Differential accumulation of transcripts for four tomato 1-aminocyclopropane-1-carboxylate synthase homologs under various conditions.
Differential accumulation of transcripts for four tomato 1-aminocyclopropane-1-carboxylate synthase homologs under various conditions.
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DOI：
10.1073/pnas.89.6.2475
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发表时间：
1992-03
影响因子：
11.1
通讯作者：
W. Yip;Thomas Moore;S. F. Yang
中科院分区：
文献类型：
--
作者：
W. Yip;Thomas Moore;S. F. Yang
简并寡核苷酸引物对应于 1-氨基环丙烷-1-羧酸酯 (ACC) 合酶 (EC 4.4.1.14) 活性位点结构域侧翼的保守区域，用于聚合酶链式反应 (PCR)，以扩增从番茄果实和番茄悬浮细胞培养物中分离的 mRNA 的 DNA 片段。 PCR 产物克隆到 Bluescript 质粒并在大肠杆菌中表达后，使用针对两个保守肽序列（TNPSNPLGTT 和 SLSKDLGLPGFRVG）的抗体筛选阳性菌落。分离出四个编码 ACC 合酶同源物的不同 cDNA 片段。虽然 pBTAS1 和 pBTAS4 是从水果 mRNA 中获得的，但细胞培养物 mRNA 产生了三个序列：pBTAS1、pBTAS2 和 pBTAS3。对这些基因片段的测序表明，pBTAS1 和 pBTAS4 与 Van Der Straeten 等人先前报道的全长序列相同。 [Van Der Straeten, D.、Van Wiemeersch, L.、Goodman, H. 和 Van Montague, M. (1990) Proc.国家。阿卡德。科学。 USA 87, 4859-4863] 和 Olson 等人。 [Olson, D. C.、White, J. A.、Edelman, J.、Harkin, R. N. 和 Kende, H. (1991) Proc.国家。阿卡德。科学。 USA 88, 5340-5344] 来自番茄果实，而 pBTAS2 和 pBTAS3 代表新序列。使用核糖核酸酶保护测定来检查这些转录物在乙烯产生增强的三种不同条件下的表达，即在果实成熟期间、响应果实组织中的机械损伤和营养组织中的生长素刺激。 pBTAS1 的转录本在成熟和损伤过程中大量积累，但对生长素处理的反应只有轻微。尽管 pBTAS4 与果实成熟相关，但它对营养组织中的生长素处理没有反应。相反，pBTAS2和pBTAS3的表达在生长素处理的营养组织中大大促进，但在果实组织中不表达。虽然 pBTAS2 的表达在一定程度上依赖于受伤，但 pBTAS3 对受伤没有反应。这些数据支持这样的观点，即 ACC 合酶由多基因家族编码，并且成员因发育、环境和激素因素而差异表达。
Degenerate oligonucleotide primers corresponding to conserved regions flanking the active-site domain of 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) were used for the polymerase chain reaction (PCR) to amplify DNA fragments from mRNA isolated from tomato fruit and tomato suspension cell culture. Antibodies raised against two conserved peptide sequences (TNPSNPLGTT and SLSKDLGLPGFRVG) were used to screen for positive colonies, after the PCR products were cloned into a Bluescript plasmid and expressed in Escherichia coli. Four distinct cDNA fragments encoding ACC synthase homologs were isolated. While pBTAS1 and pBTAS4 were obtained from fruit mRNA, cell culture mRNA yielded three sequences, pBTAS1, pBTAS2, and pBTAS3. Sequencing of these gene fragments revealed that pBTAS1 and pBTAS4 were identical to those full-length sequences previously reported by Van Der Straeten et al. [Van Der Straeten, D., Van Wiemeersch, L., Goodman, H. & Van Montague, M. (1990) Proc. Natl. Acad. Sci. USA 87, 4859-4863] and Olson et al. [Olson, D. C., White, J. A., Edelman, J., Harkin, R. N. & Kende, H. (1991) Proc. Natl. Acad. Sci. USA 88, 5340-5344] from tomato fruit, whereas pBTAS2 and pBTAS3 represent new sequences. Ribonuclease protection assays were used to examine the expression of these transcripts under three different conditions of enhanced ethylene production--namely, during fruit ripening, in response to mechanical wounding in fruit tissue, and auxin stimulation in vegetative tissue. Transcripts of pBTAS1 accumulated massively during ripening and wounding but only slightly in response to auxin treatment. Although pBTAS4 was associated with fruit ripening, it was unresponsive to auxin treatment in vegetative tissue. In contrast, the expression of pBTAS2 and pBTAS3 was greatly promoted in auxin-treated vegetative tissue but was absent from fruit tissue. While the expression of pBTAS2 was moderately dependent on wounding, pBTAS3 was unresponsive to wounding. These data support the view that ACC synthase is encoded by a multigene family and that the members are differentially expressed in response to developmental, environmental, and hormonal factors.