Cloning,Expression And Vector Construction Of Fatty Acid Acyl-CoA Reductase Gene Family Members From Brassica Napus L.

Plant cuticular waxes have a variety of physiological and ecological functions, which is believed to be initial defense system to external environment stresses. Cuticular waxes are mostly composed of very-long-chain fatty acids 、 alkanes 、aldehydes、acids、alcohols、ketones、esters and so on. Fatty acyl-CoA reductase is a functional enzyme involved in the wax primary alcohol formation. In this study, four ORF sequences of FAR gene(BnCER4-1, BnCER4-2, BnCER4-3 and Bn CER4-4) were cloned from Brassica napus, and were analyzed by bioinformatics tools. Their expression pattern and differential expression between cultivars with different wax content were also analyzed. The overexpression vector and RNAi vector of BnCER4-4 were constructed, laying the foundation for further study on gene function by genetic transformation. The main results are as follows:1. Cloning and sequence analysis of BnCER4 gene families from B. napusFour ORF sequences of B. napus FAR genes were cloned by silico cloning and RT-PCR, and they were named BnCER4-1(GenBank: KT795333) 、 BnCER4-2(GenBank: KT795334)、BnCER4-3(GenBank: KT795335)、BnCER4-4(GenBank: KT795336). The length of BnCER4-1 and BnCER4-2 ORFs were 1479 bp, which encodes a protien of 492 amino acids. The length of BnCER4-3 and BnCER4-4 ORFs were 1482 bp, which encodes a protien of 493 amino acids. Guanine and cytosine contents of BnCER4-1、BnCER4-2、BnCER4-3 and BnCER4-4 are 41.58%、41.58%、41.36% and 41.5% respectively. NCBI blastn and Multiple alignments of amino acid sequence showed that BnCER4 genes have the highest homology with FAR from B.rapa and B.oleracea, followed by Arabidopsis thaliana and Camelina sativa. Their secondary structures were mainly composed of alpha helix(38.62%-42.39%), followed by the random coil(25.20%-26.17%) and the extended strand(21.91%-25%), the least beta turn(9.53%-10.77%). The subcellular localization showed that BnCER4-1、BnCER4-2、BnCER4-3、BnCER4-4 were most likely localized in the chloroplas. Conserved Domain search detected a NAD(P)H-binding Rossmann-fold domain at the N-terminus as well as a fatty acyl-Co A reductase domain at the C-terminus within these four genes. At N-terminus, the motif GXXGXX(G/A) and the classic YXXXK active site were found in these four genes. The characteristics of these four genes were accord with a typical plant FAR.2. Expression analysis of BnCER4 gene familyOrgan-specificity of BnCER4 gene familiy members expression levels in Brassica napus L. is significantly different. BnCER4-1/2 was found to be highly expressed in leaves, followed by stems、siliques、flowers, but was very weakly in roots. The highest expression of BnCER4-3/4 was also in leaves, followed by siliques、stems、flowers. BnCER4-3 was not detected in roots, while BnCER4-4 had low expression in roots. These indicated BnCER4 family members may actively participate in the wax synthesis of aerial parts in B. napus. BnCER4-1/2 may play a major role in leaf wax deposition and BnCER4-3/4 may widely involved in wax deposition of aerial parts. In stems, the expression of BnCER4-3/4 in zhongshuang 11 is significant higher than wax mutant, while no significant difference of BnCER4-1/2 expression was detected. The expression of BnCER4-1/2 and BnCER4-3/4 are significantly reduced in leaf of wax mutant. Particularly, the expression of BnCER4-4 is is not detected in wax mutant leaves. These results indicated that the reduced wax deposition in mutants was related with transcriptional downregulation of BnCER4 family members. SA, MeJA, and ACC treatment differentially induced the expression of BnCER4-1/2 in the zhongshuang 9 and yuyou 19.3. Construction of BnCER4-4 overexpression vector and RNAi vectorPrimers with Sac I/XmaI restriction sites were designed for amplifying the full-length of BnCER4-4 ORF sequence from B.napus. After sequencing, the target sequence was cloned into the vector pCambia2301 by Xma I+SacI, forming recombinant vector pCambia2301G-BnCER4-4, then transformed into Escherichia coli DH5α and Agrobacterium tumefaciens strain LBA4404 to obtain engineering strains.According to the conserved sequences of BnCER4-3/4, primers were designed to amplify a 284 bp RNAi fragment. The antisense fragment was subcloned between the promoter and the spacer of pFGC5941 M by NocI and Aat II double digestion, and the sense fragment was subcloned between the spacer and the terminator of pFGC5941 M by BamHI and XbaI, forming the RNAi vector p FGC5941M-BCER4 I. It was successfully verified by multiple PCR checking and then transformed into Agrobacterium tumefaciens strain LBA4404 to form engineering strain.