橡胶草TkDREB2基因的克隆以及在烟草中的抗旱功能分析

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

摘要/Abstract

摘要：

为探究茉莉酸在提高植物抗旱性方面发挥的重要作用。对橡胶草外施茉莉酸甲酯（MeJA）处理的转录组进行差异表达谱分析,并筛选克隆出一个受茉莉酸诱导表达显著上调的AP2/EREBP亚家族基因DREB,命名为TkDREB2,对其进行生物信息学分析并转化烟草,进一步对TkDREB2转基因烟草植株在发育期的抗旱性功能鉴定,对转基因烟草进行自然干旱处理,并测定相关生理指标。结果显示在茉莉酸甲酯处理的转录组分析中发现与茉莉酸合成相关的基因和转录因子AP2/EREBP、MYB、MYC、NAC和WRKY的表达水平发生显著变化。筛选克隆的TkDREB2基因全长CDS序列为1 002 bp,编码333个氨基酸,属于AP2/EREBP亚家族的DREB型转录因子;系统进化树分析表明,该蛋白与苜蓿的DREB蛋白同源性最近。对转基因烟草进行自然干旱处理15 d后发现野生型叶片开始萎蔫;继续处理至22 d,野生型叶片严重萎蔫而转基因烟草仅轻微萎蔫,测定正常生长及干旱处理后野生型和转基因烟草的相关生理指标发现:在正常生长条件下,转基因烟草的超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）活性大约是野生型的2倍;在干旱处理后,转基因烟草的CAT、SOD和POD活性分别是野生型的2倍、2倍和1.5倍左右,丙二醛（MDA）含量和相对电导率仅是野生型的1/2左右。综合以上结果表明TkDREB2能够积极响应茉莉酸的诱导表达,过表达TkDREB2基因能够明显提高烟草的抗旱性,推测DREB2转录因子可能在茉莉酸信号转导调控植物抗旱性方面发挥重要作用。

关键词: 转录组, 转录因子, TkDREB2, 烟草, 抗旱性

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

张云川, 林熠轩, 曹新文, 王海楠, 闫洁. 橡胶草TkDREB2基因的克隆以及在烟草中的抗旱功能分析[J]. 生物技术通报, 2021, 37(11): 212-224.

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.

图/表 17

图1 差异表达的AP2/EREBP（A）、bHLH（B）、MYB（C）和WRKY（D）转录因子热图红色表达上调,蓝色表达下调,颜色越深代表表达量越高;MJ0_R、MJ6_R和MJ24_R表示茉莉酸处理0、6和24 h

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

表1 茉莉酸合成相关酶编码基因的差异表达情况

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

图2 TkDREB2基因的克隆 M:DNA marker Ⅲ;1-6:TkDREB2

Fig. 2 Cloning of TkDREB2 gene

图3 重组质粒pMD19T-TkDREB2酶切 M:DNA marker Ⅲ;1-2:BamHI和Pst I双酶切pMD19T-TkDREB2重组质粒;3:pMD19T-TkDREB2重组质粒

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

图4 TkDREB2蛋白跨膜结构预测

Fig. 4 Prediction of TkDREB2 protein transmembrane structure

图5 TkDREB2蛋白信号肽预测

Fig. 5 Prediction of TkDREB2 protein signal peptide

图6 TkDREB2蛋白二级结构预测蓝色:α-螺旋;红色:延伸链;绿色:β-转角;紫色:无规则卷曲

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

图7 TkDREB2蛋白质三级结构预测

Fig. 7 Prediction of the tertiary structure of TkDREB2 protein

图8 TkDREB2氨基酸序列分析方框内的序列代表AP2结构域;下划线部分表示预测的核定位信号序列

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

图9 TkDREB2氨基酸多序列比对

Fig. 9 TkDREB2 amino acid multiple sequence alignment

图10 TkDREB2蛋白系统进化树和保守motif分析 A:TkDREB2蛋白系统进化树和保守motif分析;B:5个保守motif片段。Taraxacum kok-saghyz:TkDREB2（橡胶草,ASL72071.1）;Lactuca sativa:LsDREB2A（莴苣,XP_023748594.1）;Cichorium intybus:CiDREB2（菊苣,AHJ08574.1）;Cynara cardunculus:CcDREB2A（朝鲜蓟,XP_024968974.1）;Chrysanthemum vestitum:CvDREB2（毛华菊,ABR23508.1）;Helianthus annuus:HaDREB2A（向日葵,XP_021982098.1）;Nicotiana sylvestris:NsDREB2A（美花烟草,XP_009788693.1）;Nicotiana attenuata:NaDREB2A（狼烟草,XP_019245341.1）;Nicotiana tomentosiformis:NtDREB2A（茸毛烟草,XP_009594350.1）;Nicotiana tabacum:NtDREB2A（普通烟草,XP_016461559.1）;Sesamum indicum:SiDREB2C（芝麻,XP_011084866.1）

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）

图11 重组质粒pCAMBIA2300-35S-TkDREB2酶切 M:DNA marker Ⅲ;1:pCAMBIA2300-35S-TkDREB2重组质粒;2-3:BamHI和Pst I双酶切pCAMBIA2300-35S-TkDREB2重组质粒

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

图12 PCR鉴定TkDREB2转基因植株 M:DNA marker Ⅲ;2-9:8个鉴定正确的转基因植株

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

图13 正常生长条件（A）和干旱处理22 d（B）的野生型（WT）与转基因烟草 Transgenic lines:转基因烟草;WT:野生型烟草

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

图14 正常生长条件下野生型和转基因植株的SOD、POD和CAT酶活以及MDA含量测定结果 3株野生型和3株转基因烟草各生理指标测定结果的均值作图进行比较;*表示与野生型相比差异显著（P<0.05,t-test）,下同

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

图15 干旱处理后野生型和转基因植株的SOD、POD和CAT酶活以及MDA含量测定结果

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

图16 正常生长和干旱处理后野生型和转基因植株电导率测定结果 Transline 1-3:野生型植株的电导率值是3株野生型测定结果的均值

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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