甜瓜CmLEA基因家族鉴定及抗非生物胁迫基因挖掘

Identification of CmLEA gene family and mining of abiotic stress resistant genes in melon

  • 摘要: 【目的】 鉴定甜瓜胚胎发育晚期丰富蛋白(LEA)基因家族成员并挖掘抗非生物胁迫基因,为探究CmLEA基因家族在甜瓜非生物胁迫中的作用机制及培育优良抗性甜瓜品种提供理论参考。【方法】 以耐冷型甜瓜L5283幼苗为试验材料,在幼苗3叶1心期进行盐胁迫300 mmol/L氯化钠(NaCl)处理、干旱15%聚乙二醇(PEG)6000处理、低温(昼夜15℃/6℃,12 h/12 h)处理和脱落酸(ABA)(浓度为100μmol/L)处理。基于甜瓜低温处理后的转录组测序数据挖掘CmLEA候选基因,采用生物信息学方法对CmLEA基因家族成员的染色体位置、基因结构、亚细胞定位、蛋白二级结构、蛋白三级结构、系统进化关系、蛋白互作及启动子顺式作用元件等进行预测,并通过实时荧光定量PCR检测CmLEA家族基因在不同非生物胁迫下的表达模式。【结果】 共鉴定到5个CmLEA候选基因,命名为CmLEA1CmLEA5;5个CmLEA家族基因不均匀分布在甜瓜4条染色体上,5个CmLEA家族基因编码158~315个氨基酸残基;理论等电点(pI)为4.47~9.70,分子量为16.90~32.71 kD,除CmLEA2为疏水性蛋白外,其他均为亲水性蛋白。5个CmLEA家族蛋白的结构以无规卷曲占比较高,延伸链和α-螺旋次之,β-折叠占比最低。CmLEA家族蛋白成员间保守基序差异较大,CmLEA1CmLEA2基因均有1个外显子,无内含子;CmLEA3CmLEA5基因分别含有4和2个外显子。系统发育进化分析结果显示,CmLEA基因家族成员与黄瓜亲缘关系较近,相似性最高可达100%。CmLEA家族基因的启动子顺式作用元件被分为6类,包括光响应元件、激素响应元件、厌氧诱导元件、低温响应元件、防御与胁迫响应元件及MYB结合元件。CmLEA5蛋白在蛋白互作网络中较为关键,推测其参与雄蕊的发育过程。实时荧光定量PCR检测结果显示,CmLEA1CmLEA2均受低温和ABA诱导相对表达量显著增加(P<0.05,下同);当甜瓜幼苗受到干旱胁迫和盐胁迫时,与处理0 h相比,CmLEA5基因相对表达量均在处理12 h时达到峰值,分别显著增加214倍和5212倍。【结论】 鉴定到5个甜瓜CmLEA基因家族成员,成员间结构和功能多样。筛选到对非生物胁迫响应强烈的3个基因,分别是CmLEA1CmLEA2CmLEA5,可作为后续研究甜瓜对非生物胁迫响应的分子机制及甜瓜抗性品种选育的候选基因。

     

    Abstract: 【Objective】 To identify and analyze members of the late embryogenesis abundant(LEA) protein gene family in melon,mine the abiotic stress resistant genes,which could provide theoretical foundation for investigating the role of CmLEA gene family in abiotic stress responses and breeding of stress resistant melon varieties.MethodUsing cold tolerant melon L5283 seedlings as experimental materials.At the 3-leaf-and-1-bud stage,4 abiotic stress treatments were applied:salt stress 300 mmol/L sodium chloride(NaCl),drought 15% polyethylene glycol(PEG) 6000,low temperature(15 ℃/6℃ day/night,12 h/12 h),and abscisic acid(ABA)(100 μmol/L).Based on transcriptomic data from melon seedlings treated by low temperature,CmLEA candidate genes were identified.Chromosomal locations,gene structures,subcellular localization,secondary and tertiary protein structures,phylogenetic relationships,protein interactions,and promoter cis-acting elements were predicted by bioinformatics methods.Real-time fluorescence quantitative PCR was used to detect the expression patterns of CmLEA family genes under different abiotic stresses.Result A total of 5CmLEA candidate genes were identified and named CmLEA1-CmLEA5.The 5 CmLEA family genes were unevenly distributed across 4 chromosomes of melon,they encoded 158-315 amino acids,with Theoretical isoelectric point(pI) of4.47-9.70 and molecular weight of 16.90-32.71 kD.Except CmLEA2 were hydrophobic protein,the others were hydrophilic proteins.Among the 5 CmLEA family proteins,random curl accounted for the highest proportion,followed by extended chain and α-helix,and β-fold accounted for the lowest proportion.The conserved motifs of CmLEA family proteins differed greatly.CmLEA1 and CmLEA2 genes both had 1 exon and no intron;CmLEA3 and CmLEA5 genes contained 4 residues and 2 exons respectively.Phylogenetic analysis showed that CmLEA gene family members were closely related to cucumber,and the similarity was up to 100%.The cis-acting elements of CmLEA family genes were classified into 6 categories,including light response elements,hormone response elements,anaerobic induction elements,low temperature response elements,defense and stress response elements,and MYB binding elements.CmLEA5 protein played a key role in the protein-interaction network and was presumed to be involved in the development of stamens.Real-time fluorescence quantitative PCR showed that the relative expression of CmLEA1 and CmLEA2 were significantly upregulated by low temperature and ABA induction(P<0.05,the same below).When melon seedlings were subjected to drought stress and salt stress,the relative expression of CmLEA5 gene reached its peak value at 12 h compared with 0 h,and significantly increased by 214 and 5212 times respectively.Conclusion Five CmLEA gene family members were identified,and their structure and function are diverse.Three genes,which are highly responsive to abiotic stress,are identified:CmLEA1,CmLEA2 and CmLEA5,and they can be used as candidate genes for subsequent studies on the molecular mechanism of response to abiotic stress and selection of melon resistant varieties.

     

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