Cloning And Expression Analysis Of New Sress-resistant NAC83, MYB15 And COR15a Gene From Citrus

The Citrus industries always suffer from abiotic stresses, such as drought, low temperature, high salinity and so on, which then restrict the development of those industries. So enhancing the sress-resistant ability of plant is an important target in Citrus breeding. In this research, three novel sress-resistant genes, NAC83、MYB15 and COR15 a 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(qRT-PCR)under the stress of cold, salt, drought and ABA, in order to obtain more sress-resistant gene resources of Citrus. Following are the main results:(1) Three NAC83 genes, named Cm NAC83 、 Pt NAC83 and Cl NAC83, were respectively cloned from seedlings of pomelo, trifoliate orange and lemon. 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 sequence similarity of which reached to 98.93%, and there are eight positions existing differences. The N-terminal of all the three new proteins contained a conserved sequence, which composed of 160 amino acid residues and could be devided into five substructures named A, B, C, D and E. The phylogenetic tree analysis showed that all the three proteins belonged to subtribe SENU5. And the qRT-PCR analysis indicated that the expressions of three NAC83 genes were induced by stress of ABA, drought, cold and salt, which was obvious in pomelo and lemon but gently in trifoliate orange.(2) Three MYB15 genes, named CmMYB15, PtMYB15 and Cl MYB15, were respectively cloned from seedlings of pomelo, trifoliate orange and lemon. The full length of cDNA sequence of the three genes were 994 bp、992 bp and 988 bp respectively, which coding three polypeptides of 267, 266 and 265 amino acids. And the N-terminal of the three novel proteins contained two series of incomplete repeat MYB DNA-binding domain, which composed of 50 to 55 amino acid residues, so these three genes belonged to R2R3 subclass of MYB family. The qRT-PCR analysis indicated that the expressions of three MYB15 genes were induced by stress of ABA, drought, cold and salt, such as CmMYB15 were rapidly expressed in leaves, Pt MYB15 were expessed gently, and ClMYB15 were intensely induced expressed but responsed slowly.(3) Three COR15 a genes, named CmCOR15 a, PtCOR15 a and ClCOR15 a, were respectively cloned from seedlings of pomelo, trifoliate orange and lemon. The full length of cDNA sequence of the three genes were 696 bp、701 bp and 692 bp respectively, all of them contained a 417 bp ORF encoding a polypeptide of 138 amino acids. And the sequence similarity of the three polypeptides reached to 99.03%, and there are four locuses existing differences, which located at number 84, 109, 120 and 121 respectively. And all of the three proteins contained a dehydrin structural domain located at number 19 to 78. The qRT-PCR analysis indicated that the expressions of three COR15 a genes were induced by stress of ABA, drought, cold and salt, but they were differently expressed in different citrus species.