CAJ | 학술논문

利用NCBI、CODEHOPE、Primer Premier5.0等数据库和生物软件设计兼并引物，以矮牵牛花柱cDNA为模板，利用RT-PCR扩增到1条长为417 bp的序列，在GenBank中比对发现，该序列与SX基因相似性最高。然后以SX序列为模板设计特异性引物，RT-PCR扩增到1条全长为747 bp的目的片段，测序结果分析表明，该基因核苷酸序列开放阅读框全长660 bp，编码220个氨基酸，其中包括有22个氨基酸的跨质膜信号肽区，和5个保守区(C1-C5)以及2个高变区(Hva, Hvb)，据前人研究所知，该基因具有S-核酸酶功能。同时，从番茄DNA中克隆到一长为717 bp的花粉特异性启动子LAT52。为了使SX基因能在花粉中发挥其S-核酸酶功能，去掉22个氨基酸跨质膜信号肽(命名为sx)之后，与番茄花粉特异性启动子LAT52嵌合构建植物表达载体并转化拟南芥。 TTC染色结果表明，LAT52能特异性的在花粉中调控sx基因的表达，使花粉活力很低甚至没有活力，从而使转基因植株不育。
이용NCBI、CODEHOPE、Primer Premier5.0등수거고화생물연건설계겸병인물，이왜견우화주cDNA위모판，이용RT-PCR확증도1조장위417 bp적서렬，재GenBank중비대발현，해서렬여SX기인상사성최고。연후이SX서렬위모판설계특이성인물，RT-PCR확증도1조전장위747 bp적목적편단，측서결과분석표명，해기인핵감산서렬개방열독광전장660 bp，편마220개안기산，기중포괄유22개안기산적과질막신호태구，화5개보수구(C1-C5)이급2개고변구(Hva, Hvb)，거전인연구소지，해기인구유S-핵산매공능。동시，종번가DNA중극륭도일장위717 bp적화분특이성계동자LAT52。위료사SX기인능재화분중발휘기S-핵산매공능，거도22개안기산과질막신호태(명명위sx)지후，여번가화분특이성계동자LAT52감합구건식물표체재체병전화의남개。 TTC염색결과표명，LAT52능특이성적재화분중조공sx기인적표체，사화분활력흔저심지몰유활력，종이사전기인식주불육。
A 417bp fragment was cloned from Petunia hybrida cDNA by using some internet datebases and biological softwares such as NCBI, CODEHOPE, Primer Premier 5.0 to design degenerate primers and degenerate primers RT-PCR. Through analysis in GenBank. It has the highest similarity with SX gene sequence. Designing a specific primer according to the sequence of SX gene, finally a 747 bp fragment was obtained by RT-PCR. The result showed that SX was 660 bp in size and encoded 220 amino acids, including a transmembrance signal domain of 22 amino acids, the conserved regions (C1-C5) and hyper-variable regions (HVa and HVb). And then a signal peptide of 22 amino acids was removed to make it has a function of S-RNase in pollen, the pollen-specific LAT52 promoter of tomato was used to express sx, and a plant expression vector was constructed. Transgenic Arabidopsis plants expressing sx can influence the development of Arabidopsis's floral organ, the pollen vitality is very low and even no vitality, and cause the infertility of transgenic as a result.