摘要: |
【目的】探究红光熊蜂低温胁迫不同时间、不同温度下GS、Akt2基因的表达差异,为研究红光熊蜂抵御低温胁迫的分子机制提供参考。【方法】以RT-PCR结合RACE技术,克隆、测序红光熊蜂GS、Akt2基因,通过ORF Finder、Prot Param和TBtools等生物信息学软件预测红光熊蜂GS和Akt2蛋白的结构、理化特性以及染色体定位等信息;设计GS、Akt2基因特异性引物,采用实时荧光定量PCR技术检测不同温度、不同处理时间下GS、Akt2基因相对表达量的差异。【结果】红光熊蜂GS基因全长1 417 bp,开放阅读框(ORF)1 020 bp,编码339个氨基酸;GS蛋白二级结构包括无规则卷曲(51.92%)、α-螺旋(23.89%)、延伸链(12.09%)和β-转角(12.09%),具有12个Ser、6个Thr、2个Tyr,可能为GS蛋白激酶磷酸化位点。Akt2基因全长926 bp,ORF为837 bp,编码278个氨基酸;Akt2蛋白二级结构包括α-螺旋(34.89%)、无规则卷曲(33.45%)、延伸链(23.74%)和β-转角(7.91%),具有7个Ser、8个Thr、5个Tyr,可能成为Akt2蛋白激酶磷酸化位点。亚细胞定位GS、Akt2蛋白均存在于线粒体中,为亲水性蛋白,且无信号肽序列,故均为非分泌蛋白。GS、Akt2基因分别位于红光熊蜂1H和3H号染色体上。5~25 ℃时,红光熊蜂GS基因相对表达量随温度降低而显著下调,而Akt2基因相对表达量则随温度降低呈明显上升趋势,且15 ℃时其相对表达量显著升高,5 ℃达到峰值。8 ℃低温胁迫不同时间,随胁迫时间延长,GS基因相对表达量显著下降;而Akt2基因的相对表达量显著升高,且胁迫12 h达到峰值,随后其相对表达量随胁迫时间延长而缓慢恢复。【结论】红光熊蜂GS、Akt2基因可能通过调控PI3K/Akt或Mtor/FoxO通路以抵御低温胁迫。 |
关键词: 红光熊蜂 基因克隆 低温胁迫 基因表达 |
DOI: |
分类号: |
基金项目:中央财政专项(851800501);辽宁省科学事业公益研究项目(2019N0021) |
|
Cloning and expression of GS and Akt2 gene in Bombus ignites Smith under low temperature stress |
WANG Dandan,WANG Xing,ZHOU Qiaochu
|
Abstract: |
【Objective】This study explored the differences in expression of GS and Akt2 genes in Bombus ignites Smith under low temperature stress at different processing times and temperatures to provide reference for molecular mechanism of bumblebees against low temperature.【Method】The RT-PCR combined RACE technology was used to clone GS and Akt2 genes from Bombus ignites,and bioinformatics software of ORF Finder,Prot Param and TBtools were used to predict structure,physical and chemical properties and chromosome localization of GS and Akt2 proteins and design specific primers for GS and Akt2 genes.The quantitative Real-time PCR technology was used to detect relative expression differences of GS and Akt2 genes at different processing times and temperatures.【Result】The full-length of GS gene was 1 417 bp with ORF of 1 020 bp and it encoded 339 amino acids.The secondary structure of GS protein included random coil (51.92%),α-helix (23.89%),extended strand (12.09%) and β-turn (12.09%).There were 12 Ser,6 Thr and 2 Tyr as possible phosphorylation sites of GS protein kinase.The full-length of Akt2 gene was 926 bp with ORF of 837 bp and it encodes 278 amino acids.The secondary structure of Akt2 protein included α-helix (34.89%),random coil (33.45%),extended strand (23.74%) and β-turn (7.91%).There were 7 Ser,8 Thr and 5 Tyr as possible phosphorylation sites of Akt2 protein kinase.Both proteins existed in mitochondria as hydrophilic proteins and they had no signal peptide sequence as non-secreting proteins.GS and Akt2 genes distributed on chromosome 1H and chromosome 3H,separately.At 5-25 ℃,the relative expression level of GS decreased significantly with the decrease of temperature,but that of Akt2 increased significantly with the decrease of temperature with significantly increase expression at 15 ℃ and peak value at 5 ℃.At 8 ℃,the relative expression level of GS decreased significantly with increasing time of stress,but Akt2 increased significantly with peak value at 12 h.【Conclusion】GS and Akt2 genes in Bombus ignites Smith may resist low temperature stress through regulating PI3K/Akt or Mtor/FoxO pathways. |
Key words: Bombus ignites gene cloning low temperature stress gene expression |