凌云鹤,李静,景兵,李春莲,肖恩时,王中华.向日葵3个谷胱甘肽-S-转移酶GST基因的克隆及表达模式分析[J].干旱地区农业研究,2019,37(5):92~98
向日葵3个谷胱甘肽-S-转移酶GST基因的克隆及表达模式分析
Cloning and expression analysis of three glutathione S-transferase GST genes from sunflower (Helianthus annuus)
  
DOI:10.7606/j.issn.1000-7601.2019.05.14
中文关键词:  向日葵  谷胱甘肽-S-转移酶GST基因  基因克隆  基因相对表达量
英文关键词:Helianthus annuus  glutathione S-transferase GST gene  gene clone  relative gene expression
基金项目:国家特色油料产业技术体系杨凌综合试验站项目(CARS-14-2-21);国家自然科学基金青年科学基金项目 (31601335);中央高校基本科研业务费(2452016012)
作者单位
凌云鹤 College of Agriculture,Northwest Agriculture and Forestry University,Yangling,Shanxi 71210, China 
李静 College of Agriculture,Northwest Agriculture and Forestry University,Yangling,Shanxi 71210, China 
景兵 College of Agriculture,Northwest Agriculture and Forestry University,Yangling,Shanxi 71210, China 
李春莲 College of Agriculture,Northwest Agriculture and Forestry University,Yangling,Shanxi 71210, China 
肖恩时 College of Agriculture,Northwest Agriculture and Forestry University,Yangling,Shanxi 71210, China 
王中华 College of Agriculture,Northwest Agriculture and Forestry University,Yangling,Shanxi 71210, China 
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中文摘要:
      从向日葵转录组测序结果中获得了3个对盐胁迫有响应的谷胱甘肽-S-转移酶(GST,EC2.5.1.18)GST基因,构建了系统进化树并进行分析,得知这3个基因属于Tau型GST,并将它们命名为HaGSTU26(HanXRQChr04g0127901)、HaGSTU8(HanXRQChr06g0177581)、HaGSTU27(HanXRQChr10g0316331),然后以向日葵Sk02R为试验材料,克隆这3个基因,并进行了不同组织和不同胁迫条件下的表达分析。序列分析表明:HaGSTU26基因组为1674bp,CDS(编码蛋白序列)长666bp,编码221个氨基酸,由2个外显子和1个内含子组成;HaGSTU8基因组为2271bp,CDS长654bp,编码217个氨基酸,由2个外显子和1个内含子组成; HaGSTU27基因组为663bp,CDS长663bp,编码220个氨基酸,由1个外显子组成。实时荧光定量PCR分析表明:向日葵HaGSTU26HaGSTU8HaGSTU27基因在不同组织(根、幼叶、成熟叶、茎、幼茎、苞叶)中表达量不同,其中,HaGSTU26基因和HaGSTU27基因在根中表达量最高,而HaGSTU8基因在苞叶中表达量最高,但这3个基因均在成熟茎中的表达量最低。在不同胁迫条件下,测定这3个基因在向日葵幼苗中的表达量,结果表明在盐及ABA胁迫下,基因表达量均随着处理时间的增加而呈现先增加后下降的趋势。其中,在盐胁迫下,HaGSTU26基因在12 h后相对表达量最高,HaGSTU8HaGSTU27基因表达量在3h后相对表达量最高。在ABA胁迫后,HaGSTU26HaGSTU27基因表达量在12 h后相对表达量最高,HaGSTU8在24 h后相对表达量最高。在冷胁迫下,HaGSTU26HaGSTU27基因上调表达,它们分别在3、24 h后相对表达量最高,HaGSTU8基因下调表达,其相对表达量随处理时间的延长呈现逐渐减少的趋势。在热胁迫条件下,这3个基因的相对表达量随着胁迫时间的延长而增加,均在24 h后表达量最高。以上结果说明这3个基因对不同非生物胁迫(盐、ABA、冷、热胁迫)均有响应。
英文摘要:
      The plant glutathione-S-transferase (GST, EC2.5.1.18) is a group of enzymes with multiple biological functions encoded by complex gene families that has great responses to various abiotic stresses. In this study, three GST genes responding to salt stress were obtained from sunflower transcriptome sequencing results and a phylogenetic tree were constructed for analysis. These three genes belong to Tau GST and were named as HaGSTU26 (HanXRQChr04g0127901), HaGSTU8 (HanXRQChr06g0177581) and HaGSTU27(HanXRQChr10g0316331). Next, we cloned these three genes from sunflower and performed expression analysis under different tissues and different stress conditions. Sequence analysis showed that DNA sequence of HaGSTU26 gene was 1674bp and the CDS was 666 bp, which encoded 221 amino acids and consisted of 2 exons and 1 intron. The DNA sequence of HaGSTU8 gene was 2271 bp and the CDS was 654 bp, which encoded 217 amino acids and consisted of 2 exons and 1 intron. The DNA sequence of HaGSTU27 gene and CDS were both 663bp, encoding 220 amino acids and consisting of one exon. Real-time fluorescence quantitative PCR analysis showed that the expression levels of HaGSTU26, HaGSTU8 and HaGSTU27 sunflower were different in different tissues (root, young leaf, mature leaf, stem, young stem and temporal leaf), the HaGSTU26 gene and HaGSTU27 gene had the highest expression in roots, while HaGSTU8 gene had the highest expression in the temporal leaf. However, all three genes have the lowest expression in mature stems. Under different stress conditions, the expression levels of these three genes in sunflower seedlings were determined. The results showed that under salt and ABA stress, the amount of gene expression increased first and then decreased with increasing treatment time. Under salt stress, the relative expression of HaGSTU26 gene was at peak after 12h, and the expression of HaGSTU8 and HaGSTU27 were at the highest after 3 h. After ABA stress, the expression levels of HaGSTU26 and HaGSTU27 were the highest after 12 h, and the relative expression of HaGSTU8 was the highest after 24 h. Under cold stress, HaGSTU26 and HaGSTU27 genes were up-regulated, and they had the highest relative expression after 3 h and 24 h, respectively. The expression of HaGSTU8 gene was down-regulated, and its relative expression decreased gradually with the prolongation of treatment time. Under heat stress, the relative expression of three genes increased with the prolongation of stress time, and the highest expression was obtained after 24 h. The above results indicate that these three genes respond to different abiotic stresses (salt, ABA, cold and heat stress).
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