摘要
该研究以天山雪莲(Saussurea involucrata)的转录组数据为基础,利用Premier 5.0设计1对特异性引物SiICE2-Up和SiICE2-Down,以天山雪莲cDNA为模板克隆得到天山雪莲SiICE2基因的开放阅读框(ORF),对其进行生物信息学分析;构建植物表达载体pCAMBIA2300-35S-SiICE2-Nos,利用农杆菌介导法导入番茄(Lycopersicon esculentum),通过PCR和RT-PCR对转化植株进行验证,qRT-PCR分析转SiICE2基因番茄株系SiICE2基因的相对表达量;将转SiICE2基因型和野生型番茄在0℃处理后,进行抗寒性分析。结果表明:(1)成功克隆得到天山雪莲SiICE2基因,其大小为462bp,共编码153个氨基酸,系统进化分析发现SiICE2蛋白与菜蓟(Cynara scolymus L)亲缘关系最近。(2)成功构建了植物表达载体pCAMBIA2300-35S-SiICE2-Nos,经农杆菌介导法侵染番茄,PCR鉴定表明共有9株为转SiICE2基因番茄植株。(3)膜生理指标测定结果显示,随着低温处理时间的增加,转SiICE2基因型番茄的相对电导率、丙二醛含量均显著低于野生型,在处理时间为24h时,转SiICE2基因型番茄相对电导率比野生型低31.7%,丙二醛含量比野生型低4.2μmol/g。(4)抗氧化酶活性测定结果显示,随着低温处理时间的增加,转SiICE2基因番茄植株的POD、CAT和SOD活性均呈现持续递增趋势,野生型呈先逐渐升高后降低的趋势,且各处理时间内转SiICE2基因番茄的POD、CAT和SOD活性均显著高于野生型。研究发现,天山雪莲SiICE2基因可以显著增强非低温驯化番茄的抗寒性。
The study is based on the transcriptome data of Saussurea involucrata,using Premier 5.0 designing a pair of specific primers SiICE2-Up and SiICE2-Down,Using S.involucrata cDNA as a template and the open reading frame(ORF)of the S.involucrata SiICE2 gene was cloned and analyzed with bioinformatics.Plant expression vector pCAMBIA2300-35S-SiICE2-Nos was constructed and then introduced into tomato(Lycopersicon esculentum)by using Agrobacterium-mediated transformation.Identification of transgenic plants by PCR and RT-PCR.The relative expression level of Saussurea involucrata SiICE2 gene in transgenic tomato lines was analyzed by qRT-PCR.The cold resistance analysis was performed after the transgenic and wild-type tomatoes were treated at 0℃.The results show:(1)cloning of the S.involucrata SiICE2 gene and the opening reading frame(OFR)of the Saussurea involucrata SiICE2 gene is 462 bp which encoded a deduced protein including 153 acids residues.Phylogenetic analysis revealed that the Saussurea involucrata SiICE2 gene is closely related to Cynara scolymus L.(2)Plant expression vector pCAMBIA2300-35S-SiICE2-Nos was constructed and then introduced into tomato(S.lycoper sicum)by using Agrobacterium-mediated transformation.There are 9 strains of tomato(S.lycoper sicum)plants of transgenic Saussurea involucrata SiICE2 gene detected by PCR.(3)Membrane physiological index showed that the relative electrical conductivity and MDA content of the transgenic tomato were significantly lower than that of the wild type with the increase of the low temperature treatment time.The relative conductivity ratio of the transgenic tomato was 31.7%lower and the malondialdehyde content was 4.2μmol/g lower than that of wild type at the treatment time of 24 hours.(4)Antioxidant enzyme activity assay showed that the POD,CAT and SOD activities of the transgenic tomato plants continuously increasing with the increase of the low temperature treatment time.The activities of transgenic POD,CAT and SOD were significantly higher than those of the wild type at each treatment time,and the wild type showed a trend of gradually increasing and then decreasing.The study found that the S.involucrata SiICE2 gene of Saussurea involucrat a can significantly enhance the cold resistance of non-cold domesticated tomatoes.
作者
陈建权
张梦恬
熊意
张向前
张尧
王爱英
祝建波
CHEN Jianquan;ZHANG Mengtian;XIONG Yi;ZHANG Xiangqian;ZHANG Yao;WANG Aiying;ZHU Jianbo(Laboratory of Agricultural Biotechnology,College of Life Science of Shihezi University,Shihezi,Xinjiang 832003,China)
出处
《西北植物学报》
CAS
CSCD
北大核心
2018年第7期1194-1202,共9页
Acta Botanica Boreali-Occidentalia Sinica
基金
国家转基因生物新品种培育重大专项(2016ZX08005-004)
