小热激蛋白基因(hsp23.5)在小麦BNS雄性不育系和转换系中的差异表达

摘要

23.5 kD小热激蛋白(sHSP)是BNS(Bainong sterility)型雄性不育系和转换系花药的一个重要差异表达蛋白.为了探讨该蛋白与BNS不育性的联系,本研究利用荧光实时定量PCR方法,在不育发生的3个关键时期,四分体期、单核期和二核初期,定量检测该蛋白的基因hsp23.5在不育系及其转换系花药中的mRNA表达水平.结果显示,hsp23.5基因在转换系花药中从四分体到二核期呈持续上升趋势,但在不育系中表达量则显著下调,3个时期的表达量分别比同时期转换系下调3.8、20.0和4.6倍.克隆的hsp23.5片段序列,经BLAST比对,与来源于普通小麦(Triticmaes tiv um)的hsp23.6和硬粒小麦(T.turgidum)的hsp23.5小热激蛋白序列一致性最大,为94％,进化树分析遗传距离也最近.同时发现该基因在单核向二核期发育期间由于较高气温出现,表达量表现回复上调.这些结果表明,hsp23.5基因在BNS花药中组成型表达,同时也有温度诱导表达特性,在不育系中表达量显著下调.提示hsp23.5基因与BNS雄性不育发生有重要联系.%23.5 kD small heat shock protein is an important anther differentially expressed protein between male sterile line and its conversion line in wheat BNS (Bainong sterility), In order to validate the relationship between this protein and BNS male sterility, the differentially expressed mRNA of this protein's gene, hsp23.5, was examined by means of fluorescent Real-time quantitative PCR taking the anthers of BNS as materials at three key developmental stages(tetrad phase, uninucleated period and binucleated period). The results showed that the expression quantities of the hsp23.5 mRNA in three developmental stages were continuously rised in rnconversion line, but down-regulated significantly in sterile line. The amount of down-regulated in sterile line was as many as the times of 3.8, 20.0 and 4.6 in the three developmental periods comparing to that in conversion line, respectively. The result of sequencing indicated that the numbers of bases of hsp23.5 cDNA segment cloned by RT-PCR were 82.56% homologous wheat sequences from NCBI. By BLAST alignment and phylogenetic tree analysis, there were the biggest segment sequence identities of 94%, and the nearest genetic distance of <0.05, between the BNS hsp23.5 and common wheat (Triticm aestivum) hsp23.6 , and between the BNS hsp23.5 and cone wheat (T. Turgidum) hsp23.5. It was also found that the mRNA expression quantity of the gene was up-regulated in sterile line in binucleated period because of higher atmosphere temperature appearance in this period. These results suggested that the hsp23.5 gene expressed constitutive in BNS conversion line, and inducible by higher temperiture in sterile line. It may be the characteristic of sensitive-temperature that regulated the down-regulated expression of the gene in sterile line. It also implied that hsp23.5 may be one of the important relation sterile gene.