红苋R104叶绿体基因文库的构建及1,5-二磷酸核酮糖羧化/加氧酶大亚基基因的克隆

CONSTRUCTION OF CHLOROPLAST GENOMIC LIBRARY OF AMARANTHUS CRUENTUS R104 AND CLONING OF THE GENE FOR THE LARGE SUBUNIT OF RIBU-LOSE-1,5-DISPHOSPHATE CARBOXYLASE/OXYGENASE

本实验以红苋(Amaranthus cruentus)R104为材料,对其叶绿体DNA进行了BamHⅠ、EcoRⅠ、BgⅢ、PstⅠ、SalⅠ 5种限制性内切酶酶切分析,估算出其叶绿体DNA分子总长度为140kb,并以 pBR322为克隆载体,构建了红苋140kb叶绿体DNA BamHⅠ片段基因文库,通过分子杂交,从中筛选到含1,5-二磷酸核酮糖羧化/加氧酶大亚基基因的克隆pAB592,对此克隆的初步分析表明,红苋的rbcL基因与C_4植物玉米的对应基因可能结构相近。此外,还对红苋R104的pSbA基因不能被克隆的原因进行了讨论。

Grain amaranth is an annual food and forage crop and C_4-plant with characteristics of rich nutrients, high photosynthesis efficiency and strong stress-resistance. Chloroplast DNA (ctDNA) was prepared from Amaranthus cruentus R104. The ctDNA was digested with res- triction enzymes Bam HI, EcoRI, Bglll. PstI and Sall, and the fragments thus obtained were electrophoresed in 0.7% agarose gel. The length of the doublestranded ctDNA was measured to be 140 kb or so. BamHI-digested fragments of R104 ctDNA were inserted into pBR322 and a chloroplast genomic library has been constructed. The recombinant clone containing rbcL gene has been identified and selected our from the library. The restriction analysis of the clo- ne showed that the rbcL gene of A. cruenus R104 has the structure similar to that of C_4-plant corn. Moreover the reason of unsuccessful cloning of 5.9 kb BamHI fragment containing psbA gene is discussed.