Cloning and Expression Analysis of StDof5 Gene in Potato

doi:10.13560/j.cnki.biotech.bull.1985.2023-0960

Abstract

Abstract:

【Objective】 The DNA binding with one finger（Dof）gene family is a plant-specific transcription factor family that plays important roles in plant growth, development and stress response. Studying the function and mechanism of Dof transcription factors responding to abiotic stress, especially salt stress in potato, provides some theoretical and technical basis for obtaining new varieties resistant to salt stress.【Method】 StDof5 was cloned, and it was analyzed via bioinformatics. StDof5 overexpression vector was constructed by homologous recombination, potato was transformed to obtain positive plants lines, and their expressions were analyzed.【Result】 CDS of StDof5 is 498 bp, it encodes 165 amino acids, and the molecular formula is C₈₀₀H₁₂₃₆N₂₄₂O₂₄₆S₉. The number of StDof5 atoms is 2 533, the relative molecular weight is 18 468.63 kD, the isoelectric point is 8.47, the aliphatic index is 51.45, and the instability coefficient is 45.16, the protein is predicted to be an unstable protein. The predicted number of StDof5 transmembrane helices is 0, thus it is a non-transmembrane protein. The average value of hydrophilicity of StDof5 protein is -0.876, predicting that it is hydrophilic. The predicted secondary structure of StDof5 protein showed it has 20% α-helices, 0 β-turn, 9.7% extended strands and 66.06% random coils. Salt stress phenotype experiments showed that the plant heights and fresh weights of overexpressing plants（OPs）were significantly higher than those of wild type（DES）plants, but there was no significant difference in root length, root number and leaf number between them. 【Conclusion】 Overexpressing plants（OPs）can resist salt stress and demonstrate desensitization phenotype to salt stress. StDof5 has significant responses to salt stress and tissue specificity.

Key words: potato, StDof5 gene, vector construction, genetic transformation

MEI Xian-jun, SONG Hui-yang, LI Jing-hao, MEI Chao, SONG Qian-na, FENG Rui-yun, CHEN Xi-ming. Cloning and Expression Analysis of StDof5 Gene in Potato[J]. Biotechnology Bulletin, 2024, 40(3): 181-192.

Figures/Tables 12

References 48

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引物名称Primer name	引物序列Primer sequence（5'-3'）
Actin-F	GGGATGGAGAAGTTTGGTGGTGG
Actin-R	CTTCGACCAAGGGATGGTGTAGC
Dof-F.2	GCTTGACTTTGATGGAGTGGTGG
Dof-R.2	AAGATTGGCTATCGGAGGTGC
ABF4-F	CTTCTGCACATACAAGGATATTCAA
ABF4-R	TGCCTAGCCAAAGGGAAATCA
MYB2-F	ATGACATGGACCTCCGACGA
MYB2-R	CGTTCGCTTTAGACCAGCAC
SnRK2s-F	TTGCTGTAGAGAAGGTGGGT
SnRK2s-R	GTCCTACTGTCACTGCCGTC
DREB2A-F	CACAGAAGAAAAGAGAAATGGCT
DREB2A-R	TCCACTATAATCCAACGGCAGA
ABI3-F	TGCTGACATGAAGTGTGGCA
ABI3-R	TTTTGCCCTCCGACTTTGGT
ABI5-F	TAAAACAGGCCCTGGCGGAG
ABI5-R	TTGCGCCTTTGTTTGAGCTT

引物名称Primer name	引物序列Primer sequence（5'-3'）
Actin-F	GGGATGGAGAAGTTTGGTGGTGG
Actin-R	CTTCGACCAAGGGATGGTGTAGC
Dof-F.2	GCTTGACTTTGATGGAGTGGTGG
Dof-R.2	AAGATTGGCTATCGGAGGTGC
ABF4-F	CTTCTGCACATACAAGGATATTCAA
ABF4-R	TGCCTAGCCAAAGGGAAATCA
MYB2-F	ATGACATGGACCTCCGACGA
MYB2-R	CGTTCGCTTTAGACCAGCAC
SnRK2s-F	TTGCTGTAGAGAAGGTGGGT
SnRK2s-R	GTCCTACTGTCACTGCCGTC
DREB2A-F	CACAGAAGAAAAGAGAAATGGCT
DREB2A-R	TCCACTATAATCCAACGGCAGA
ABI3-F	TGCTGACATGAAGTGTGGCA
ABI3-R	TTTTGCCCTCCGACTTTGGT
ABI5-F	TAAAACAGGCCCTGGCGGAG
ABI5-R	TTGCGCCTTTGTTTGAGCTT

测量指标Measurement index	盐浓度Salting strength/（mol·L^-1）	品种名称Variety name
测量指标Measurement index	盐浓度Salting strength/（mol·L^-1）	DES	OP-6	OP-7	OP-9	OP-10	OP-16	OP-24
株高Plant height/cm	0	6.87±0.38a	7.10±0.17a	5.57±0.40a	5.43±0.15a	6.53±0.85a	7.37±0.75a	7.83±1.01a
	50	3.70±010b	4.83±0.65b	3.70±0.36b	4.37±0.21b	3.97±0.35b	6.00±0.72b	4.13±0.45b
	100	2.13±0.76c	2.97±0.42c	2.00±0.53c	1.77±0.31c	2.80±0.36c	3.00±0.44c	2.93±0.15c
鲜重Fresh weight/g	0	0.21±0.01a	0.23±0.01a	0.19±0.01a	0.21±0.02a	0.24±0.02a	0.19±0.02a	0.35±0.01a
	50	0.14±0.01b	0.19±0.02b	0.12±0.02b	0.16±0.02b	0.16±0.02b	0.16±0.01a	0.28±0.02b
	100	0.09±0.01c	0.12±0.02c	0.09±0.01c	0.09±0.01c	0.12±0.01c	0.12±0.02b	0.15±0.02c
根长Root length/cm	0	12.13±0.25a	12.20±0.46a	12.23±2.03a	14.70±0.62a	17.63±0.35a	12.90±1.49a	12.03±0.72a
	50	9.00±0.26b	9.73±0.12b	10.93±1.79ab	13.07±0.31b	13.73±0.31b	10.53±0.61b	11.67±0.31a
	100	7.53±0.25c	8.93±060b	6.70±2.74b	4.00±0.72c	10.1±0.20c	9.73±0.31b	8.60±0.53b
根数Root	0	6.33±1.15a	7.33 ±2.31a	8.00±0.00a	7.00±1.00a	8.67±1.53a	6.00±0.00a	9.67±2.08a
	50	3.67±2.08a	5.67±2.31a	4.00±1.00b	5.33±2.31a	4.67±1.53b	6.67±2.89a	5.67±1.15b
	100	3.33±1.53a	5.00±1.00a	3.33±2.08b	1.00±0.00b	4.33±2.31b	1.67±0.58b	3.00±1.73b
叶片数Number of leaves	0	7.33±0.58a	8.33±0.58a	7.67±0.58a	6.67±0.58a	7.00±0.00a	7.00±1.00a	6.67±0.58a
	50	6.33±0.58a	7.00±0.00a	6.33±1.15a	6.00±1.00ab	7.00±0.00a	7.00±1.00a	6.66±0.58a
	100	7.00±1.00a	7.00±1.00a	6.67±0.58a	5.00±0.00b	6.33±1.15a	6.00±1.00a	6.67±0.58a

测量指标Measurement index	盐浓度Salting strength/（mol·L^-1）	品种名称Variety name
测量指标Measurement index	盐浓度Salting strength/（mol·L^-1）	DES	OP-6	OP-7	OP-9	OP-10	OP-16	OP-24
株高Plant height/cm	0	6.87±0.38a	7.10±0.17a	5.57±0.40a	5.43±0.15a	6.53±0.85a	7.37±0.75a	7.83±1.01a
	50	3.70±010b	4.83±0.65b	3.70±0.36b	4.37±0.21b	3.97±0.35b	6.00±0.72b	4.13±0.45b
	100	2.13±0.76c	2.97±0.42c	2.00±0.53c	1.77±0.31c	2.80±0.36c	3.00±0.44c	2.93±0.15c
鲜重Fresh weight/g	0	0.21±0.01a	0.23±0.01a	0.19±0.01a	0.21±0.02a	0.24±0.02a	0.19±0.02a	0.35±0.01a
	50	0.14±0.01b	0.19±0.02b	0.12±0.02b	0.16±0.02b	0.16±0.02b	0.16±0.01a	0.28±0.02b
	100	0.09±0.01c	0.12±0.02c	0.09±0.01c	0.09±0.01c	0.12±0.01c	0.12±0.02b	0.15±0.02c
根长Root length/cm	0	12.13±0.25a	12.20±0.46a	12.23±2.03a	14.70±0.62a	17.63±0.35a	12.90±1.49a	12.03±0.72a
	50	9.00±0.26b	9.73±0.12b	10.93±1.79ab	13.07±0.31b	13.73±0.31b	10.53±0.61b	11.67±0.31a
	100	7.53±0.25c	8.93±060b	6.70±2.74b	4.00±0.72c	10.1±0.20c	9.73±0.31b	8.60±0.53b
根数Root	0	6.33±1.15a	7.33 ±2.31a	8.00±0.00a	7.00±1.00a	8.67±1.53a	6.00±0.00a	9.67±2.08a
	50	3.67±2.08a	5.67±2.31a	4.00±1.00b	5.33±2.31a	4.67±1.53b	6.67±2.89a	5.67±1.15b
	100	3.33±1.53a	5.00±1.00a	3.33±2.08b	1.00±0.00b	4.33±2.31b	1.67±0.58b	3.00±1.73b
叶片数Number of leaves	0	7.33±0.58a	8.33±0.58a	7.67±0.58a	6.67±0.58a	7.00±0.00a	7.00±1.00a	6.67±0.58a
	50	6.33±0.58a	7.00±0.00a	6.33±1.15a	6.00±1.00ab	7.00±0.00a	7.00±1.00a	6.66±0.58a
	100	7.00±1.00a	7.00±1.00a	6.67±0.58a	5.00±0.00b	6.33±1.15a	6.00±1.00a	6.67±0.58a