马尾松NAC转录因子基因PmNAC8的克隆及表达分析

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

摘要/Abstract

摘要：

NAC（NAM,ATAF1/2,CUC2）家族基因参与调控植物生长发育、响应非生物胁迫及激素信号转导。研究马尾松中NAC转录因子在非生物胁迫中的应答,为阐明基因功能提供理论依据。以马尾松（Pinus massoniana）为试验材料,通过RT-PCR及RACE技术克隆一个NAC家族基因,并对其进行生物信息学分析。运用qRT-PCR检测其组织表达特性及对逆境胁迫的响应情况;以基因组DNA为模板,运用染色体步移（genome walking）技术克隆该基因上游启动子序列;通过农杆菌注射本氏烟草（Nicotiana benthamiana）,观察其亚细胞定位。该基因全长1 726 bp,开放阅读框为1 209 bp,编码402个氨基酸,将其命名为PmNAC8,GenBank登录号为MZ291447;亚细胞定位结果显示,该基因定位于细胞核;其密码子使用偏性较弱,偏好使用A/T结尾的密码子,且烟草与酵母更适合作为PmNAC8的异源表达受体;qRT-PCR显示PmNAC8在根部表达量最高,且受机械损伤及植物激素GA、ABA的诱导;克隆获得PmNAC8上游1 492 bp的启动子区域,含有多个胁迫诱导元件、赤霉素调控元件和光响应元件。PmNAC8参与多种逆境胁迫,可能在赤霉素信号途径中发挥重要作用。

关键词: 马尾松, NAC, 密码子偏性, 亚细胞定位, 非生物胁迫, 启动子

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

镐青青, 姚圣, 刘佳禾, 陈佩珍, 张梦洋, 季孔庶. 马尾松NAC转录因子基因PmNAC8的克隆及表达分析[J]. 生物技术通报, 2022, 38(4): 202-216.

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.

图/表 14

参考文献 39

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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