盐爪爪液泡膜H^+-ATPase B亚基基因克隆及盐胁迫液泡膜相关基因的表达  

Cloning of a Vacuolar H^+-ATPase B Gene from the Halophyte Kalidium foliatum and Expression Analysis of Vacuolar Related Genes under Salt Stress

作　　者：易小娅[1] 杨瑞瑞[1] 曾幼玲[1] 

机构地区：[1]新疆大学生命科学与技术学院/新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《新疆农业科学》2015年第5期902-907,共6页Xinjiang Agricultural Sciences

基　　金：新疆大学博士启动基金(BS080123);新疆生物资源基因工程重点实验室开放课题(XJDX0201-2005-05);国家自然科学基金(31160186)

摘　　要：【目的】研究藜科盐生植物盐爪爪(Kalidium foliatum)V-H+-ATPase B亚基基因的克隆与液泡膜相关基因的表达分析。【方法】采用RT-PCR和RACE技术克隆盐爪爪V-H+-ATPase B亚基基因(VHAB),利用实时荧光定量PCR(qRT-PCR)技术分析液泡膜相关基因Kf VHA-B、Na+/H+逆向转运蛋白基因Kf NHX的表达情况。【结果】Kf VHA-B全长1 881 bp,含1 467 bp的ORF阅读框,命名为Kf VHA-B(Gen Bank登录号:EF114316),编码488个氨基酸,推测的蛋白分子量大小为54.01 k D,理论等电点为4.68,含一个保守的ATP结合位点‘323-SGSIT-327’和两个推测的多聚腺苷酸化的信号保守序列;基因编码蛋白的系统进化树分析表明,盐爪爪VHA-B与盐穗木的VHA-B聚类关系最近;qRT-PCR检测了液泡膜相关基因Kf VHA-B、Na+/H+逆向转运蛋白基因Kf NHX的表达,100、500 mmol/L Na Cl处理下,Kf VHA-B在转录水平表达量始终保持较稳定,Kf NHX基因在转录水平随盐浓度的增加其表达量呈现递增趋势,而且100 mmol/L Na Cl处理下72 h时其表达量达到最大。【结论】Kf VHA-B可能在盐爪爪应对盐胁迫中起着重要的作用。【Objective】To clone the V- H^＋- ATPase B（ VHA- B） from Kalidium foliatum and carry out expression analysis.【Method】The full- length c DNA of the V- H^＋- ATPase B（ VHA- B） from Kalidium foliatum was cloned by RT- PCR and RACE,an expression pattern of vacuolar related gene Kf VHA- B and Kf NHX were analyzed by qRT- PCR.【Result】The full- length c DNA of the V- H＋- ATPase B（ VHA- B） was of 1 881 bp,opening reading frame（ ORF） was 1 467 bp,which was named as Kf VHA- B（ GenBank： EF114316）. And the ORF was encoded 488 amino acids,suggesting a molecular weight of 54. 01 k D,theoretical isoelectric point was 4. 68,which contained a conservative ATP binding site of ＇323- SGSIT- 327＇ ＇323- SGSIT- 327＇ and two putative polyadenylation signal consensus sequences. Phylogenetic tree analysis revealed that this gene of broccoli had closer relationship with that from Halostachys caspica. The expression ofvacuolar related gene Kf VHA- B and Kf NHX by qRT- PCR suggested that Kf VHA- B was stable in transcription level under 100 mmol / L and 500 mmol / L Na Cl,Kf NHX was induced with the salt concentration increased,and reached maximum under 100 mmol / L Na Cl at 72 h.【Conclusion】Kf VHA- B may play an important role in Kalidium foliatum under salt stress.

关 键 词：盐爪爪 液泡膜H^+-ATPASE B亚基 盐胁迫 克隆 表达分析 

分 类 号：S188.2[农业科学—农业基础科学]