柑橘抗逆基因NAC83的克隆与表达分析

Cloning and Expression Analysis of New Stress-resistant NAC83 Gene from Citrus

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。

In the present study,three novel NAC genes,CmNAC83,PtNAC83 and ClNAC83,were cloned by in silico cloning and RT-PCR approaches from seedlings of pomelo[Citrus maxima(Burm.)Merr.],trifoliate orange[Poncirus trifoliata(L.)Raf. ]and lemon[Citrus limon(L.)Burm. f.]. The expression patterns of these genes were analyzed by Real-time quantitative PCR(q RT-PCR)under the stress of ABA,drought,cold and salt. The results showed that the full length of cDNA sequence of all the three genes were 841 bp and the open reading frame(ORF)were 750 bp which encoded three polypeptides of 249 amino acids. The molecular weight of the three proteins were 24.18,28.00 and 28.15 kD,with isoelectric point about 9.02,9.31 and 9.30 respectively. The three novel proteins contained N-terminal conserved domains of NAC family. The phylogenetic analysis showed that the three proteins have the highest similarity with NAC22 from Malus × domestica,and Its belongs to the SENU5 subfamily in NAC family. The q RT-PCR analysis indicated that the expressions of three NAC83 genes were induced by stress of ABA,drought,cold and salt,but they were differently expressed in different citrus species. The results of this paper suggested that CmNAC83,PtNAC83 and ClNAC83 were new members of the NAC gene family,and they might play important roles in citrus in response to abiotic stress.