Cloning and Expression Analysis of NAC Transcription Factor PmNAC8 in Pinus massoniana

doi:10.13560/j.cnki.biotech.bull.1985.2021-0830

Abstract

Abstract:

NAC（NAM,ATAF1/2,CUC2）family of genes are involved in regulation of plant growth and development,abiotic stress response and hormone signal transduction. Studying the responses of NAC transcription factors in Pinus massoniana to abiotic stress would provide theoretical basis for elucidating gene function. A NAC family’s gene from P. massoniana was cloned by RT-PCR and RACE techniques,and then analyzed by bioinformatics. And the expression patterns in different tissues and its responses to stresses were detected by qRT-PCR. Using genomic DNA as a template,the upstream promoter region of the gene was cloned by the genome walking techniques. By agrobacterium injection of Nicotiana benthamiana,the subcellular localization was observed. The full length of the gene was 1 726 bp and open reading frame was 1 209 bp,encoding 402 amino acids,and named as PmNAC8. GenBank accession number is MZ291447. Subcellular localization showed that the gene was located in the nucleus and the codon usage bias was low,it preferred to use the codon ending in A/T,and tobacco and Accharomyces cerevisiae are more suitable as heterologous expression receptor materials for PmNAC8. The qRT-PCR demonstrated that PmNAC8 was expressed in all tissues of P. massoniana,and the highest expression was found in the root,and was induced by mechanical damage and plant hormones GA and ABA. The 1 492 bp promoter region upstream of PmNAC8 contained multiple stress components,gibberellin control components and light response components. These results suggest that PmNAC8 is a transcription factor involved in various stresses and may play an important role in gibberellin signaling.

Key words: Pinus massoniana, NAC, codon bias, subcellular localization, abiotic stress, promoter

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.

Figures/Tables 14

References 39

[1]	Shinozaki K, Yamaguchi-Shinozaki K, Seki M. Regulatory network of gene expression in the drought and cold stress responses[J]. Curr Opin Plant Biol, 2003, 6(5):410-417. pmid: 12972040
[2]	Rejeb IB, Pastor V, Mauch-Mani B. Plant responses to simultaneous biotic and abiotic stress:molecular mechanisms[J]. Plants-Basel, 2014, 3(4):458-475. doi: 10.3390/plants3040458 pmid: 27135514
[3]	Liu H, Zhou Y, Li H, et al. Molecular and functional characterization of ShNAC1, an NAC transcription factor from Solanum habrochaites[J]. Plant Sci, 2018, 271:9-19. doi: 10.1016/j.plantsci.2018.03.005 URL
[4]	Long L, et al. GbMPK3, a mitogen-activated protein kinase from cotton, enhances drought and oxidative stress tolerance in tobacco[J]. Plant Cell Tissue Organ Cult, 2014, 116(2):153-162. doi: 10.1007/s11240-013-0392-1 URL
[5]	Shao H, Wang H, Tang X. NAC transcription factors in plant multiple abiotic stress responses:progress and prospects[J]. Front Plant Sci, 2015, 6:902.
[6]	Duval M, Hsieh TF, Kim SY, et al. Molecular characterization of AtNAM:a member of the Arabidopsis NAC domain superfamily[J]. Plant Mol Biol, 2002, 50(2):237-248. doi: 10.1023/A:1016028530943 URL
[7]	Tran LS, et al. Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive Cis-element in the early responsive to dehydration stress 1 promoter[J]. Plant Cell, 2004, 16(9):2481-2498. doi: 10.1105/tpc.104.022699 URL
[8]	Chen X, Wang Y, Lv B, et al. The NAC family transcription factor OsNAP confers abiotic stress response through the ABA pathway[J]. Plant Cell Physiol, 2014, 55(3):604-619. doi: 10.1093/pcp/pct204 pmid: 24399239
[9]	Xue GP, Way HM, et al. Overexpression of TaNAC69 leads to enhanced transcript levels of stress up-regulated genes and dehydration tolerance in bread wheat[J]. Mol Plant, 2011, 4(4):697-712. doi: 10.1093/mp/ssr013 URL
[10]	Lu M, Ying S, Zhang DF, et al. A maize stress-responsive NAC transcription factor, ZmSNAC1, confers enhanced tolerance to dehydration in transgenic Arabidopsis[J]. Plant Cell Rep, 2012, 31(9):1701-1711. doi: 10.1007/s00299-012-1284-2 URL
[11]	Puranik S, Sahu PP, et al. NAC proteins:regulation and role in stress tolerance[J]. Trends Plant Sci, 2012, 17(6):369-381. doi: 10.1016/j.tplants.2012.02.004 pmid: 22445067
[12]	Mahmood K, El-Kereamy A, Kim SH, et al. ANAC032 positively regulates age-dependent and stress-induced senescence in Arabidopsis thaliana[J]. Plant Cell Physiol, 2016, 57(10):2029-2046. pmid: 27388337
[13]	Mahmood K, Xu Z, El-Kereamy A, et al. The Arabidopsis transcription factor ANAC032 represses anthocyanin biosynjournal in response to high sucrose and oxidative and abiotic stresses[J]. Front Plant Sci, 2016, 7:1548. pmid: 27790239
[14]	Oda-Yamamizo C, Mitsuda N, Sakamoto S, et al. The NAC transcription factor ANAC046 is a positive regulator of chlorophyll degradation and senescence in Arabidopsis leaves[J]. Sci Rep, 2016, 6:23609. doi: 10.1038/srep23609 pmid: 27021284
[15]	蒙露露, 何冠谛, 田维军, 等. 马铃薯StNAC2基因的克隆及其在镉胁迫下的表达分析[J]. 分子植物育种, 2020, 18(22):7293-7300.
	Meng LL, He GD, Tian WJ, et al. Cloning and expression analysis of StNAC2 gene in potato(Solanum tuberosum L.)under cadmium stress[J]. Mol Plant Breed, 2020, 18(22):7293-7300.
[16]	Jeong JS, Kim YS, Redillas MCFR, et al. OsNAC5 overexpression enlarges root diameter in rice plants leading to enhanced drought tolerance and increased grain yield in the field[J]. Plant Biotechnol J, 2013, 11(1):101-114. doi: 10.1111/pbi.12011 pmid: 23094910
[17]	岳文冉, 岳俊燕, 张秀娟, 等. 中间锦鸡儿CiNAC1基因促进转基因拟南芥叶片的衰老[J]. 中国生物工程杂志, 2018, 38(4):24-29.
	Yue WR, Yue JY, Zhang XJ, et al. The CiNAC1 from Caragana intermedia promotes transgenic Arabidopsis leaf senescence[J]. China Biotechnol, 2018, 38(4):24-29.
[18]	Fang L, Su L, Sun X, et al. Expression of Vitis amurensis NAC26 in Arabidopsis enhances drought tolerance by modulating jasmonic acid synjournal[J]. J Exp Bot, 2016, 67(9):2829-2845. doi: 10.1093/jxb/erw122 URL
[19]	武肖琦, 冯涛, 刘艳军, 等. 桃PpNAC72的克隆及表达分析[J]. 生物技术通报, 2019, 35(6):16-23. doi: 10.13560/j.cnki.biotech.bull.1985.2018-0891
	Wu XQ, Feng T, et al. Cloning and expression analysis of the PpNAC72 in peach[J]. Biotechnol Bull, 2019, 35(6):16-23.
[20]	徐向华, 丁贵杰. 马尾松适应低磷胁迫的生理生化响应[J]. 林业科学, 2006, 42(9):24-28.
	Xu XH, Ding GJ. Physiological and biochemical responses of Pinus massoniana to low phosphorus stress[J]. Sci Silvae Sin, 2006, 42(9):24-28.
[21]	王晓锋, 何卫龙, 蔡卫佳, 等. 马尾松转录组测序和分析[J]. 分子植物育种, 2013, 11(3):385-392.
	Wang XF, He WL, Cai WJ, et al. Analysis on transcriptome sequenced for Pinus massoniana[J]. Mol Plant Breed, 2013, 11(3):385-392.
[22]	Kumar S, Stecher G, Li M, et al. MEGA X:molecular evolutionary genetics analysis across computing platforms[J]. Mol Biol Evol, 2018, 35(6):1547-1549. doi: 10.1093/molbev/msy096 URL
[23]	赵春丽, 彭丽云, 王晓, 等. 苋菜AtGAI基因密码子偏好性与进化分析[J]. 中国农业大学学报, 2019, 24(12):10-22.
	Zhao CL, Peng LY, Wang X, et al. Codon bias and evolution analysis of AtGAI in Amaranthus tricolor L[J]. J China Agric Univ, 2019, 24(12):10-22.
[24]	薛拥志, 王宝地, 王连荣. 杏扁PaCBF1基因密码子偏好性分析[J]. 分子植物育种, 2018, 16(15):4890-4898.
	Xue YZ, Wang BD, Wang LR. Analysis of codon bias of PaCBF1 gene in Prunus armeniaca L[J]. Mol Plant Breed, 2018, 16(15):4890-4898.
[25]	李尊强, 王春军, 杨爱国, 等. 烟草DXS基因的克隆及其亚细胞定位分析[J]. 安徽农业科学, 2013, 41(30):11957-11960.
	Li ZQ, Wang CJ, Yang AG, et al. Cloning and subcellular localization of DXS gene in tobacco[J]. J Anhui Agric Sci, 2013, 41(30):11957-11960.
[26]	Yao S, Wu F, Hao QQ, et al. Transcriptome-wide identification of WRKY transcription factors and their expression profiles under different types of biological and abiotic stress in Pinus massoniana lamb[J]. Genes, 2020, 11(11):1386. doi: 10.3390/genes11111386 URL
[27]	Zhu PH, Ma YY, Zhu LZ, et al. Selection of suitable reference genes in Pinus massoniana lamb. under different abiotic stresses for qPCR normalization[J]. Forests, 2019, 10(8):632. doi: 10.3390/f10080632 URL
[28]	朱灵芝, 朱沛煌, 李荣, 等. 马尾松PmDXR基因密码子偏好性分析[J]. 林业科学研究, 2021, 34(2):102-113.
	Zhu LZ, Zhu PH, Li R, et al. Analysis on codon bias of PmDXR gene in Pinus massoniana lamb[J]. For Res, 2021, 34(2):102-113.
[29]	Ondati Evans. 陆地棉GhNAC18和GhNAC20基因在叶片衰老和胁迫应答中的功能分析[D]. 北京:中国农业科学院, 2016.
	Ondati E. Functional analysis of GhNAC18 and GhNAC20 during leaf senescence and stress responses in cotton(Gossypium hirsutum L.)[D]. Beijing:Chinese Academy of Agricultural Sciences, 2016.
[30]	Jiang G, Yan H, Wu F, et al. Litchi fruit LcNAC1 is a target of LcMYC2 and regulator of fruit senescence through its interaction with LcWRKY1[J]. Plant Cell Physiol, 2017, 58(6):1075-1089. doi: 10.1093/pcp/pcx054 URL
[31]	张海娟, 吴剑锋, 胡帅, 等. 芜菁NAC转录因子BcNAC2基因的分离及其表达[J]. 园艺学报, 2011, 38(6):1089-1096.
	Zhang HJ, Wu JF, Hu S, et al. Isolation and expression analysis of BcNAC2, a NAC transcription factor gene in turnip[J]. Acta Hortic Sin, 2011, 38(6):1089-1096.
[32]	Yang X, Hu YX, Li XL, et al. Molecular characterization and function analysis of SlNAC2 in Suaeda liaotungensis K[J]. Gene, 2014, 543(2):190-197. doi: 10.1016/j.gene.2014.04.025 URL
[33]	Yang SD, Seo PJ, Yoon HK, et al. The Arabidopsis NAC transcription factor VNI2 integrates abscisic acid signals into leaf senescence via the COR/RD genes[J]. Plant Cell, 2011, 23(6):2155-2168. doi: 10.1105/tpc.111.084913 URL
[34]	荣玉萍, 唐彬, 李鹏, 等. 苦荞NAC转录因子FtNAC17的鉴定及表达分析[J]. 生物技术通报, 2021, 37(1):174-181. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0556
	Rong YP, Tang B, Li P, et al. Identification and expression of NAC transcription factor FtNAC17 in Tartary buckwheat[J]. Biotechnol Bull, 2021, 37(1):174-181.
[35]	孙欣, 上官凌飞, 房经贵, 等. 葡萄NAC转录因子家族生物信息学分析[J]. 基因组学与应用生物学, 2011, 30(2):229-242.
	Sun X, Shangguan LF, Fang JG, et al. Bioinformatics analysis of the NAC transcription factor family in grapevine[J]. Genom Appl Biol, 2011, 30(2):229-242.
[36]	朱沛煌, 陈妤, 朱灵芝, 等. 马尾松转录组密码子使用偏好性及其影响因素[J]. 林业科学, 2020, 56(4):74-81.
	Zhu PH, Chen Y, Zhu LZ, et al. Codon usage bias and its influencing factors in Pinus massoniana transcriptome[J]. Sci Silvae Sin, 2020, 56(4):74-81.
[37]	杨晓娜, 赵昶灵, 李云, 等. 启动子序列克隆和功能分析方法的研究进展[J]. 云南农业大学学报:自然科学版, 2010, 25(2):283-290.
	Yang XN, Zhao CL, Li Y, et al. Research advances in the methods of cloning and function-analyzing of promoters[J]. J Yunnan Agric Univ:Nat Sci, 2010, 25(2):283-290.
[38]	颜静宛, 林智敏, 周淑芬, 等. 水稻胚特异表达基因Os08PTS启动子的克隆及分析[J]. 福建农业科技, 2021, 52(3):6-10.
	Yan JW, Lin ZM, Zhou SF, et al. Cloning and analysis of the promoter of rice embryo-specific expression gene Os08PTS[J]. Fujian Agric Sci Technol, 2021, 52(3):6-10.
[39]	Puranik S, Bahadur RP, Srivastava PS, et al. Molecular cloning and characterization of a membrane associated NAC family gene, SiNAC from foxtail millet[Setaria italica(L.)P. Beauv][J]. Mol Biotechnol, 2011, 49(2):138-150. doi: 10.1007/s12033-011-9385-7 pmid: 21312005

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

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

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
Phe	UUC	0.46	3	7.444	0.231	Tyr	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	TER	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	His	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	Gln	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	Asn	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	Lys	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	Asp	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	Glu	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	Cys	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	Ser	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	Arg	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	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
Phe	UUC	0.46	3	7.444	0.231	Tyr	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	TER	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	His	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	Gln	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	Asn	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	Lys	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	Asp	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	Glu	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	Cys	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	Ser	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	Arg	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
Phe	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
Tyr	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
His	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
Gln	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
Asn	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
Lys	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
Asp	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
Glu	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
Cys	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
Ser	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
Arg	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