碱胁迫应答基因GsARHP的克隆及转基因紫花苜蓿的耐碱性分析  

Isolation of GsARHPfrom Glycine soja and its functional analysis in transgenic alfalfa under alkaline stress

作　　者：陈冉冉 朱娉慧 贾博为 宋雪薇[1] 王子君 李佶娜 李强[1] 丁晓东[1] 朱延明[1] 

机构地区：[1]东北农业大学农业生物功能基因重点实验室，黑龙江 哈尔滨 150030

出　　处：《草业学报》2017年第9期92-103,共12页Acta Prataculturae Sinica

基　　金：国家自然科学基金项目(31171578);黑龙江省高校科技创新团队建设计划项目(2011TD005);东北农业大学学科团队建设项目-团队1资助

摘　　要：本研究基于实验室前期野生大豆碱胁迫转录组数据,筛选出一个碱胁迫下上调表达的假定蛋白基因,暂命名为GsARHP(alkali stress related hypothetical protein gene)。首先利用Real-time PCR方法验证了GsARHP基因受碱胁迫诱导表达。生物信息学分析表明,该基因编码一个含有130个氨基酸的亲水蛋白,含有信号肽但无跨膜结构域;构建了GsARHP植物超量表达载体,利用农杆菌介导的子叶节侵染法转化肇东紫花苜蓿,通过PCR,Southern Blot和RT-PCR方法检测获得了3个超量表达GsARHP基因的转基因株系,并对其耐碱性进行了分析。结果表明,在0,100和150mmol/L NaHCO3处理14d后,非转基因株系明显萎蔫、黄化甚至死亡,而转基因株系则长势良好;进一步分析其生理指标显示,相对质膜透性与丙二醛含量均显著低于非转基因株系(P<0.01),而叶绿素含量与CAT活性显著高于非转基因株系(P<0.01),说明GsARHP基因的超量表达可以增强紫花苜蓿的耐碱能力。GsARHP which encodes h y p o th e t ic a l p ro te in u p - re g u la te d by a lk al in e s t r e s s , was ident ified from RNA-seq data of wild soybean （Glycine soja） a t th e early s ta g e of th e sa l t s t r e s s re sp o n se . R e a l- t im e PC R a n a -lyses showed that GsARHP was induced by alkali s tre s s . A b io in fo rm a tic s an a ly sis sh ow ed th a t th e gene en -codes a hydrophilic protein consisting of 130 amino acids, with a signal peptide but no transmembrane domain. A plant overexpression vector was constructed and transformed into alfalfa by Agrobacterium tumefaciens-me-diated cotyledonary node infection. Three transgenic lines were identified by PCR, Southern blotting, and RT -PCR analyses. The non-transgenic lines became wilted and yellow or even died under 0, 100, and 150mmol/L NaHC 03 treatment for 14 days , w h i l e t h e t r a n s g e n i c lines g r e w w e l l u n d e r t h e s a m e c o n d i t i o n s. F u r -ther analyses showed that t he t r a n s g e n i c lines ha d significantly l o w e r relative p l a s m a m e m b r a n e p e r m e a b i l i t y and malondialdehyde （MDA ） c ontent than did n o n - t r a n s g e n i c lines （P 〈0. 01） a n d significantly h i g h e r c h l o r o-phyll content and CAT activity （P 〈0. 01）. These results indic a t e d th a t o v e r e x p r e s s i o n of GsARHP c o u l d e n -hance the alkali resistance of alfalfa.

关 键 词：野生大豆 GsARHP基因 碱胁迫 紫花苜蓿 遗传转化 

分 类 号：Q943.2[生物学—植物学] S541.9[农业科学—作物学]