摘要
为了验证铝耐受型丹波黑大豆根尖14-3-3a基因（soybean 14-3-3a,SGF14a）在植物应答铝胁迫中的作用,本研究利用35S组成型启动子和大豆SGF14a的编码区构建植物表达载体pK-35S-SGF14a,在野生型（wild type,WT）烟草中过量表达SGF14a获得3个转基因株系（S11、S19和S23）;并用50μmol/L铝处理WT和转基因烟草,分析过量表达SGF14a对烟草铝耐受性的影响.结果表明：过量表达SGF14a的转基因烟草经铝胁迫处理后根的相对生长量比WT高约30%-40%;此外,在没有铝胁迫的正常生长条件下,3株转基因烟草根中可溶性蛋白的含量都比WT高,而用50μmol/L铝胁迫24h后WT烟草和3株转基因烟草根中可溶性蛋白的含量都下降,但转基因烟草根中可溶性蛋白含量仍显著高于WT,并且过量表达SGF14a还可显著提高过氧化物酶、过氧化氢酶以及抗坏血酸过氧化物酶的活性,降低铝胁迫下烟草根中过氧化氢（H2O2）的积累以及氧化胁迫的水平;同时,转基因烟草在低酸土壤中的生长状况也明显优于WT.这说明过量表达SGF14a可增强烟草对铝胁迫的耐受性及其适应酸性土壤生长的能力.
Summary Approximately 30% of the global arable land in the world is acidic soils（pH5）and Chinas acidic soil accounts for more than 21% of the area.Aluminum（Al）toxicity is a major limiting factor which limited crop production in acid soils.Al firstly inhibits the growth and development of plant roots.Consequently,it decreased the absorption of water and nutrients,which results in poor growth and production of plants.Therefore,in recent years,many researchers dedicated to study Al tolerance mechanisms and bred high Al-tolerant and acid soil-resistant crops by genetic engineering.14-3-3proteins are a group of highly conserved regulatory proteins found in eukaryotic cells,and have roles in regulating plant development and stress responses.A lot of 14-3-3gene family members are isolated in different plants.14-3-3proteins regulate various physiological activities and functions by interacting withphosphorylated or non-phosphorylated target proteins in plants.The expression levels of certain 14-3-3 gene isoforms can be adjusted directly by environmental stimuli. Our previous study showed that Al stress induced the expression of 14-3-3a（SGF14a）in the Al resistant soybean（Glycine max）root tips.In order to further validate the role of SGF14 ain response to Al stress in plants,plant expression vectors of SGF14 a were constructed.Now,the 35 Sconstitutive promoter was used to overexpress SGF14 a.We constructed pK-35S-SGF14 aplant expression vectors using gateway technology,then introduced the SGF14 a expression vectors into Agrobacterium pMP105, which was transformed into tobacco plants via transformation method. The plants were selected by genomic polymerase chain reaction（PCR）,reverse transcriptase-PCR and Western blot analysis.Finally we got three transgenic lines（S11,S19,and S23）.The wild type（WT）and transgenic tobacco plants（S11,S19,S23）were treated with 50μmol/L Al to analyze the effect of SGF14 a overexpression on the Al-resistance of tobacco. Changes of the relative root growth, H2O2,malondialdehyde（MDA）,soluble protein contents as well as antioxidant enzyme activities in transgenic plants were compared. The results showed that the relative root growth of the transgenic plants increased approximately 1.5-fold as compared with the WT. Moreover,the soluble protein contents in the transgenic tobacco roots increased significantly compared with the WT plants,and antioxidant enzyme activities（peroxidase,catalase,and ascorbate peroxidase）in roots of transgenic tobacco plants also increased when exposed to 50μmol/L Al.Moreover,the H2O2 accumulation and oxidative stress level in transgenic tobacco roots were reduced under the Al stress.The growth status of the transgenic tobacco was better than that of WT when grown in acidic soil.In summary,the evidences suggest that overexpression of SGF14 aan enhance the Al-tolerance of transgenic tobacco and its ability to adapt to acidic soil.
出处
《浙江大学学报（农业与生命科学版）》
CAS
CSCD
北大核心
2015年第3期285-292,共8页
Journal of Zhejiang University:Agriculture and Life Sciences
基金
国家自然科学基金(31260063
31260297)
关键词
14-3-3蛋白
过量表达
转基因烟草
铝胁迫
耐铝性
14-3-3 protein
overexpression
transgenic tobacco
aluminum stress
aluminum tolerance
作者简介
第一作者联系方式：杨志丽，E—mail：yangzhili668@126．com
通信作者：陈丽梅，E—mail：chenlimeikm@126．com
