【作者】 李彤；

【导师】 郭东林；

【作者基本信息】 哈尔滨师范大学 ， 遗传学， 2023， 硕士

【摘要】 ABC转运蛋白是一类多结构域跨膜蛋白,利用ATP水解获得的能量转运底物。ABCG亚家族是植物ABC转运蛋白家族最大的亚族,ABCG亚族成员在维持植物金属稳态和激素合成转运中发挥重要作用。本研究利用分子生物学手段验证了大豆GmABCG5L基因在铁转运调控中的功能。研究结果如下:1.GmABCG5L蛋白的亚细胞定位构建p BI121-GmABCG5L-GFP表达载体,瞬时转化洋葱表皮细胞,激光共聚焦荧光显微镜观察显示,GmABCG5L蛋白定位于细胞质膜上。2.GmABCG5L基因表达特性荧光定量PCR结果表明,GmABCG5L在绥农37大豆中的表达水平从高到低为:花>荚>种子>茎>根>叶。与正常处理相比,缺铁胁迫下GmABCG5L在根、茎、叶中的表达量均显著升高,表明GmABCG5L受缺铁诱导表达。叶中GmABCG5L的表达在叶中受脱落酸诱导。3.GmABCG5L基因启动子特性将pBI121-GmABCG5Lpro::GUS植物表达载体转入拟南芥中。GUS染色表明,缺铁胁迫下,拟南芥幼苗根部、叶片中出现蓝色;ABA胁迫下,拟南芥叶中染色面积变大,表明GmABCG5L启动子响应缺铁和脱落酸胁迫。4.过表达和RNAi GmABCG5L的大豆毛状根分析筛选获得长度为350 bp的RNA干扰片段,构建了p ZH01-GmABCG5L RNAi植物表达载体。分别将p ZH01-GmABCG5L、p ROKⅡ-GmABCG5L侵染大豆,获得过表达GmABCG5L、RNA干扰GmABCG5L和对照的大豆复合体。在缺铁胁迫下,过表达GmABCG5L基因的大豆复合体叶片的黄化程度、MDA和过氧化氢含量极显著低于对照;而RNA干扰的大豆复合体显著高于对照,过表达GmABCG5L大豆复合体叶绿素含量、抗氧化酶和铁还原酶活性极显著高于对照,而RNA干扰的大豆复合体显著低于对照。结果表明GmABCG5L基因具有提高植物缺铁胁迫抵抗能力的作用。5.转GmABCG5L基因拟南芥的铁分布检测低铁胁迫下,皮尔斯染色表明,与野生型相比,过表达GmABCG5L基因拟南芥根部皮尔斯染色浅,叶中皮尔斯染色更深。结果表明过表达GmABCG5L拟南芥根中铁积累较少,在叶中积累较多的铁。预示GmABCG5L转运蛋白可能参与铁的转运。更多还原

【Abstract】 ABC transporters are a class of multi-domain transmembrane proteins that use energy obtained from ATP hydrolysis to transport substrates.ABCG subfamily is the largest subfamily of plant ABC transporters,and ABCG members play an important role in maintaining metal homeostasis and hormone synthesis and transport in plants.In this study,molecular biological methods were used to verify the function of iron transport regulation of soybean GmABCG5 L.The results are as follows:1.Subcellular localization of GmABCG5 L proteinThe expression vector p BI121-GmABCG5L-GFP was constructed,and the onion epidermal cells were transformed instantaneously.Laser confocal fluorescence microscopy obsrevation showed that GmABCG5 L protein was located on the plasma membrane of the cells.2.Gene expression analysis of GmABCG5LQuantitative PCR results showed that the expression levels of GmABCG5 L in Suinong 37 soybean were as follows: flower > pod > seed > stem > root > leaf.Compared with normal treatment,the expression levels of GmABCG5 L gene in roots,stems and leaves were significantly increased by iron deficiency,indicating that the expression of GmABCG5 L was induced by iron deficiency.The expression of GmABCG5 L in leaves was induced by abscisic acid.3.Characteristic analysis of GmABCG5 L gene promoterThe plant expression vector p BI121-GmABCG5Lpro::GUS was transferred into Arabidopsis Thaliana.The GUS staining showed that blue appeared in roots and leaves of Arabidopsis seedlings under excess iron stress,and the stained area increased in leaves under ABA stress,indicating that GmABCG5 L promoter responded to iron stress and abscisic acid stress.4.Analysis on overexpression RNAi GmABCG5 L soybean hairy rootRNA interference fragments with length of 350 bp were screened to construct p ZH01-GmABCG5 L RNAi plant expression vector.The p ZH01-GmABCG5 L RNAi and overexpressed GmABCG5 L vector p ROK Ⅱ-GmABCG5 L were transformed into Agrobacterium K599 by freeze-thawing method,and then infected soybean with needle.Soybean complexes with overexpression of GmABCG5 L,RNA interference GmABCG5 L and K599 control were obtained.Under iron deficiency,the degree of yellowing,MDA and hydrogen peroxide contents in GmABCG5 L overexpressed soybean complex were significantly lower than the control and those of GmABCG5 L RNAi soybean complex were significantly higher than the control.The chlorophyll content,antioxidant enzyme and iron reductase activities were in GmABCG5 L overexpressed soybean complex were significantly higher than the control and those of GmABCG5 L RNAi soybean complex were significantly lower than the control.The results indicated that GmABCG5 L function on improvement the plants resistance to iron deficiency.5.Iron distribution detection of transgenic GmABCG5 L plantspROKⅡ-GmABCG5 L transformed Arabidopsis thaliana plants were treated with low iron.Pierce staining showed that,the root of GmABCG5 L overexpressed Arabidopsis thaliana was lighter staining,and the leaves was deeper staining than that of the wild type.The results indicated that less iron was accumulated in the roots while more iron was accumulated in the leaves of GmABCG5 L overexpressed Arabidopsis thaliana.The results suggest that the GmABCG5 L transporter might involove in iron transport.更多还原