STUDIES ON CHLAMYDOMONAS CHLOROPLAST TRANSFORMATION - FOREIGN DNA CAN BE STABLY MAINTAINED IN THE CHROMOSOME
STUDIES ON CHLAMYDOMONAS CHLOROPLAST TRANSFORMATION - FOREIGN DNA CAN BE STABLY MAINTAINED IN THE CHROMOSOME
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DOI：
10.1105/tpc.1.1.123
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发表时间：
1989-01-01
期刊：
影响因子：
11.6
通讯作者：
SANFORD, JC
中科院分区：
文献类型：
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作者：
BLOWERS, AD;BOGORAD, L;SANFORD, JC
正如Boynton及其同事（Boynton, j.e., Gillham, n.w., Harris, e.h., Hosler, j.p., Johnson, a.m., Jones, a.r., Randolph-Anderson, b.l., Robertson， D., Klein, t.m., Shark, k.b., and Sanford， J.C.[1988]）最初所示。Science 240, 1534-1538)，一种非光合作用，需要醋酸盐的reinhardtii衣藻突变株，在叶绿体Bam10限制性片段区域缺失2.5千碱基对，去除3‘’一半的atpB基因和一部分反向重复，可以在涂有野生型Bam10 DNA的微弹丸轰炸后转化为光合能力。我们发现含有野生型atpB基因的各种其他圆形质粒、单链DNA圈或线性双链DNA分子也可以补充相同的突变体。所有这些转化体的DNA凝胶杂交分析表明，互补DNA已经整合到atpB位点的染色体上，并表明在反向重复序列之间运行的复制纠正机制保持了该区域的序列一致性。当外源DNA只含有缺失序列的一部分时，可以从完整的反向重复序列中招募序列来恢复不完整的拷贝。此外，叶绿体DNA序列两侧的外源、未选择的嵌合基因可以在叶绿体染色体中整合并稳定维持。融合到玉米叶绿体启动子核酮糖-1,5-二磷酸羧化酶（rbcL）大亚基基因的细菌新霉素磷酸转移酶结构基因已被整合到Bam10限制性片段的反向重复区。已鉴定出与引入的外源基因杂交的RNA转录本。
As shown originally by Boynton and co-workers (Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M., Shark, K.B., and Sanford, J.C. [1988]. Science 240, 1534-1538), a nonphotosynthetic, acetate-requiring mutant strain of Chlamydomonas reinhardtii with a 2.5-kilobase pair deletion in the chloroplast Bam 10 restriction fragment region that removes the 3'' half of the atpB gene and a portion of one inverted repeat can be transformed to photosynthetic competency following bombardment with microprojectiles coated with wild-type Bam10 DNA. We have found that assorted other circular plasmids, single-strand DNA circles, or linear, duplex DNA molecules containing the wild-type atpB gene can also complement the same mutant. DNA gel blot hybridization analysis of all such transformants indicates that the complementing DNA has integrated into the chromosome at the atpB locus and suggests that a copy-correct in mechanism operating between the inverted repeats maintains sequence identity in this region. Sequences from the intact inverted repeat may be recruited to restore the incomplete copy when exogenous DNA with only a portion of the deleted sequence is introduced. Furthermore, a foreign, unselected-for, chimeric gene flanked by chloroplast DNA sequences can be integrated and maintained stably in the chloroplast chromosome. The bacterial neomycin phosphotransferase structural gene fused to the maize chloroplast promoter for the large subunit gene of ribulose-1,5-biphosphate carboxylase (rbcL) has been integrated into the inverted repeat region of the Bam10 restriction fragment. RNA transcripts that hybridize to the introduced foreign gene have been identified.