11.4 | 22.7 | 0.72 | 0.72 | 1.41 | 1.52 | 0.77 | |
CUC | 9.926 | 16.1 | 12.3 | 5.4 | 10.5 | 15.6 | 0.62 | 0.81 | 1.84 | 0.95 | 0.64 | |
CUA | 4.963 | 9.9 | 9.4 | 13.4 | 3.9 | 7.6 | 0.50 | 0.53 | 0.37 | 1.27 | 0.65 | |
CUG | 0.000 | 9.8 | 10.2 | 10.5 | 51.1 | 8.5 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |
Ile | AUU | 24.814 | 21.5 | 27.8 | 30.1 | 29.7 | 28.1 | 1.15 | 0.89 | 0.82 | 0.84 | 0.88 |
AUC | 9.926 | 18.5 | 13.9 | 17.2 | 23.9 | 14.5 | 0.54 | 0.71 | 0.58 | 0.42 | 0.68 | |
AUA | 17.370 | 12.6 | 14.0 | 17.8 | 5.5 | 15.0 | 1.38 | 1.24 | 0.98 | 3.16 | 1.16 | |
Met | AUG | 17.370 | 24.5 | 25.0 | 20.9 | 27.2 | 23.6 | 0.71 | 0.69 | 0.83 | 0.64 | 0.74 |
Val | GUU | 12.407 | 27.2 | 26.8 | 22.1 | 18.1 | 39.1 | 0.46 | 0.46 | 0.56 | 0.69 | 0.32 |
GUC | 7.444 | 12.8 | 11.1 | 11.8 | 14.8 | 10.8 | 0.58 | 0.67 | 0.63 | 0.50 | 0.69 | |
GUA | 7.444 | 9.9 | 11.4 | 11.8 | 10.9 | 9.4 | 0.75 | 0.65 | 0.63 | 0.68 | 0.79 | |
GUG | 12.407 | 17.4 | 16.7 | 10.8 | 26.2 | 19.8 | 0.72 | 0.74 | 1.15 | 0.47 | 0.63 | |
Ser | UCU | 17.370 | 25.2 | 20.0 | 23.5 | 8.7 | 18.3 | 0.69 | 0.87 | 0.74 | 2.00 | 0.95 |
UCC | 4.963 | 11.2 | 10.2 | 14.2 | 8.9 | 11.8 | 0.44 | 0.49 | 0.35 | 0.56 | 0.42 | |
UCA | 32.258 | 18.3 | 17.6 | 18.7 | 8.1 | 21.2 | 1.76 | 1.83 | 1.73 | 3.98 | 1.52 | |
UCG | 0.000 | 9.3 | 5.3 | 8.6 | 8.8 | 8.3 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |
Pro | CCU | 27.295 | 18.7 | 18.7 | 13.5 | 7.2 | 15.8 | 1.46 | 1.46 | 2.02 | 3.80 | 1.73 |
CCC | 9.926 | 5.3 | 6.6 | 6.8 | 5.6 | 8.0 | 1.87 | 1.50 | 1.46 | 1.77 | 1.24 | |
CCA | 32.258 | 16.1 | 19.8 | 18.3 | 8.4 | 16.1 | 2.00 | 1.63 | 1.76 | 3.84 | 2.00 | |
CCG | 0.000 | 8.6 | 5.0 | 5.3 | 22.4 | 7.4 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |
Thr | ACU | 24.814 | 17.5 | 20.3 | 20.3 | 9.1 | 19.7 | 1.42 | 1.22 | 1.22 | 2.73 | 1.26 |
ACC | 2.481 | 10.3 | 9.7 | 12.7 | 22.8 | 11.7 | 0.24 | 0.26 | 0.20 | 0.27 | 0.21 | |
ACA | 24.814 | 15.7 | 17.4 | 17.8 | 8.1 | 20.6 | 1.58 | 1.43 | 1.39 | 3.06 | 1.20 | |
ACG | 9.926 | 7.7 | 4.5 | 8.0 | 15.0 | 3.4 | 1.29 | 2.21 | 1.24 | 0.66 | 2.92 | |
Ala | GCU | 7.444 | 28.3 | 31.2 | 21.2 | 15.4 | 26.8 | 0.26 | 0.24 | 0.35 | 0.48 | 0.28 |
GCC | 2.481 | 10.3 | 12.5 | 12.6 | 25.2 | 12.0 | 0.24 | 0.20 | 0.20 | 0.10 | 0.21 | |
GCA | 22.333 | 17.5 | 23.1 | 16.2 | 20.7 | 23.5 | 1.28 | 0.97 | 1.38 | 1.08 | 0.95 | |
GCG | 2.481 | 9.0 | 5.8 | 6.2 | 32.3 | 6.9 | 0.28 | 0.43 | 0.40 | 0.08 | 0.36 | |
Tyr | UAU | 14.888 | 14.6 | 17.8 | 18.8 | 16.5 | 16.5 | 1.02 | 0.84 | 0.79 | 0.90 | 0.90 |
UAC | 2.481 | 13.7 | 13.5 | 14.8 | 12.3 | 9.7 | 0.18 | 0.18 | 0.17 | 0.20 | 0.26 | |
His | CAU | 27.295 | 13.8 | 13.4 | 13.6 | 12.8 | 13.5 | 1.98 | 2.04 | 2.01 | 2.13 | 1.51 |
CAC | 9.926 | 8.7 | 8.7 | 7.8 | 9.4 | 8.3 | 1.14 | 1.14 | 1.27 | 1.06 | 1.20 | |
Gln | CAA | 27.295 | 19.4 | 20.7 | 27.3 | 14.7 | 18.1 | 1.41 | 1.32 | 1.00 | 1.86 | 1.16 |
CAG | 22.333 | 15.2 | 15.0 | 12.1 | 29.4 | 16.7 | 1.47 | 1.49 | 1.85 | 0.76 | 1.34 | |
Asn | AAU | 29.777 | 22.3 | 28.0 | 35.7 | 19.2 | 23.9 | 1.34 | 1.06 | 0.83 | 1.55 | 1.25 |
AAC | 24.814 | 20.9 | 17.9 | 24.8 | 21.7 | 15.4 | 1.19 | 1.39 | 1.00 | 1.14 | 1.61 | |
Lys | AAA | 27.295 | 30.8 | 32.6 | 41.9 | 34.0 | 26.4 | 0.89 | 0.84 | 0.65 | 0.80 | 1.03 |
AAG | 29.777 | 32.7 | 33.5 | 30.8 | 11.0 | 29.5 | 0.91 | 0.89 | 0.97 | 2.71 | 1.01 | |
Asp | GAU | 54.591 | 36.6 | 36.9 | 37.6 | 32.8 | 42.1 | 1.49 | 1.52 | 1.45 | 1.66 | 1.30 |
GAC | 14.888 | 17.2 | 16.9 | 20.2 | 19.2 | 14.5 | 0.87 | 0.88 | 0.74 | 0.78 | 1.07 | |
Glu | GAA | 54.591 | 34.3 | 36.0 | 45.6 | 39.3 | 35.1 | 1.59 | 1.52 | 1.20 | 1.39 | 1.56 |
GAG | 34.739 | 32.2 | 29.4 | 19.2 | 18.7 | 27.4 | 1.08 | 1.18 | 1.81 | 1.86 | 1.27 | |
Cys | UGU | 24.814 | 10.5 | 9.8 | 8.1 | 5.2 | 13.4 | 2.36 | 2.53 | 3.06 | 4.77 | 1.85 |
UGC | 7.444 | 7.2 | 7.2 | 4.8 | 6.4 | 9.4 | 1.03 | 1.03 | 1.55 | 1.16 | 0.79 | |
Trp | UGG | 14.888 | 12.5 | 12.2 | 10.4 | 15.3 | 11.5 | 1.19 | 1.22 | 1.43 | 0.97 | 1.29 |
Arg | CGU | 12.407 | 9.0 | 7.5 | 6.4 | 20.2 | 5.7 | 1.38 | 1.65 | 1.94 | 0.61 | 2.18 |
CGC | 0.000 | 3.8 | 3.9 | 2.6 | 20.8 | 4.0 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |
CGA | 4.963 | 6.3 | 5.3 | 3.0 | 3.8 | 4.6 | 0.79 | 0.94 | 1.65 | 1.31 | 1.08 | |
CGG | 2.481 | 4.9 | 3.7 | 1.7 | 6.2 | 1.8 | 0.51 | 0.67 | 1.46 | 0.40 | 1.38 | |
Ser | AGU | 7.444 | 14.0 | 13.3 | 14.2 | 9.4 | 14.5 | 0.53 | 0.56 | 0.52 | 0.79 | 0.51 |
AGC | 13.370 | 11.3 | 10.0 | 9.8 | 16.0 | 10.6 | 1.18 | 1.34 | 1.36 | 0.84 | 1.26 | |
Arg | AGA | 12.407 | 19.0 | 16.0 | 21.3 | 2.9 | 10.9 | 0.65 | 0.78 | 0.58 | 4.28 | 1.14 |
AGG | 12.407 | 11.0 | 12.2 | 9.2 | 1.8 | 15.0 | 1.13 | 1.02 | 1.35 | 6.89 | 0.83 | |
Gly | GGU | 13.370 | 22.2 | 22.3 | 23.9 | 24.2 | 22.9 | 0.06 | 0.60 | 0.56 | 0.55 | 0.58 |
GGC | 7.444 | 9.2 | 11.2 | 9.8 | 28.1 | 11.6 | 0.81 | 0.66 | 0.76 | 0.26 | 0.64 | |
GGA | 32.258 | 24.2 | 23.2 | 10.9 | 8.9 | 17.1 | 1.33 | 1.39 | 2.96 | 3.62 | 1.89 | |
GGG | 22.333 | 10.2 | 10.5 | 6.0 | 11.8 | 14.9 | 2.17 | 2.13 | 3.72 | 1.89 | 1.50 | |
TER | *UAA | 0.000 | 0.9 | 1.1 | 1.1 | 2.0 | 1.1 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
*UAG | 0.000 | 0.5 | 0.5 | 0.5 | 0.3 | 0.0 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |
*UGA | 2.481 | 1.2 | 1.0 | 0.7 | 1.1 | 1.6 | 2.07 | 2.48 | 3.54 | 2.26 | 1.55 |
Table 4 Comparison of codon usage preference between PmNAC8 and other model organisms
氨基酸 Amino acid | 密码子 Codon | 马尾松PmNAC8 | 拟南芥基因组At | 烟草基因组Nt | 酵母基因组Sc | 大肠杆菌基因组Ec | 欧洲山杨基因组Pt | PmNAC8/At | PmNAC8/Nt | PmNAC8/Sc | PmNAC8/Ec | PmNAC8/Pt |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Phe | UUU | 24.814 | 21.8 | 25.1 | 26.1 | 22.2 | 28.2 | 1.14 | 0.99 | 0.95 | 1.12 | 0.10 |
UUC | 7.444 | 20.7 | 18.0 | 18.4 | 15.9 | 17.3 | 0.36 | 0.36 | 0.40 | 0.47 | 0.43 | |
Leu | UUA | 14.888 | 12.7 | 13.4 | 26.2 | 13.8 | 14.0 | 1.17 | 1.11 | 0.57 | 1.08 | 1.06 |
UUG | 12.407 | 20.9 | 22.3 | 27.2 | 13.0 | 26.3 | 0.59 | 0.56 | 0.55 | 0.95 | 0.47 | |
CUU | 17.370 | 24.1 | 24.0 | 12.3 | 11.4 | 22.7 | 0.72 | 0.72 | 1.41 | 1.52 | 0.77 | |
CUC | 9.926 | 16.1 | 12.3 | 5.4 | 10.5 | 15.6 | 0.62 | 0.81 | 1.84 | 0.95 | 0.64 | |
CUA | 4.963 | 9.9 | 9.4 | 13.4 | 3.9 | 7.6 | 0.50 | 0.53 | 0.37 | 1.27 | 0.65 | |
CUG | 0.000 | 9.8 | 10.2 | 10.5 | 51.1 | 8.5 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |
Ile | AUU | 24.814 | 21.5 | 27.8 | 30.1 | 29.7 | 28.1 | 1.15 | 0.89 | 0.82 | 0.84 | 0.88 |
AUC | 9.926 | 18.5 | 13.9 | 17.2 | 23.9 | 14.5 | 0.54 | 0.71 | 0.58 | 0.42 | 0.68 | |
AUA | 17.370 | 12.6 | 14.0 | 17.8 | 5.5 | 15.0 | 1.38 | 1.24 | 0.98 | 3.16 | 1.16 | |
Met | AUG | 17.370 | 24.5 | 25.0 | 20.9 | 27.2 | 23.6 | 0.71 | 0.69 | 0.83 | 0.64 | 0.74 |
Val | GUU | 12.407 | 27.2 | 26.8 | 22.1 | 18.1 | 39.1 | 0.46 | 0.46 | 0.56 | 0.69 | 0.32 |
GUC | 7.444 | 12.8 | 11.1 | 11.8 | 14.8 | 10.8 | 0.58 | 0.67 | 0.63 | 0.50 | 0.69 | |
GUA | 7.444 | 9.9 | 11.4 | 11.8 | 10.9 | 9.4 | 0.75 | 0.65 | 0.63 | 0.68 | 0.79 | |
GUG | 12.407 | 17.4 | 16.7 | 10.8 | 26.2 | 19.8 | 0.72 | 0.74 | 1.15 | 0.47 | 0.63 | |
Ser | UCU | 17.370 | 25.2 | 20.0 | 23.5 | 8.7 | 18.3 | 0.69 | 0.87 | 0.74 | 2.00 | 0.95 |
UCC | 4.963 | 11.2 | 10.2 | 14.2 | 8.9 | 11.8 | 0.44 | 0.49 | 0.35 | 0.56 | 0.42 | |
UCA | 32.258 | 18.3 | 17.6 | 18.7 | 8.1 | 21.2 | 1.76 | 1.83 | 1.73 | 3.98 | 1.52 | |
UCG | 0.000 | 9.3 | 5.3 | 8.6 | 8.8 | 8.3 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |
Pro | CCU | 27.295 | 18.7 | 18.7 | 13.5 | 7.2 | 15.8 | 1.46 | 1.46 | 2.02 | 3.80 | 1.73 |
CCC | 9.926 | 5.3 | 6.6 | 6.8 | 5.6 | 8.0 | 1.87 | 1.50 | 1.46 | 1.77 | 1.24 | |
CCA | 32.258 | 16.1 | 19.8 | 18.3 | 8.4 | 16.1 | 2.00 | 1.63 | 1.76 | 3.84 | 2.00 | |
CCG | 0.000 | 8.6 | 5.0 | 5.3 | 22.4 | 7.4 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |
Thr | ACU | 24.814 | 17.5 | 20.3 | 20.3 | 9.1 | 19.7 | 1.42 | 1.22 | 1.22 | 2.73 | 1.26 |
ACC | 2.481 | 10.3 | 9.7 | 12.7 | 22.8 | 11.7 | 0.24 | 0.26 | 0.20 | 0.27 | 0.21 | |
ACA | 24.814 | 15.7 | 17.4 | 17.8 | 8.1 | 20.6 | 1.58 | 1.43 | 1.39 | 3.06 | 1.20 | |
ACG | 9.926 | 7.7 | 4.5 | 8.0 | 15.0 | 3.4 | 1.29 | 2.21 | 1.24 | 0.66 | 2.92 | |
Ala | GCU | 7.444 | 28.3 | 31.2 | 21.2 | 15.4 | 26.8 | 0.26 | 0.24 | 0.35 | 0.48 | 0.28 |
GCC | 2.481 | 10.3 | 12.5 | 12.6 | 25.2 | 12.0 | 0.24 | 0.20 | 0.20 | 0.10 | 0.21 | |
GCA | 22.333 | 17.5 | 23.1 | 16.2 | 20.7 | 23.5 | 1.28 | 0.97 | 1.38 | 1.08 | 0.95 | |
GCG | 2.481 | 9.0 | 5.8 | 6.2 | 32.3 | 6.9 | 0.28 | 0.43 | 0.40 | 0.08 | 0.36 | |
Tyr | UAU | 14.888 | 14.6 | 17.8 | 18.8 | 16.5 | 16.5 | 1.02 | 0.84 | 0.79 | 0.90 | 0.90 |
UAC | 2.481 | 13.7 | 13.5 | 14.8 | 12.3 | 9.7 | 0.18 | 0.18 | 0.17 | 0.20 | 0.26 | |
His | CAU | 27.295 | 13.8 | 13.4 | 13.6 | 12.8 | 13.5 | 1.98 | 2.04 | 2.01 | 2.13 | 1.51 |
CAC | 9.926 | 8.7 | 8.7 | 7.8 | 9.4 | 8.3 | 1.14 | 1.14 | 1.27 | 1.06 | 1.20 | |
Gln | CAA | 27.295 | 19.4 | 20.7 | 27.3 | 14.7 | 18.1 | 1.41 | 1.32 | 1.00 | 1.86 | 1.16 |
CAG | 22.333 | 15.2 | 15.0 | 12.1 | 29.4 | 16.7 | 1.47 | 1.49 | 1.85 | 0.76 | 1.34 | |
Asn | AAU | 29.777 | 22.3 | 28.0 | 35.7 | 19.2 | 23.9 | 1.34 | 1.06 | 0.83 | 1.55 | 1.25 |
AAC | 24.814 | 20.9 | 17.9 | 24.8 | 21.7 | 15.4 | 1.19 | 1.39 | 1.00 | 1.14 | 1.61 | |
Lys | AAA | 27.295 | 30.8 | 32.6 | 41.9 | 34.0 | 26.4 | 0.89 | 0.84 | 0.65 | 0.80 | 1.03 |
AAG | 29.777 | 32.7 | 33.5 | 30.8 | 11.0 | 29.5 | 0.91 | 0.89 | 0.97 | 2.71 | 1.01 | |
Asp | GAU | 54.591 | 36.6 | 36.9 | 37.6 | 32.8 | 42.1 | 1.49 | 1.52 | 1.45 | 1.66 | 1.30 |
GAC | 14.888 | 17.2 | 16.9 | 20.2 | 19.2 | 14.5 | 0.87 | 0.88 | 0.74 | 0.78 | 1.07 | |
Glu | GAA | 54.591 | 34.3 | 36.0 | 45.6 | 39.3 | 35.1 | 1.59 | 1.52 | 1.20 | 1.39 | 1.56 |
GAG | 34.739 | 32.2 | 29.4 | 19.2 | 18.7 | 27.4 | 1.08 | 1.18 | 1.81 | 1.86 | 1.27 | |
Cys | UGU | 24.814 | 10.5 | 9.8 | 8.1 | 5.2 | 13.4 | 2.36 | 2.53 | 3.06 | 4.77 | 1.85 |
UGC | 7.444 | 7.2 | 7.2 | 4.8 | 6.4 | 9.4 | 1.03 | 1.03 | 1.55 | 1.16 | 0.79 | |
Trp | UGG | 14.888 | 12.5 | 12.2 | 10.4 | 15.3 | 11.5 | 1.19 | 1.22 | 1.43 | 0.97 | 1.29 |
Arg | CGU | 12.407 | 9.0 | 7.5 | 6.4 | 20.2 | 5.7 | 1.38 | 1.65 | 1.94 | 0.61 | 2.18 |
CGC | 0.000 | 3.8 | 3.9 | 2.6 | 20.8 | 4.0 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |
CGA | 4.963 | 6.3 | 5.3 | 3.0 | 3.8 | 4.6 | 0.79 | 0.94 | 1.65 | 1.31 | 1.08 | |
CGG | 2.481 | 4.9 | 3.7 | 1.7 | 6.2 | 1.8 | 0.51 | 0.67 | 1.46 | 0.40 | 1.38 | |
Ser | AGU | 7.444 | 14.0 | 13.3 | 14.2 | 9.4 | 14.5 | 0.53 | 0.56 | 0.52 | 0.79 | 0.51 |
AGC | 13.370 | 11.3 | 10.0 | 9.8 | 16.0 | 10.6 | 1.18 | 1.34 | 1.36 | 0.84 | 1.26 | |
Arg | AGA | 12.407 | 19.0 | 16.0 | 21.3 | 2.9 | 10.9 | 0.65 | 0.78 | 0.58 | 4.28 | 1.14 |
AGG | 12.407 | 11.0 | 12.2 | 9.2 | 1.8 | 15.0 | 1.13 | 1.02 | 1.35 | 6.89 | 0.83 | |
Gly | GGU | 13.370 | 22.2 | 22.3 | 23.9 | 24.2 | 22.9 | 0.06 | 0.60 | 0.56 | 0.55 | 0.58 |
GGC | 7.444 | 9.2 | 11.2 | 9.8 | 28.1 | 11.6 | 0.81 | 0.66 | 0.76 | 0.26 | 0.64 | |
GGA | 32.258 | 24.2 | 23.2 | 10.9 | 8.9 | 17.1 | 1.33 | 1.39 | 2.96 | 3.62 | 1.89 | |
GGG | 22.333 | 10.2 | 10.5 | 6.0 | 11.8 | 14.9 | 2.17 | 2.13 | 3.72 | 1.89 | 1.50 | |
TER | *UAA | 0.000 | 0.9 | 1.1 | 1.1 | 2.0 | 1.1 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
*UAG | 0.000 | 0.5 | 0.5 | 0.5 | 0.3 | 0.0 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |
*UGA | 2.481 | 1.2 | 1.0 | 0.7 | 1.1 | 1.6 | 2.07 | 2.48 | 3.54 | 2.26 | 1.55 |
Fig.7 PmNAC8 expression level in the different tissues of P. massoniana F:Whole flowers containing pollen. NL:New leaves of the current year. OL:Old leaves. NS:Immature stem of current year. OS:Mature stem current year. R:Root. M:Xylem. P:Phloem. The young leaf tissue is used as a control. Different lowercase letters in the same column indicate a significant difference,P<0.05. The same below
Fig.8 PmNAC8 expression of P. massonniana under different stress All the above treatments are treated with 0 h as control.(a):10 mmol/L MeJA;(b):50 μmol/L ETHs;(c):10 mmol/L H2O2;(d):2 mmol/L GA;(e):1 mmol/L SA;(f):15% PEG6000;(g):mechanical injury stress;(h):400 μmol/L ABA
元件Element | 功能Function | 核心序列Core sequence | 数量Amount |
---|---|---|---|
Box 4 | 参与光响应部分保守DNA组件 Part of a conserved DNA module involved in light responsiveness | ATTAAT | 2 |
G-Box | 参与光响应的顺式调控元件 Cis-acting regulatory element involved in light responsiveness | CACGAC | 2 |
GT1-motif | 光响应元件Light responsive element | GGTTAAT | 1 |
MRE | 涉及光反应的MYB结合位点 MYB binding sites involved in light reactions | AACCTAA | 1 |
MBS | 参与干旱诱导MYB结合位点 MYB binding site involved in drought induction | CAACTG | 1 |
ARE | 抗氧化反应元件 Antioxidant reaction element | AAACCA | 2 |
O2-site | 参与玉米醇溶蛋白的代谢调节 Cis-acting regulatory element involved in zein metabolism regulation | GTTGACGTGA | 1 |
TATA-box | 转录起始-30处核心启动元件 Core promoter element around -30 of transcription start | TACAAAA | 21 |
HD-Zip 3 | 蛋白结合位点 Protein binding site | GTAAT(G/C)ATTAC | 3 |
CAAT-box | 启动子和增强子区域共有顺式作用元件 Common cis-acting element in promoter and enhancer regions | CAAT/CAAAT/CCAAT/TGCCAAC/ | 34 |
P-box | 赤霉素响应顺式作用元件 Cis-acting element involved in gibberellin responsiveness | CCTTTTG | 1 |
Table 5 Cis-acting elements in the PmNAC8 promoter
元件Element | 功能Function | 核心序列Core sequence | 数量Amount |
---|---|---|---|
Box 4 | 参与光响应部分保守DNA组件 Part of a conserved DNA module involved in light responsiveness | ATTAAT | 2 |
G-Box | 参与光响应的顺式调控元件 Cis-acting regulatory element involved in light responsiveness | CACGAC | 2 |
GT1-motif | 光响应元件Light responsive element | GGTTAAT | 1 |
MRE | 涉及光反应的MYB结合位点 MYB binding sites involved in light reactions | AACCTAA | 1 |
MBS | 参与干旱诱导MYB结合位点 MYB binding site involved in drought induction | CAACTG | 1 |
ARE | 抗氧化反应元件 Antioxidant reaction element | AAACCA | 2 |
O2-site | 参与玉米醇溶蛋白的代谢调节 Cis-acting regulatory element involved in zein metabolism regulation | GTTGACGTGA | 1 |
TATA-box | 转录起始-30处核心启动元件 Core promoter element around -30 of transcription start | TACAAAA | 21 |
HD-Zip 3 | 蛋白结合位点 Protein binding site | GTAAT(G/C)ATTAC | 3 |
CAAT-box | 启动子和增强子区域共有顺式作用元件 Common cis-acting element in promoter and enhancer regions | CAAT/CAAAT/CCAAT/TGCCAAC/ | 34 |
P-box | 赤霉素响应顺式作用元件 Cis-acting element involved in gibberellin responsiveness | CCTTTTG | 1 |
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