青钱柳bHLH基因家族的鉴定及盐胁迫响应分析
Genome-wide identification of bHLH transcription factors and their response to salt stress in Cyclocarya paliurus
[目的]在青钱柳全基因组范围内鉴定 bHLH 家族成员,分析基因和蛋白序列结构特点及在盐胁迫下的表达特征,筛选响应盐胁迫的 bHLH 基因,为进一步研究它们在盐胁迫响应分子途径中的功能提供参考,以期推动青钱柳耐盐性遗传育种的进展,为在东南沿海盐碱区发展青钱柳人工林提供理论依据。[方法]利用 bHLH 蛋白序列的隐马尔科夫模型(pfam00010)鉴定具有特定保守结构域的bHLH基因,对获得的青钱柳bHLH基因利用 MEGA X、ClustalX、TBtools、GSDS2.0、MEGA、MEME、CD-Search等软件进行基因结构、定位分析、蛋白理化性质、序列特征分析以及进化树构建,及蛋白互作网络分析。采用一年生青钱柳幼苗进行盐胁迫水培实验,并提取叶RNA进行转录组测序分析青钱柳bHLH基因在盐胁迫下的表达特征,筛选重点基因。随后对两年生青钱柳幼苗进行土培盐胁迫实验,采取叶片对筛选出的差异表达基因进行qPCR分析。[结果]共鉴定到 159个bHLH基因,根据其在染色体上的位置将其命名为CpbHLH1～159.159 个bHLH基因个在各染色体上均有分布,内含子数目在 1～28之间,蛋白分子质量在 10.45～175.49 kDa 之间。所有CpbHLH均含有保守的bHLH结构域。根据进化树分析将 159 个bHLH基因分为 26 个亚类,根据命名法命名为Ia～XV亚类。基于对保守基序和内含子/外显子的分析,结果显示系统发育进化树相同亚家族中的CpbHLHs在遗传和基序结构上相似,进一步证实了系统进化树分类的准确性。GO分析和启动子结构分析表明CpbHLH蛋白可能具有多种生物学功能,并鉴定出大量响应胁迫的顺式作用元件。根据转录组分析筛选 12个高度响应盐胁迫的基因。通过qRT-PCR分析及蛋白互作网络预测最终筛选出 3个响应盐胁迫的候选基因。[结论]基于青钱柳的全基因组,本研究系统地鉴定了 159个青钱柳bHLH基因,将其分为26个亚家族。除了研究它们的结构和DNA结合能力外,还进行了盐处理下盆栽和水培实验的基因表达和调控网络分析,以筛选出高度响应青钱柳盐胁迫的基因。通过盆栽实验PCR实验和蛋白质相互作用网络分析,共筛选出 12个候选基因,进一步证实其中 3个基因(CpbHLH36/68/146)参与调控青钱柳的耐盐性。我们的研究结果不仅为进一步了解bHLH转录因子的调控机制提供了基础,而且为青钱柳耐盐性的遗传改良提供理论依据。
As a highly valued and multiple function tree species,the leaves of Cyclocarya paliurus are enriched in diverse bioactive substances with healthy function.To meet the requirement for its leaf production and medical use,the land with salt stress would be a potential resource for developing C.paliurus plantations due to the limitation of land resources in China.The basic helix-loop-helix(bHLH)transcription factor protein family,the second largest protein family in plants,has been found to play essential roles in the response to multiple abiotic stresses,especially salt stress.However,the bHLH gene family in C.paliurus has not been investigated.In this study,159 CpbHLH genes were successfully identified from the whole-genome sequence data,and were classified into 26 subfamilies.Meanwhile,the 159 members were also analyzed from the aspects of protein sequences alignment,evolution,motif prediction,promoter cis-acting elements analysis and DNA binding ability.Based on transcriptome profiling under a hydroponic experiment with four salt concentrations(0%,0.15%,0.3%,and 0.45%NaCl),9 significantly up-or down-regulated genes were screened,while 3 genes associated with salt response were selected in term of the GO annotation results.Totally 12 candidate genes were selected in response to salt stress.Moreover,based on expression analysis of the 12 candidate genes sampled from a pot experiment with three salt concentrations(0%,0.2%and 0.4%NaCl),CpbHLH36/68/146 were further verified to be involved in the regulation of salt tolerance genes,which is also confirmed by protein interaction network analysis.This study was the first analysis of the transcription factor family at the genome-wide level of C.paliurus,and our findings would not only provide insight into the function of the CpbHLH gene family members involved in salt stress but also drive progress in genetic improvement for the salt tolerance of C.paliurus.
张子洁;
南京林业大学,南京,210037;
Wheel wingnut bHLH family genes salt tolerance expression analysis regulation network
2229-2244 / 16
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