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采用RT-PCR方法从小麦品种豫教2号的发育籽粒中克隆出AGPase胞质型大亚基基因(large subunit,LSU I)的cDNA全长(1947 bp,GenBank No.DQ839506),同源性比较结果表明,与GenBank上已报道的LSU I基因同源性达99%,虽与其有2处碱基不同(与Z21969相比,分别在851 bp处的T变为C和926 bp处的G变为A),但他们的氨基酸序列相同,故不影响其功能.以pBI121质粒为基础,通过中间载体pBSK,构建了由35S启动子调控的LSU I基因的正义表达载体pBI121LSUⅠS;将该基因反向置于pBI121质粒的CaMV35S启动子之后,构建了LSU I的反义表达载体pBI121LSUⅠA.同时还克隆出LSU I基因的250 bp片段,以pFGC5941质粒为基础,构建了RNAi干扰载体pFGCLsuⅠ,这为研究该基因的功能打下了很好的基础.
The full-length cDNA (1947 bp, GenBank No.DQ839506) of AGPase large subunit gene (LSU I) was cloned from the developmental kernels of wheat cultivar Yujiao 2 by RT-PCR. The homology The results showed that the homology was 99% with the reported LSU I gene in GenBank, although it had 2 bases different from that of Z21969 (T at 851 bp and C at 926 bp, respectively) And changed to A), but their amino acid sequences were the same, so their function was not affected.Based on the pBI121 plasmid, a sense expression vector pBI121LSUIS of the LSU I gene regulated by the 35S promoter was constructed through the intermediate vector pBSK. After the CaMV35S promoter was inserted into pBI121 plasmid, the antisense expression vector pBI121LSUⅠA of LSU I was constructed and a 250 bp fragment of LSU I gene was cloned. Based on the pFGC5941 plasmid, the RNAi interference vector pFGCLsuⅠ was constructed, The function of the gene laid a good foundation.