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

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 puntip ennis dzungarica)中克隆获得抗冻蛋白(Antifreeze protein)基因Mp AFP149.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.

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

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 transferred into cotton by Pollentube 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.