荔枝基因枪转化及其GUS瞬时表达研究  

Transformation Using Particle Bombardment and Its GUS Transient Expression Assay in Litchi chinensis Sonn.

作　　者：桑庆亮[1,2] 赖钟雄[1] 林玉玲[1] 陈裕坤[1] 

机构地区：[1]福建农林大学园艺植物生物工程研究所,福建福州350002 [2]泉州师范学院化学与生命科学学院,福建泉州362000

出　　处：《热带作物学报》2014年第11期2223-2229,共7页Chinese Journal of Tropical Crops

基　　金：国家教育部霍英东教育基金会高等学校青年教师基金(No.71026);国家自然科学基金(No.39870544);国家"948"计划(No.991029)资助项目的部分内容

摘　　要：以幼胚来源的荔枝EC作为受体材料,采用基因枪轰击的方法将外源gus基因转入荔枝,并对不同条件下的GUS瞬时表达进行了详细研究。结果表明:在其它参数不变的条件下,轰击距离、真空度、可裂膜片压力、金粉用量、质粒DNA用量等理化参数显著影响GUS瞬时表达;渗透处理可显著促进GUS瞬时表达。因此,荔枝EC基因枪转化的适宜条件为在轰击距离6 cm、真空度84.66 k Pa、可裂膜片压力7 584.23 k Pa、轰击1次的情况下,每次轰击用1μg质粒DNA包裹600μg金粉对事先用0.25 mol/L前处理4 h的荔枝EC轰击,并在轰击后渗透处理20 h。另外,经50 mg/L潮霉素筛选得到荔枝抗性愈伤组织,并通过体胚发生途径获再生植株,且GUS染色显示获得了稳定表达GUS蛋白的细胞系和植株。In the paper, plasmid pCAMBIA 1301 DNA harboring the gus gene encoding fl-glucuronidase, coated on gold particles, was delivered into cultured EC via particle bombardment and transformation was monitored by analyzing GUS transient expression in Litchi chinensis Sonn. The results showed that GUS transient expression level was significantly influenced by physical and chemical factors, such as distance to targets, vocuum pressure, helium pressure, gold particle dosage, plasmid DNA dosage, and vitality of litchi EC cells, which affected by growth stages and osmatic treatment. A suitable particle bombardment procedure was proposed. Litchi EC which was pre--cultured on MS medium with 0.25 mol/L mannitol for 4 hours and post-cultured for 20 hours after bombardment was bombarded once using 600 μg gold particles coated with 1 μg plasmid DNA with a vacuum degree of 84.66 kPa, a bombarded distance of 6 cm, and a rupture disk value of 7 584.23 kPa. Hygromycin- resistant embryonic calli （HREC） and plantlets regenerated from HREC were also obtained and GUS stable expression in HREC and leaves were also examined to confirm transformation.

关 键 词：荔枝 胚性愈伤组织 基因枪 遗传转化 瞬时表达 

分 类 号：S667.1[农业科学—果树学]