转天麻抗真菌蛋白基因提高棉花对黄萎病抗性

Transgenic Upland Cotton Lines of Gastrodia Antifungal Protein Gene and Their Performance of Resistance to Verticillium Wilt

黄萎病是全球植棉业的主要病害,严重缺乏抗黄萎病资源导致棉花抗黄萎病育种至今未取得显著进展。采用农杆菌介导法,以下胚轴为受体,将天麻抗真菌蛋白基因转化陆地棉品系苏研060,通过愈伤组织诱导、胚性愈伤组织分化和植株再生,获得大量再生苗。分别以NPT II基因、gafp基因、35S启动子-gafp基因特异引物对再生苗进行PCR检测,将95个阳性再生苗嫁接到海7124幼苗砧木上,获得37个成活植株。RT-PCR检测发现,31个植株出现预期的540 bp扩增片段。盆栽花铃期花粉育性鉴定表明,9个转基因植株雄性败育,22个植株花粉育性正常,通过人工自交得到T1代种子。通过转基因作物隔离区种植、特异引物PCR检测、卡那霉素抗性筛选、自交纯合与选择,培育获得22个T3代转基因纯系。Southern blotting检测发现,转基因植株体内有2个gafp基因拷贝。GAFP蛋白免疫印迹表明,16个转基因纯系出现分子量为14 k D的GAFP蛋白,另6个纯系表现出转录后水平上的基因沉默。黄萎病抗性鉴定结果证实,获得3个抗落叶型黄萎病的棉花株系TG09、TG16和TG23。

Verticillium wilt is a major disease in worldwide cotton production. Up to now there are less progresses achieved by using conventional cotton breeding for controlling Verticillium wilt of upland cotton owing to lack of broad-spectrum resistant germplasm. The most effective approach of controlling cotton Verticillium wilt could be to create new strains with broad-spectrum resistance and then develop cultivars with improved horizontal resistance through transgenic manipulation. Transferring the gene of gastrodia antifungal protein （gafp） into upland cotton variety Suyan 060 was carried out by agrobacterium-mediated genetic transformation. Many regenerated plantlets were gained through embryogenic callus induction and plant regeneration by using hypocotyls as explants. PCR detection of plantlets using special primers from functional region of the target gene was performed and 95 positive plantlets were acquired. Thirty seven transgenic plants were eventually generated by grafting of all plantlets to Hai 7124 stock, and the survival rate of grafting reached 38.95%. RT-PCR results indicated that the characteristic band of 540 bp amplified fragment appeared in 31 plants while disappeared in the rest six plants. Pollen fertility identification in blossom stage showed that nine transgenic plants displayed male sterile, 22 plants performed male fertile and self-fertilized to T1 seed. Twenty two pure T3 transgenic gafp lines were identified by cultivating in the isolation area specific to transgenic crops, PCR detection using primers from functional region of gafp gene, kanamycin-resistance screening, self-fertilized purification and selection. Southern blotting showed that there were two copies ofgafp gene integrated into the cotton genome. Western blotting confirmed that 16 pure transgenic lines had GAFP protein with molecular weight of 14 kD, the other six lines lacked the protein due to posttranscriptional gene silencing. Identification of resistance to Verticillium wilt indicated that three pure transgenic gafp lines TG09, TG 16, TG23 were resistant to defoliated strain V991 of Verticillium wilt.