CAJ | 학술논문

植物果糖激酶(FRK)在果糖磷酸化中起重要作用.通过PCR技术从温州蜜柑(Citrus unshiu Marc.)基因组中扩增得到编码果糖激酶基因的2个基因组DNA片段,分别命名为Cufrk1、Cufrk2,利用RT-PCR从果实中分离到了与Cufrk1外显子序列一致的cDNA序列,并通过RACE技术分离到这个基因的全长cDNA序列,命名为CuFRK1(GenBank号:AY561840).Cufrk1与Cufrk2编码氨基酸序列相似性为68%.CuFRK1 cDNA全长为1 459 bp,5'端和3'端的非翻译区分别为167 bp和239 bp,该序列含有一个完整的开放读码框,编码350个氨基酸,蛋白质分子量约为37.5 kD,等电点为5.03,含有2个果糖激酶糖特异结合域及3个ATP结合域,其氨基酸序列与其他植物中已分离的果糖激酶基因相似性在62%～78%.Northern分析显示,CuFRK1(Cufrk1)与Cufrk2在柑橘幼叶、发育初期果实中表达量较高,在果皮和茎中不表达,在花瓣及成熟果实中表达模式有一定差异.酶活性分析表明,果实中的果糖激酶活性随果实的发育而降低,同时,果实中的果糖不断积累,在果实整个发育过程中果糖含量与果糖激酶活性呈极显著负相关.
식물과당격매(FRK)재과당린산화중기중요작용.통과PCR기술종온주밀감(Citrus unshiu Marc.)기인조중확증득도편마과당격매기인적2개기인조DNA편단,분별명명위Cufrk1、Cufrk2,이용RT-PCR종과실중분리도료여Cufrk1외현자서렬일치적cDNA서렬,병통과RACE기술분리도저개기인적전장cDNA서렬,명명위CuFRK1(GenBank호:AY561840).Cufrk1여Cufrk2편마안기산서렬상사성위68%.CuFRK1 cDNA전장위1 459 bp,5'단화3'단적비번역구분별위167 bp화239 bp,해서렬함유일개완정적개방독마광,편마350개안기산,단백질분자량약위37.5 kD,등전점위5.03,함유2개과당격매당특이결합역급3개ATP결합역,기안기산서렬여기타식물중이분리적과당격매기인상사성재62%～78%.Northern분석현시,CuFRK1(Cufrk1)여Cufrk2재감귤유협、발육초기과실중표체량교고,재과피화경중불표체,재화판급성숙과실중표체모식유일정차이.매활성분석표명,과실중적과당격매활성수과실적발육이강저,동시,과실중적과당불단적루,재과실정개발육과정중과당함량여과당격매활성정겁현저부상관.
Fructokinase (FRK) is of primary importance in phosphorylation of fructose in plants. Two genomic DNA fragments encoding putative fructokinase gene were isolated from Citrus unshiu Marc. using PCR, which were named Cufrk1 and Cufrk2. A cDNA sequence same to the exons' sequences of Cufrk1was amplified by RT-PCR and the full-length cDNA sequences of this gene was isolated using rapid amplification of cDNA ends (RACE) from mature fruit, named CuFRK1 (GenBank number: AY561840). Sequencing analysis showed that the amino acid sequences were 68% identical between Cufrk1and Cufrk2. The full-length cDNA of CuFRK1 was 1 459 bp in length and contained a complete ORF from 168 to 1 220 bp,encoding 350 amino acids with a calculated molecular weight of 37.5 kD and a predicted pl of 5.03. The deduced amino acids of CuFRK1 possessed two sugar-binding domains, three ATP-binding domains and were 62% to 78% identical to the previously characterized fructokinase genes of other plants. Northern analysis showed that transcripts of CuFRK1(Cufrk1) and Cufrk2 were detected at a high level in leaves,fruit at early developmental stages, but undetectable in peels and stems. Their expression patterns were distinct in petals and mature fruit. Enzymatic analysis showed that the activity of citrus fructokinase was decreasing with the fruit development, coincided with the accumulation of fructose in fruit. There was a significantly negative correlation between the decreasing fructokinase activity and the increasing fructose content during fruit development.