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为了研究番茄LCYE基因对其果实中番茄红素含量的影响机制,培育高品质番茄品种,在Gen Bank中根据已知的LCYE(ID:544 129)扩增出337 bp的保守序列;根据KJ 561 284.1扩增出2 203 bp的果实特异性启动子E8;查找番茄LCYE的内含子,选择第三个长112 bp的Intron片段;利用同尾酶构建出载体p Ri E8-LCYE,与p CAMBIA1301连接构建出果实特异性RNAi表达载体p CRi E8-LCYE,测序表明载体构建正确。农杆菌介导,叶盘法转化番茄,PCR检测表明得到了转基因番茄植株。与同样条件下番茄LCYB基因干扰载体的遗传转化过程相比,表明:对LCYE基因的干扰使番茄子叶开始褐化时间提前,且褐化率更高,说明筛选培养时子叶的褐化与所干扰的基因也有关系。
In order to study the mechanism of tomato LCYE gene affecting lycopene content in its fruit, high-quality tomato cultivars were cultivated and a 337 bp conserved sequence was amplified from GenBank by known LCYE (ID: 544 129). According to KJ 561 284.1 was amplified a 2 203 bp fruit-specific promoter E8; Find intron of LCYE tomato, select the third 112-bp Intron fragment; the use of homologous enzyme to construct the vector p Ri E8-LCYE, p CAMBIA1301 Linkage construct fruit-specific RNAi expression vector p CRi E8-LCYE, sequencing showed that the vector was constructed correctly. Agrobacterium-mediated transformation of tomato by leaf disk method, PCR test showed that transgenic tomato plants. Compared with the genetic transformation of tomato LCYB gene under the same conditions, the results showed that the interference of LCYE gene led to the earlier browning of tomato cotyledons and the higher browning rate, indicating the browning and interference of cotyledons during screening culture The genes are also related.