摘要
本研究利用RT-PCR技术从抗霜霉病哈密瓜(Cucumis melo L.)品种MR-1中克隆到AT2基因,其整编码区为1 229 bp,编码401个氨基酸,同源比对结果为99%,将其编码的丝氨酸乙醛酸转氨酶蛋白序列与多种物种进行系统进化树分析,结果显示,甜瓜与黄瓜SGT蛋白同源性较高,遗传距离最近,推测在同属中该蛋白进化相对保守。在线分析表明SGT蛋白不含信号肽及跨膜区,即该蛋白为非跨膜蛋白。RT-PCR分析表明AT2基因随着植株生长时期不同表达量不同,开花期表达量最高,幼苗期至开花期表达量呈上升趋势;且不同组织中,叶片表达量显著高于茎、根。构建了p CAMBIA2300-AT2植物表达载体,运用农杆菌介导法转化烟草,抗性筛选获得7株阳性转基因烟草;经q RT-PCR检测,AT2基因在不同株系转基因烟草T0代中表达量均高于野生型。通过对转基因烟草进行抗病性鉴定,转基因烟草对靶斑病、赤星病的抗性高于野生型烟草。本研究表明,过表达AT2基因能够提高烟草对部分真菌性病害的抗性。
In this study, AT2 gene was cloned from MR-l, a variety of Cucumis melo L. which was resistant to downy mildew using RT-PCR technology. Its complete coding region was 1229 bp, encoding 401 amino acids and the result of homologous alignment was 99%. Phylogenetic tree analysis of the serine glyoxylate aminotransferase protein sequence and a variety of species showed that the AT2 protein shared high similarity with SGT proteins from cucumber and the two had the shortest genetic distance, indicating that the evolution of this protein was relatively conservative in the same genus. The online analysis suggested that the SGT protein contained no signal peptide and transmembrane zone, which meant that the protein was non-transmembrane protein. The results of RT-PCR analysis indicated that the expression of AT2 gene varied in different growth stages of plants. It had the highest expression in flowering period, and the expression was on the rise from the seedling stage to the flowering stage. Moreover, the expression of AT2 gene in leaves was significantly higher than that in stem and root. The expression vector of pCAMBIA2300-A T2 was constructed, and it was transferred into tobacco via Agrobacterium-mediated transformation and 7 positive transgenic tobacco were obtained by resistance screening. The detection of qRT-PCR found that the expression of A T2 gene in different plants of T0 generation of transgenic tobacco was higher than that in wildtype. Through the identification of resistance of transgenic tobacco, we identified that transgenic tobacco was more resistant to target spot disease and red star disease than wildtype tobacco. This study implied that the overexpression of AT2 gene could improve the resistance of tobacco to some fungal diseases.
作者
张生翾
李思怡
王旭辉
李冠
Zhang Shengxuan;Li Siyi;Wang Xuhui;Li Guan(College of Life Science and Technology,Xinjiang University,Urumqi,830046;Academy of Agricultural Sciences Institute of Biomass Energy,Urumqi,830091)
出处
《分子植物育种》
CAS
CSCD
北大核心
2018年第15期4853-4862,共10页
Molecular Plant Breeding
基金
国家自然科学基金(31260658)
国家自然科学基金项目资助(31660297)
自治区自然科学基金项目(2016D-01C066)共同资助
