橡胶草TkDREB2基因的克隆以及在烟草中的抗旱功能分析

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

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1002 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。

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 1002 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.