花粉管介导的准噶尔小胸鳖甲抗冻蛋白转基因棉花的筛选  

Screening of Transgenic Cotton with an Insect(Microdera puntipennis dzungarica) Antifreezing Protein Gene via Pollen-tube Pathway

作　　者：马纪[1] 李春平[2] 邱立明[1] 徐建辉[2] 赵干[1] 李素丽[1] 

机构地区：[1]新疆大学生命科学与技术学院／新疆生物资源基因工程重点实验室,乌鲁木齐830046 [2]新疆农科院经作所,乌鲁木齐830091

出　　处：《新疆农业科学》2008年第3期381-385,共5页Xinjiang Agricultural Sciences

基　　金：国家教育部“春晖”计划项目(Z2004-2-65040);新疆生物资源基因工程重点实验室开放课题(XJDX0201-200503)

摘　　要：利用新疆荒漠昆虫抗冻蛋白基因进行遗传转化是培育新疆棉花抗寒品种的新途径。研究采用RT-PCR结合RACE技术从新疆荒漠昆虫准噶尔小胸鳖甲(Microdera puntipennis dzungarica)中克隆获得抗冻蛋白(Antifreeze protein)基因MpAFP149。将MpAFP149克隆至pBI121上,获得重组质粒后采用电转化法将重组质粒转入农杆菌EHA105内,PCR筛选鉴定重组子,并测序确定抗冻蛋白植物表达载体构建正确。将pBI121-MpAFP通过田间的花粉管导入技术转化棉花,收取转化的棉花种子,经检测证明在转化10 000朵花中,卡那霉素抗性筛选出13株,PCR检测有2株阳性植株。实验结果为进一步开展抗冻蛋白基因转化棉花的研究奠定了基础。It is a new way to breed cold tolerant cotton by gene transformation with antifreezing protein gene （AFP） from desert insect. An insect antifreezing gene MpAFP149 from Microdera puntipennis dzungarica was cloned with RT- PCR combined with 3＇ and 5＇ - RACE techniques. MpAFP149 gene was cloned on plant expressive vector pBI121 to form the recombinant plasmid pBI121 - MpAFP for the plant genetic transformation. The recombinant plasmid was transformed in to Agrobacterium tumefaciens （EHA105） by electroporation, and the positive clones were screened by PCR and sequencing. Then pBI121 - MpAFP was wansferred into cotton by Pollen - tube Pathway method, and positive transgenic cotton was screened and determined first by kanamycin resistant followed PCR analysis. 13 cottons out of ten thousand transformed plants displayed kanamycin resistant, and two out of the 13 were PCR positive. The test results laid the foundation of further developing study of transgenic cotton of antifeezing protein gene.

关 键 词：准噶尔小胸鳖甲 抗冻蛋白 基因转化 花粉管导入 棉花 

分 类 号：S562[农业科学—作物学] S188