TkDREB2 Clone from Taraxacum kok-saghyz and Drought Tolerance Analysis of Transgenic Nicotiana tabacum

doi:10.13560/j.cnki.biotech.bull.1985.2021-0108

Abstract

Abstract:

In order to explore the important role of jasmonic acid on the drought resistance of plants,the differential expression profile of the transcriptome of Taraxacum kok-saghyz externally treated with methyl jasmonate（MeJA）was conducted,and an AP2/EREBP subfamily gene DREB,whose expression was significantly up-regulated by jasmonic acid,was screened and cloned,named as TkDREB2. It was subjected to bioinformatics analysis and transformed to tobacco. For further identifying the drought resistance function of TkDREB2 transgenic tobacco plants during the development period,the transgenic tobacco was subjected to natural drought treatment and related physiological indicators were measured. The results showed that the expression levels of genes related to jasmonic acid synthesis and transcription factors AP2/EREBP,MYB,MYC,NAC and WRKY changed significantly in the transcriptome analysis of methyl jasmonate treatment. The full-length CDS sequence of the cloned TkDREB2 gene was 1 002 bp,encoding 333 amino acids,and belonged to the DREB type transcription factor of the AP2/EREBP subfamily. The phylogenetic tree analysis showed that this protein had the closest homology to the DREB protein of alfalfa. After 15 d of natural drought treatment for genetically modified tobacco,it was found that wild-type leaves began to wilt;continuing treatment to 22 d,wild-type leaves severely wilted and the leaves of genetically modified tobacco only slightly wilted. The physiological indicators of the wild-type and the genetically modified tobacco after normal growth and drought treatment were determined. Under normal growth conditions,the superoxide dismutase（SOD）,peroxidase（POD）and catalase（CAT）activities of the genetically modified tobacco were approximately twice that of wild-type. After drought treatment,the activities of CAT,SOD and POD in the genetically modified tobacco were about 2 times,2 times and 1.5 times that of wild-type,respectively,and the content and relative conductivity of malondialdehyde were only about 1/2 of that of wild type. The above results indicate that TkDREB2 can actively respond to the induced expression of jasmonic acid. Overexpression of the TkDREB2 gene can significantly improve the drought resistance of tobacco. It is speculated that DREB2 transcription factor may play an important role in regulating the drought resistance of Taraxacum kok-saghyz by jasmonic acid signal transduction.

Key words: transcriptome, transcription factor, TkDREB2, Nicotiana tabacum, drought tolerance

ZHANG Yun-chuan, LIN Yi-xuan, CAO Xin-wen, WANG Hai-nan, YAN Jie. TkDREB2 Clone from Taraxacum kok-saghyz and Drought Tolerance Analysis of Transgenic Nicotiana tabacum[J]. Biotechnology Bulletin, 2021, 37(11): 212-224.

Figures/Tables 17

Fig. 1 Heat map of differentially expressed AP2/EREBP（A）,bHLH（B）,MYB（C）and WRKY（D）transcription factors Red expression is up-regulated,blue expression is down-regulated,the darker the color,the higher the expression level. MJ0_R,MJ6_R and MJ24_R indicate jasmonic acid treatment 0,6 and 24 h

Table 1 Differential expression of genes encoding enzymes related to jasmonic acid synthesis

基因信息Genetic information		茉莉酸处理时间Treatment time by jasmonic acid			差异表达倍数Log₂FoldChange
基因名称 Gene name	基因Gene ID	MJ6	MJ0	MJ24	6 VS 0	P value	24 VS 0	P value
OPR	c49640_g1	25 210.72	956.65		4.72	1.71E-35
			1 017.64	16 350.86			4.01	1.77E-31
	c49640_g2	15 311.67	589.34		4.70	3.61E-35
			626.95	10 019.59			4.00	3.15E-33
	c52666_g1	2 642.99	268.00		3.30	1.82E-19
			284.96	2 252.14			2.98	4.39E-20
AOS	c39329_g1	1 825.74	17.73		6.69	2.28E-17
			18.87	10 973.08			9.18	3.60E-25
	c47723_g1		603.38	7 806.60			3.69	1.46E-06
LOX	c53508_g1	21 905.05	359.65		5.93	4.42E-10
			382.62	49 808.79			7.02	2.75E-13
	c51038_g1		294.97	895.27			1.60	1.62E-05
AOC	c44998_g1	369.99	146.00		1.34	0.000 269
			155.26	737.86			2.25	9.16E-12

Fig. 2 Cloning of TkDREB2 gene

Fig. 3 Recombinant plasmid pMD19T-TkDREB2 digestion M: DNA marker Ⅲ. 1-2: BamHI and Pst I double digestion pMD19T-TkDREB2 recombinant plasmid. 3: pMD19T-TkDREB2 recombinant plasmid

Fig. 4 Prediction of TkDREB2 protein transmembrane structure

Fig. 5 Prediction of TkDREB2 protein signal peptide

Fig. 6 Prediction of the secondary structure of TkDREB2 protein Blue:α-helix;red:extended chain;green:β-turn;purple:random curl

Fig. 7 Prediction of the tertiary structure of TkDREB2 protein

Fig. 8 TkDREB2 amino acid sequence analysis The sequence in the square represents the AP2 domain, and the underlined part represents the predicted nuclear localization signal sequence

Fig. 9 TkDREB2 amino acid multiple sequence alignment

Fig. 10 Phylogenetic tree and conservative motif analysis of TkDREB2 protein A:Phylogenetic tree and conservative motif analysis of TkDREB2 protein. B:5 conservative motif fragments. Taraxacum kok-saghyz:TkDREB2（Taraxacum kok-saghyz,ASL72071.1）; Lactuca sativa:LsDREB2A（Lactuca sativa,XP_023748594.1）; Cichorium intybus:CiDREB2（Cichorium intybus,AHJ08574.1）; Cynara cardunculus:CcDREB2A（Cynara cardunculus,XP_024968974.1）; Chrysanthemum vestitum:CvDREB2（Chrysanthemum vestitum,ABR23508.1）; Helianthus annuus:HaDREB2A（Helianthus annuus,XP_021982098.1）; Nicotiana sylvestris:NsDREB2A（Nicotiana sylvestris,XP_009788693.1）; Nicotiana attenuata:NaDREB2A（Nicotiana attenuata,XP_019245341.1）; Nicotiana tomentosiformis:NtDREB2A（Nicotiana tomentosiformis,XP_009594350.1）; Nicotiana tabacum:NtDREB2A（Nicotiana tabacum,XP_016461559.1）; Sesamum indicum:SiDREB2C（Sesamum indicum,XP_011084866.1）

Fig. 11 Recombinant plasmid pCAMBIA2300-35S-TkDREB2 digestion M: DNA marker Ⅲ; 1: pCAMBIA2300-35S-TkDREB2 recombinant plasmid;2-3: BamHI and Pst I double digestion pCAMBIA2300-35S-TkDREB2 recombinant plasmid

Fig. 12 PCR identification of TkDREB2 transgenic plants M: DNA marker Ⅲ; 2-9: 8 correctly identified transgenic plants

Fig. 13 Wild-type（WT）and genetically modified tobacco under normal growth conditions（A）and 22 d of drought treatment（B） Transgenic lines:Genetically modified tobacco;WT:wild type tobacco

Fig. 14 SOD,POD and CAT enzyme activities and MDA content measurement results of wild-type and transgenic plants under normal growth conditions The mean measurement results of the physiological indexes of 3 wild-type and 3 transgenic tobaccos are compared by plotting. * indicates significant difference compared with wild-type（P<0.05,t-test）,the same below

Fig. 15 SOD,POD and CAT enzyme activities and MDA content measurement results of wild-type and transgenic plants after drought treatment

Fig. 16 Electrical conductivity measurement results of wild-type and transgenic plants after normal growth and drought treatment Transline 1-3:3 strains of genetically modified tobacco. The electrical conductivity of the wild-type plant is the mean value of the measurement results of 3 wild-type plants

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