异戊烯基酰基转移酶基因IPT是细胞分裂素合成途径中的关键基因。本研究从玉米自交系郑58中克隆ZmIPT2基因并构建单子叶植物表达载体，通过农杆菌侵染萌动胚方法将其转入玉米自交系K10中，对转基因后代进行分子检测和功能验证。结果表明，ZmIPT2基因cDNA全长969 bp，成功构建其单子叶植物表达载体pCAMBIA5300-ubi-ZmIPT2；农杆菌侵染萌动胚法共转化K10种子5163粒，获得T0代PCR阳性幼苗48株，其中13株结实收获种子；获得的3个T2代株系PCR阳性率符合3:1的分离比，且RT-PCR检测呈阳性；2个T2代转基因株系的成熟期时的叶片叶绿素含量和细胞分裂素含量极显著高于对照，相对绿叶面积和叶面积持绿期极显著或显著高于对照，百粒重和小区产量显著高于对照。上述结果初步证明ZmIPT2基因在玉米中的过表达可延缓叶片衰老，提高玉米产量。
英文摘要:
Isopentenyl transferase (IPT) gene was a key gene in synthetic pathway of cytokinin. In this study ZmIPT2 gene was cloned from maize inbred line Zheng58 and then was constructed into the expression vector of monocotyledon. The gene was introduced into maize inbred lines K10, and the molecular test and functional verification were employed to analyze transgenic progeny. The results showed that ZmIPT2 gene with 969bp length was cloned and introduced into monocot expression vector pCAMBIA5300-ubi-ZmIPT2. The gene was transformed into germinating embryo of K10 by Agrobacterium-mediated transformation. We treated 5163 germinating embryo of maize inbred lines K10, got 48 PCR positive seedlings in T0 generation, and 13 plants of them were harvested. The segregation ratio of PCR was 3:1 in 3 T2 lines and they were positive in RT-PCR test totally. The CTK and chlorophyll content of two transgenic lines were significantly higher than that of control line at mature stage. The relative green leaf area (RGLA), leaf area to green time (LAD), 100 seed weight and plot yield of transgenic lines were significantly higher or very significantly higher than that of control. The results proved that the overexpression of ZmIPT2 gene in maize could delay leaf-senescence and increased yield.
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