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为研究蝴蝶兰Rubisco活化酶基因(RCA)及其在低温胁迫下的响应机制,本研究利用RT-PCR及RACE技术从蝴蝶兰叶片中克隆了一个Rubisco活化酶基因PhRCAα的c DNA序列(登录号KU187968)该基因全长1 642 bp,编码473个氨基酸,包含P-loop_NTPase超家族结构域,预测其成熟蛋白的分子质量为46.16 k D,等电点为5.73,属于RCA基因的α亚基。实时荧光定量PCR分析显示,在13℃/8℃的昼夜温度条件下,蝴蝶兰PhRCAα基因的转录表达在处理3 d、6 d、9 d和15 d时明显下降,恢复正常温度条件后,该基因的表达升高;在4℃低温条件下,PhRCAα基因的转录表达在处理0.5 h、1 h和2 h内逐渐升高,而在处理4 h时其表达量下降,一直到低温处理48 h时,其表达量维持在较低水平。结果表明PhRCAα对驯化低温(13℃/8℃)和冷胁迫(4℃)具有不同的响应机制,PhRCAα对短期的冷胁迫有一定的防御作用。
In order to study the Rubisco activase gene (RCA) of Phalaenopsis and its response mechanism under low temperature stress, we cloned a DNA sequence of Rubisco activator gene PhRCAα from Phalaenopsis leaves by RT-PCR and RACE (Accession No. KU187968) This gene is 1 642 bp in length and encodes a protein of 473 amino acids. It contains the P-loop_NTPase superfamily domain. The predicted molecular mass of the mature protein is 46.16 kD and the isoelectric point is 5.73, which belongs to the α subunit of RCA gene. Real-time PCR analysis showed that the transcriptional expression of PhRCAα in Phalaenopsis decreased significantly at day 3, day 6, day 9 and day 15 at day / night temperature of 13 ℃ / 8 ℃. After returning to normal temperature, The expression of PhRCAαgene increased gradually at 0.5 h, 1 h and 2 h after treatment at 4 ℃, but decreased at 4 h, When its expression level is maintained at a low level. The results showed that PhRCAαhad a different response mechanism to acclimation at low temperature (13 ℃ / 8 ℃) and cold stress (4 ℃), and PhRCAαhad a certain defensive effect on short-term cold stress.