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以柚[Citrus maxima(Burm.)Merr.]、枳[Poncirus trifoliata(L.)Raf.]和柠檬[C.limon(L.)Burm.f.]实生苗为试材,使用电子克隆和RT-PCR的方法从中克隆到3个新的NAC蛋白基因,分别命名为CmNAC83、PtNAC83和ClNAC83;并用qRT-PCR技术检测了该基因在ABA、干旱、低温和高盐胁迫处理下的时空表达。结果显示:CmNAC83、PtNAC83和ClNAC83的cDNA序列全长均为841 bp,都含有750 bp的开放阅读框(ORF),编码249个氨基酸;推测其蛋白分子质量分别为24.18、28.00和28.15 kD,等电点分别是9.02、9.31和9.30,且均含有NAC家族的N端保守结构域;系统进化树分析表明,该3个蛋白均属于SENU5亚族,与苹果NAC22亲缘关系较近。实时定量qRT-PCR分析显示,NAC83基因能被ABA、干旱、低温和高盐胁迫诱导表达,且在不同的柑橘种类中存在表达差异。可见柚CmNAC83、枳PtNAC83和柠檬ClNAC83是NAC基因家族的成员,可能在柑橘响应非生物胁迫的过程中起了重要作用。
Seedlings of Citrus maxima (Burm.) Merr., Poncirus trifoliata (L.) Raf. And lemon [C.limon (L.) Burm.f.] Were used as experimental materials, -PCR. Three novel NAC protein genes were cloned and named as CmNAC83, PtNAC83 and ClNAC83 respectively. The spatial and temporal expression of this gene under ABA, drought, low temperature and high salt stress was detected by qRT-PCR. The results showed that the full-length cDNA sequences of CmNAC83, PtNAC83 and ClNAC83 were 841 bp in length, all of which contained a 750 bp open reading frame (ORF) encoding a protein of 249 amino acids. The predicted protein molecular weights were 24.18, 28.00 and 28.15 kD, respectively The electric loci were 9.02, 9.31 and 9.30, respectively, and all contained the N-terminal N-terminal conserved domain. Phylogenetic tree analysis indicated that these three proteins belonged to the SENU5 subfamily and had a close genetic relationship with apple NAC22. Real-time quantitative qRT-PCR analysis showed that NAC83 gene was induced by ABA, drought, low temperature and high salt stress, and there were differences in expression among different citrus species. Visible grapefruit CmNAC83, trifoliate PtNAC83 and lemon ClNAC83 NAC gene family members, may play an important role in citrus in response to abiotic stress.