| Plant cuticular waxes are believed to be the initial defenses to the external environment stress. MAH1 gene is classified as a member of the CYP96 family of CYP 450. In Arabidopsis thaliana, MAH1 is considered as alkane hydroxylase involveing in the decarbonylation pathway of plant cuticular wax, which can catalyze the hydroxylation reaction leading from alkanes to secondary alcohols and ketones. The rresearch on MAH1 in Brassica nupas has not been reported. In this study, two ORF sequences of Brassica nupas MAH1 genes(Bn MAH1-1 and Bn MAH1-2) were cloned by insilico cloning and RT-PCR, and their sequences were analyzed using bioinformatics method. The expression patterns of Bn MAH1 in various organs and the induced expression by exogenous hormones were detected. The differential expression of Bn MAH1 in cultivars with wax and without wax was also detected to reveal the function of Bn MAH1 in wax deposition. Meanwhile, the overexpression vector and RNAi vector of Bn MAH1-1 were constructed for functional research by genetic transformation. The main results are followed:1. Cloning and sequence analysis of Brassica nupas alkane hydroxylase gene Bn MAH1-1 and Bn MAH1-2Using the Arabidopsis gene MAH1 sequence as probe, two ORF sequences were obtained from the Brassica napus genome databases( http:// www.ncbi.nlm.nih. gov/genome/203). The two ORFs were isolated by PCR and RT-PCR from B. nupas, named Bn MAH1-1(Accession: KT795344) and Bn MAH1-2(Accession: KT795345). The sequences of both Bn MAH1-1 and Bn MAH1-2 ORF are 1491 bp, and no inron was found in Bn MAH1-1 and Bn MAH1-2. The contents of guanine and cytosine are 43.1% in both Bn MAH1-1 and Bn MAH1-2. Bn MAH1-1 and Bn MAH1-2 ORF share 92.4% identities on nucleotide level to each other. Bn MAH1-1 shows 85% of identities to Arabidopsis thaliana MAH1, while Bn PAL1-2 shows 86.4% of identities. Bn MAH1-1 and Bn MAH1-2 share 90.9% identities and 94.0% similarities on protein level to each other. NCBI blastn, multiple alignments of amino acid sequence indicate Bn MAH1-1 and Bn MAH1-2 have the highest homology with predicted Brassica oleracea MAH1, followed by Brassica rapa and Arabidopsis. The phylogenetic tree of P450 s indicates Bn MAH1-1 and Bn MAH1-2 have the highest homology with Arabidopsis MAH1/CYP96A15The deduced Bn MAH1-1 protein is 496-aa in length, with a Mw of 57.06 k Da and a p I of 8.76. The Bn MAH1-2 is also 496-aa in length, with a Mw of 56.92 k Da and a p I of 8.33. The predicted Bn MAH1-1 and Bn MAH1-2 protein were hydrophilic, and both have a signal peptide sequence. The predicted secondary structure of Bn MAH1-1 and Bn MAH1-2 are mainly composed of alpha helix(38.10%, 40.12%) and random coil(35.28%, 32.66%). The beta turn(8.27%, 8.06) and extended strand(18.35%, 19.15%) dispersed throughout the proteins. The predicted tertiary structure indicates Bn MAH1-1 and Bn MAH1-2 protein belong to Cytochrome P450 protein, containing a conserved heme binding domain F436 xx Gx Rx Cx G445.2. Expression analysis of Bn MAH1-1 and Bn MAH1-2The results of Real-time PCR and semi-quantitative indicated that Bn MAH 1-1 and Bn MAH1-2 were expressed in roots, stems, leaves, flowers, and siliques of B. napus. The expression of Bn MAH1-1 and Bn MAH1-2 were the highest in the leaves, followed by in stems and siliques, and the lowest were in the roots. It indicates that Bn MAH1-1 and Bn MAH1-2 may actively participate in the wax synthesis of aerial parts in B. napus, especially in leaves. Analysis of differential expression of Bn MAH1-1 and Bn MAH1-2 in cutivars with wax and without wax showed that the expression of Bn MAH1 in stems and leaves with wax is much higher than that without wax, and Bn MAH1-1 and Bn MAH1-2 were little expressed in leaves without wax, which indicated that wax reduction was related to the down-regulation of MAH1 transcription. After induced by exogenous Me JA for 6h, the expressions of Bn MAH1 and the marker gene PDF1-2 in JA signaling pathway were significantly up-regulated in yuyou 19, while downregulated in zhongshuang 9. ACC, as an ethylene activator, significantly induced the expression of the marker gene ERF2 in ethylene signaling pathway. ACC did not change the expression of Bn MAH1 in yuyou19, while down-regulated the expression of Bn MAH1 in zhongshuang 9. After induced by exogenous SA for 6h, marker gene PR1 of SA signaling pathway was significantly upregulated, but Bn MAH1 was downregulated in both cultivars.3. Construction of Bn MAH1-1 overexpression vector and RNAi vectorPrimers with Sac I/Xma I restriction sites were designed for amplifying the fulllength of Bn MAH1 ORF sequence from B. napus. After sequencing, the target sequence was cloned into the vector p Cambia2301 by Xma I+Sac I, forming recombinant vector p Cambia2301G-Bn MAH1-1, then transformed into Escherichia coli DH5α and Agrobacterium tumefaciens strain LBA4404 to obtain engineering strains.According to the conserved region of MAH1-1 gene, primers were designed for amplifying Bn MAH1-1 RNAi segment by PCR. The antisense fragment and the sense fragment were respectively subcloned into between the promoter and the spacer of p FGC5941 M by Noc I and Aat II, and the spacer and the terminator of p FGC5941 M by Bam HI and Xba I, forming the RNAi vector p FGC5941M-Bn MAH1-1I. The recombinant vector was then transformed into E. coli strain DH5α and A. tumefaciens strain LBA4404 to obtain engineering strain. These vectors can be used to elucidate the functional of the Bn MAH1 through genetic transformation.4. Genetic transformation of Bn MAH 1-1 overexpression vectorWith Agrobactenium-mediated technique, the overexpression vector p Cambia2301 GBn MAH1-1 was used to transform hypocotyls segments of B. napus cv Zhongshuang 11, and nineteen Kan-resistant regenerated plants were obtained. The results of PCR detection showed that 8 transgenic positive plants were finally obtained. |
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