Meiotic transmission of an in vitro-assembled autonomous maize minichromosome.
Meiotic transmission of an in vitro-assembled autonomous maize minichromosome.
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DOI：
10.1371/journal.pgen.0030179
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发表时间：
2007-10
期刊：
影响因子：
4.5
通讯作者：
Preuss, Daphne
中科院分区：
文献类型：
--
作者：
Carlson, Shawn R.;Rudgers, Gary W.;Zieler, Helge;Mach, Jennifer M.;Luo, Song;Grunden, Eric;Krol, Cheryl;Copenhaver, Gregory P.;Preuss, Daphne
自主染色体在酵母（酵母人工染色体）和人纤维肉瘤细胞（人人工染色体）中通过引入纯化的DNA片段产生，所述DNA片段使动粒成核、复制并分离到子细胞。这些自主微型染色体便于操纵和递送含有多个基因的DNA片段。相比之下，转基因作物的商业化生产依赖于将一个或几个基因整合到宿主染色体中的方法;广泛筛选以鉴定具有所需表达水平、拷贝数、结构和基因组位置的插入;以及长期育种计划以产生携带多个转基因的品种。作为提高转基因作物产量的一步，我们报告自主玉米微型染色体（MMCs）的发展。我们通过将DsRed和npt II标记基因与7-190 kb的基因组玉米DNA片段组合构建了环状MMC，所述基因组玉米DNA片段含有通常在着丝粒中发现的卫星、逆转录元件和/或其他重复序列，并使用粒子轰击将这些构建体递送到胚性玉米组织中。我们选择转化细胞，再生植物，并在没有选择的情况下繁殖它们的后代多代。荧光原位杂交和分离分析表明，自主MMC可以维持有丝分裂和减数分裂。这里描述的MMC显示接近孟德尔遗传的减数分裂分离比率：93%作为二体传递（预期为100%），39%作为与野生型杂交的单体传递（预期为50%），59%在自交中传递（预期为75%）。MMC上的荧光报告基因DsRed经过四代表达，Southern杂交分析表明编码基因是完整的。这种用于植物转化的新方法可以通过以下方式促进作物生物技术：（i）将几个性状基因组合在单个DNA片段上，（ii）将基因排列在确定的序列背景中以获得更一致的基因表达，以及（iii）提供可以快速渗入到各种种质中的独立连锁群。转基因玉米的生产传统上使用将DNA片段整合到宿主染色体中的技术。这可能会破坏重要的天然基因或可能导致添加的基因表达不良;因此，必须筛选大量的转基因植物以找到适合商业用途的转基因植物。此外，可以整合的DNA数量有限，因此很难一次添加多个基因。在这里，我们描述了一个新的系统，提供基因玉米。我们构建了一个微型染色体载体，当引入玉米细胞时，它与植物的染色体保持分离或自主。这些微型染色体是由玉米着丝粒中天然存在的DNA序列构建的，着丝粒是遗传所需的染色体区域。我们通过四代来表征玉米微型染色体1（MMC 1）的行为，表明它是有效遗传的，并且它所携带的基因得到了表达。这项工作使得设计携带多个基因的微型染色体成为可能，增强了工程植物过程的能力，包括提高抗病性，耐旱性或生产复杂的生化物质。
Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7–190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms. The production of transgenic maize has traditionally used techniques that integrate DNA fragments into a host chromosome. This can disrupt important native genes or can lead to poor expression of the added gene; consequently, large numbers of transgenic plants must be screened to find one suitable for commercial use. Further, there is a limit to the amount of DNA that can be integrated, making it difficult to add multiple genes at one time. Here, we describe a new system for delivering genes to maize. We constructed a minichromosome vector that remains separate, or autonomous, from the plant's chromosomes when introduced into maize cells. These minichromosomes were constructed from DNA sequences that naturally occur in maize centromeres, the chromosomal regions needed for inheritance. We characterized the behavior of Maize Minichromosome 1 (MMC1) through four generations, showing that it is efficiently inherited and that the genes it carries are expressed. This work makes it possible to design minichromosomes that carry several genes, enhancing the ability to engineer plant processes, including improving disease resistance, drought tolerance, or the production of complex biochemicals.
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影响因子：
2.6
作者：
Co, DO;Borowski, AH;Drayer, JI
通讯作者：
Drayer, JI
影响因子：
3.3
作者：
Hall, SE;Luo, S;Preuss, D
通讯作者：
Preuss, D
影响因子：
5.3
作者：
COTTAREL, G;SHERO, JH;HEGEMANN, JH
通讯作者：
HEGEMANN, JH
影响因子：
2.6
作者：
Frame, BR;Zhang, HY;Wang, K
通讯作者：
Wang, K
DOI：
10.1073/pnas.94.8.3524
发表时间：
1997-04-15
影响因子：
11.1
作者：
Ananiev, EV;RieraLizarazu, O;Phillips, RL
通讯作者：
Phillips, RL