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Vector construction and plant transformation. DNA fragments 2 kb upstream of the DEP1 transcription start site and 900 bp downstream of its termination site were amplified and cloned into the binary vector pCAMBIA1300 to generate the pDEP1:3￠-UTR expression cassette. Full-length dep1 and DEP1 cDNA were separately inserted into the binary vectors pDEP1:3￠-UTR, pCaMV 35S: NOS and pActin:OCS. A pDEP1: RNAi-DEP1 construct was based on the sequence of a 300-bp fragment of the N terminus of the DEP1 cDNA sequence, which shows no significant homology with any other sequences in the rice genome. A 250-bp fragment was amplified from the bread wheat variety Ni982105 to generate the construct pUbi: RNAi-TaDEP1. Transgenic lines of wheat and rice were created by Agrobacterium-mediated transformation22,23. The relevant PCR primer sequences are given in Supplementary Table 3 online.
载体构建与植物遗传转化。DEP1转录起始点的DNA片段2kb上游序列与终止点900bp下游序列被扩增并复制进入双元载体pCAMBIA 1300，产生pDEP1:3′-UTR的表达框。dep1和DEP1的cDNA全长被分开导入双元载体pDEP 1:3′－UTR， pCaMV 35S：NOS 和 pActin：OCS。一个pDEP 1:RNAi－DEP1结构是基于DEP1cDNA 序列的N终端的300bp序列的基础上的，与水稻基因组的任何其他序列没有显著的同源性。来自面包小麦品种Ni982105的一个250bp的序列被扩增产生pUbi：RNAi－TaDEP1。通过农杆菌介导法产生了小麦和水稻的基因品系。有关PCR引物序列在补足的表3中（在线）给出。 |
[论文翻译]Vector construction and plant transformation