Cloning And Expression Analysis Of Three WD40 Related Genes Of Narcissus Tazetta Var. Chinensis Cv.Huanghua Ⅱ

As the triploid plants of Narcissus, Bulbs are used for the asexual reproduction of Narcissus tazetta var. chinensis for a long time. The degradation is obvious as resistance reduced in cultivars. And fertile fields for narcissus are reducing with the advancement of urbanization. Therefore, to improve the resistance of the Chinese narcissus and enable it to adapt to more complex cultivation environment is crucial for the future of Narcissus tazetta. In this research three gene fragments of WD40 family have been screened from curb cuts hybridization cDNA library of Narcissus tazetta established by the Narcissus research group from horticulture college, FAFU named NtWD1, NtWD2 and NtWD3.Cloning their full-length cDNA through RT-PCR and RACE. The expression of these three genes and an uncharacterized NtWD40 under seven kinds of abiotic stress and tissue specificity were examined by qPCR, which contributes to the improvement of germplasm and beeding of hardy cultivars.Cloning of NtWD1,NtWD2,NtWD3 Full-length of NtWDl and 5’parts of NtWD 2 and NtWD3 were cloned from petals of Narcissus tazetta var. chinensis cv.Huanghua Ⅱ.The open reading frame of NtWDl encompassed 3378bp encoding a polypeptide of 1125 amino acids. It contains two dimerization motifs, the LisH (lissencephaly homology) and CTLH (C-terminal to LisH) domain, at the N terminus, as well as 11 centrally located WD40 domains and the C-terminal WD40 domains. All these are same as the secondary structure of the TPL/TPR (topless/topless-related) co-repressors. Three-dimensional conformation simulations show two intersecting 7-fold β-propellers.On the basis of phylogenetic analysis, NtWD1 can be grouped with TPL/TPR proteins of Phoenix dactylifera, Elaeis guineensis and Musa acuminata subsp. malaccensis.5’parts of NtWD2 and NtWD3 were cloned. Sequence spliced a length of 741bp NtWD2 gene fragment and 1639bp NtWD3 gene fragment.Expression analysis of NtWDl, NtWD2, NtWD3, NtWD40 Analyzed by BLAST (basic local alignment search tool), NtWDl is similar to TPL/TPR co-repressors; nevertheless, NtWD2, NtWD3 and NtWD40 are uncharacterized. Expression of these four genes above under seven kinds of abiotic stress (drought, high temperature, mechanical injury, MeJA, ABA,2,4-D, high salinity) as well as different parts of the plant were examined by qPCR. Results demonstate that NtWD40 hardly expressed in root, NtWD3 expressed stably in root, leaf, petals and sub-corolla. NtWDl and NtWD2 have a slightly higher expression in the subcorollas than petals. Notably, the NtWDl gene is expressed at high levels under MeJA, ABA and 2,4-D treatment, salt-stress and mechanical injury. Moreover, NtWD2 shows the most significant upregulating under salt-stress. NtWD3 shows significant upregulating under both salt-stress and ABA treatment. Interestingly, NtWD40 is upregulated under ABA and MeJA treatment, but remains stable in mechanical injury. In addition, NtWD40 has an expression peak after NtWDl under ABA treatment.