红麻 RPT4全长cDNA的克隆及其在红麻花药中的表达

摘要

[目的]克隆红麻细胞质雄性不育系与保持系花药中差异表达蛋白RPT4相应基因的cDNA全长,分析该基因在花药中的表达.[方法]采用同源克隆法和RACE技术相结合,从保持系L23B花药总RNA中克隆RPT4全长cDNA.通过半定量RT-PCR检测花粉小孢子败育前,败育期间和败育晚期该基因在不育系和保持系间花药中的表达差异.[结果]克隆得到1 596 bp的cDNA全长,最长开放阅读框(ORF)1 197 bp,编码398个氨基酸的肽序列;该蛋白与蓖麻中直系同源蛋白的相似性最高,达91.93%,内含3个模体Walker A、Walker B和arginine finger的保守域,暗示其具有复杂多样的重要功能.RPT4的mRNA在红麻不育系和保持系花药中均有表达,但在四分体前(败育前),不育系和保持系中的表达量一致;在小孢子单核期(败育期),不育系花药中的表达量比保持系中的低;而小孢子双核期(败育晚期)的表达变化则相反.[结论]RPT4参与红麻小孢子的发育过程,可能具有与红麻细胞质雄性不育相关的复杂分子功能.%[ Objective ] This study aims to clone the full-length cDNA of the corresponding gene of RPT4 that with differential expression between cytoplasmic male sterile (CMS) lines and their maintainers in kenaf (Hibiscus cannabinus L.) and to analyze its expression in anthers. [ Method ] With homology cloning approaches coupling with RACE techniques, the full-length cDNA of RPT4 was cloned from total RNA of the anthers of the maintainer line L23B in kenaf. Then the expressions of mRNA in the anthers before abortive stage, at abortive stage, and late abortive stage were analyzed by semi-quantitative RT-PCR. [Result] The longest 1197 bp ORF of RPT4 was detected from the cloned sequence with full-length of cDNA of 1 596 bp. The encoded peptide of 398 amino acids shares 91.93％ homology with the one in Ricinus communis. It also contains conserved domain consisting of three motifs- Walker A, Walker B and arginine finger, which suggests that the domain has various important functions. RTP4 was confirmed to express in anthers of both CMS line L23A and its maintainer. The expressing level of RPT4 in the CMS line L23A was identical to that in the maintainer L23B at the tetrad stage. However, the expressing level of RPT4 in L23A was lower at the uninucleate (abortive) stage, and higher at the binucleate (late abortive) stage than that of L23B. [Conclusion] RPT4 is involved in the development ofmicrospores in kenaf and may be related to CMS with complex molecular function.