异源四倍体油菜HAKs家族核心基因的鉴定及其功能初步解析

Identification of the core member of the HAKs family and primary analysis on their functions in allotetraploid rapeseed(Brassica napus L.)

[目的]HAKs家族基因编码高亲和力K^(+)转运蛋白,在K^(+)吸收和运输过程中发挥关键作用。鉴定甘蓝型油菜HAKs家族核心基因,研究不同养分胁迫下核心基因表达的响应,不仅可加深对HAKs功能的理解,也可为HAKs介导油菜营养胁迫抗性的遗传改良提供基因资源参考。[方法]对甘蓝型油菜HAKs基因家族进行全基因组鉴定和分析,其中包括系统发育关系、基因结构、保守基序、染色体定位、顺式作用元件。利用不同养分胁迫下的转录组数据对HAKs家族基因进行差异基因表达分析,并用Cytoscape构建共表达网络图。以低钾抗性品系“H280”和低钾敏感品系“L49”为材料,在低钾胁迫下进行水培试验,使用ICP-MS分析了“H280”和“L49”根部K+含量,RT-qPCR以及亚细胞定位分析了低钾胁迫下BnaA7.HAK5的响应和功能。[结果]在甘蓝型油菜中共鉴定到40个HAKs家族成员。对其进行进化关系分析,发现它们的Ka/Ks值均小于1.0。顺式作用元件分析表明,MYB和激素响应顺式作用调控元件ABRE在HAKs家族基因的启动子区域高度富集。共线性分析表明在进化过程中,甘蓝型油菜中绝大多数的HAKs基因保存完整。差异表达基因分析发现,大部分HAKs家族基因的表达水平受低钾胁迫的显著诱导。共表达网络分析表明,BnaA7.HAK5响应油菜低钾胁迫。RT-qPCR结果显示,BnaA7.HAK5在抗低钾品系“H280”根中的表达量显著高于低钾敏感品系“L49”。亚细胞定位结果显示,BnaA7.HAK5定位在细胞质膜上。[结论]甘蓝型油菜共有40个BnaHAKs基因,BnaHAKs基因响应低钾或盐等养分胁迫。定位在细胞质膜上的BnaA7.HAK5在低钾条件下的表达量显著上调,且在“H280”和“L49”油菜品系根部的表达量差异显著,表明BnaA7.HAK5在油菜高亲和性钾离子的吸收和转运中发挥重要功能。

[Objectives]The HAKs(high-affinity K transporter)family genes encode high-affinity potassium(K^(+))transporters,and play key roles in the absorption and transport of K^(+).Identification of the core genes of the HAKs family in rapeseed,and the study of their expression responses when exposure to different nutrient stresses,will help fully understanding of the functions of HAKs,and also provide valuable genetic resource references for the genetic improvement of HAKs-mediated nutrient resistance in rapeseed.[Methods]The genome-wide identification and analysis of the rapeseed HAKs gene family were made from aspects of phylogenetic relationships,gene structure,conserved motifs,chromosomal localization,cis-acting elements.Differential expression of HAKs family genes were analyzed using transcriptomic data under different nutrient stresses,and the co-expression network was constructed by Cytoscape.Low-K-resistant line“H280”and low-K-sensitive line“L49”were subjected to K stress in a hydroponic experiment,the K+contents in the roots of line“H280”and“L49”were analyzed using ICP-MS.The response and function of BnaA7.HAK5 under low-K+stress were analyzed by RT-qPCR and subcellular localization.[Results]A total of 40 HAKs family members were identified through genome-wide identification.Evolutionary relationship analysis of the HAKs family genes showed that their Ka/Ks values were all less than 1.0.The MYB and the hormone response cis-acting regulatory element ABRE were highly enriched in the promoter region of the HAKs family genes.Collinear analysis showed that most of the HAKs genes in Brassica napus were intact during the evolutionary process.Differential expression gene analysis revealed that the expression levels of most HAKs family genes were significantly induced by low potassium stress.The co-expression network analysis showed that BnaA7.HAK5 responded to low potassium stress in oilseed rape.The RT-qPCR results showed that the expression level of BnaA7.HAK5 in the roots of line“H280”was significantly higher than that of line“L49”.The results of subcellular localization showed that BnaA7.HAK5 was localized on the plasma membrane of cells.[Conclusions]There are 40 BnaHAKs genes in rapeseed,and the BnaHAKs genes respond to nutrient stresses such as low potassium or salt.BnaA7.HAK5,localized in the plasma membrane,is significantly up-regulated under low-potassium conditions,and showed differential expressions in the roots between“H280”and“L49”oilseed rape lines.It could be concluded that BnaA7.HAK5 plays an important role in the high-affinity uptake and transport of potassium ions in rapeseed.