矮牵牛自交不亲和性基因sx的克隆及功能初步分析  

Cloning and Preliminary Functional Analysis of sx Gene in Petunia hybrida

作　　者：祝建波[1] 何黎[1] 邱红林[1] 陈泉[1] 闫甜甜[1] 程晨[1] 

机构地区：[1]石河子大学农业生物技术重点实验室,石河子832000

出　　处：《分子植物育种》2013年第5期578-584,共7页Molecular Plant Breeding

基　　金：兵团博士基金(2009jc01)资助

摘　　要：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。A 417 bp 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 A rabidopsis 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.

关 键 词：矮牵牛 SX基因 克隆 功能分析 

分 类 号：S681.6[农业科学—观赏园艺]