橡胶草bZIP基因家族鉴定及胶乳高表达基因功能分析

Identification of bZIP gene family of Taraxacum kok-saghyz Rodin and functional analysis of highly expressed genes in latex

  • 摘要: 【目的】 探究橡胶草bZIP基因家族在天然橡胶生物合成中的调控功能,为解析bZIP转录因子在天然橡胶生物合成中的调控机制打下基础。【方法】 采用生物信息学方法从橡胶草全基因组中鉴定出bZIP基因家族成员,对其基因结构及编码蛋白保守基序、保守结构域进行预测分析,基于转录组测序数据筛选出在胶乳组织中较高表达且具有组织表达特异性的候选基因,PCR克隆候选基因的编码区(CDS)序列,并对其编码蛋白进行亚细胞定位、转录自激活活性和蛋白互作分析。将构建的基因敲除载体pCAMBIA1300-pYAO-cas9-TkbZIPs和超表达载体pCAMBIA1300-35S-TkbZIPs-GFP进行遗传转化,筛选获得TkbZIPs基因敲除株系和超表达株系。【结果】 从橡胶草中共鉴定到53个bZIP基因家族成员,根据不同组织的转录组数据筛选出4个候选基因TkbZIP1TkbZIP2TkbZIP3TkbZIP4,其CDS序列长度为468、564、465和1092 bp,分别编码155、187、154和363个氨基酸残基。通过实时荧光定量PCR验证发现,仅TkbZIP3TkbZIP4基因在胶乳中表达量较高,其中TkbZIP4基因在胶乳中具有特异性表达,而TkbZIP1TkbZIP2在胶乳中的相对表达量较低,无明显的组织表达特异性。亚细胞定位结果显示,4个TkbZIPs蛋白均定位在细胞核。酵母转录自激活试验结果显示,TkbZIP1、TkbZIP2和TkbZIP3具有转录自激活活性,而TkbZIP4无转录自激活活性。双分子荧光互补(BiFC)试验结果显示,TkbZIP1、TkbZIP2、TkbZIP3和TkbZIP4蛋白之间相互作用形成异源二聚体,且4个TkbZIPs蛋白自身也能相互作用形成同源二聚体。通过遗传转化获得TkbZIP2TkbZIP3基因超表达株系和TkbZIP1、TkbZIP2TkbZIP3基因敲除株系。【结论】 TkbZIP1、TkbZIP2、TkbZIP3TkbZIP4基因在胶乳中高表达,可能在天然橡胶生物合成中发挥重要的调控功能。4个胶乳中高表达的TkbZIPs蛋白之间能形成同源二聚体或异源二聚体,推测bZIP蛋白以二聚体形式调控下游基因表达。成功获得了TkbZIP2TkbZIP3基因超表达材料和TkbZIP1、TkbZIP2TkbZIP3基因敲除材料,可用于后续bZIP基因家族调控天然橡胶生物合成研究。

     

    Abstract: 【Objective】 This study aimed to explore the role of bZIP gene family of Taraxacum kok-saghyz Rodin in regulating natural rubber biosynthesis, so as to provide foundations for elucidating the regulation mechanism of natural rubber biosynthesis of bZIP transcription factor. 【Method】 Members of the bZIP gene family were identified from the whole genome of Taraxacum kok-saghyz Rodin using bioinformatical methods to predict and analyze their gene structures, conserved motifs, and domains of encoded protein. Based on the data of transcriptome sequencing, candidate genes in latex tissues with high expression and tissue-specific expression were identified. The coding sequences (CDSs) of candidate genes was cloned using PCR, and subcellular localization, transcriptional self-activation activity, and protein interaction analyses were performed. Genetic transformations of the established gene knockout vector pCAMBIA1300-pYAO-cas9-TkbZIPs and overexpressed vector pCAMBIA1300-35S-TkbZIPs-GFP were conducted to identified TkbZIPs gene knockout lines and overexpressed lines. 【Result】 A total of 53 bZIP gene family members were identify from Taraxacum kok-saghyz Rodin. Based on transcriptome data of different tissues, four target genes of TkbZIP1TkbZIP2TkbZIP3, and TkbZIP4 were identified, and their CDS lengths were 468, 564, 465, and 1092 bp, encoding 155, 187, 154, and 363 amino acid residues, respectively. Based on real-time fluorescence quantitative PCR, TkbZIP3 and TkbZIP4 genes had high expression in latex, with TkbZIP4 being specifically expressed in latex, and TkbZIP1 and TkbZIP2 genes showed lower expression in latex without obvious tissue-specific expression. Subcellular localization analysis showed four TkbZIPs proteins localized in the nucleus. Yeast transcriptional self-activation assays indicated that TkbZIP1, TkbZIP2, and TkbZIP3 possessed transcriptional self-activation activity, while TkbZIP4 did not have possessed transcriptional self-activation activity. Bimolecular fluorescence complementation (BiFC) assays demonstrated that TkbZIP1, TkbZIP2, TkbZIP3, and TkbZIP4 proteins could form heterodimers through interaction, and the four TkbZIPs proteins could form homodimers through self-interaction. Genetic transformation yielded TkbZIP2 and TkbZIP3 overexpressed lines and TkbZIP1TkbZIP2, and TkbZIP3 knockout lines. 【Conclusion】 TkbZIP1TkbZIP2TkbZIP3, and TkbZIP4 genes exhibit high expressions in latex, suggesting their pivotal regulatory roles in natural rubber biosynthesis. The four TkbZIPs proteins with high expressions in latex could form both homodimers or heterodimers, indicating that bZIP proteins may regulate downstream gene expression in dimeric forms. Gene overexpression materials from TkbZIP2 and TkbZIP3, as well as knockout materials from TkbZIP1TkbZIP2, and TkbZIP3, were successfully obtained for further investigation of the regulatory role of bZIP gene family in natural rubber biosynthesis.

     

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