Expression Profile And Functional Characterization Of KCS Gene Family In Brassica Napus

KCS gene family encode β-ketoacyl CoA synthase, a rate-limiting enzyme in the synthesis of very long chain fatty acid (VLCFA), catalyzing the condensation reaction of VLCFA elongation process. Cloning of KCSgenes help to elucidate the function of the KCSgenes in the synthesis of VLCFAs and wax, and provide a new source of genes for improving rapeseed fatty acid composition.In this study, FAE1gene sequences were isolated from high erucic acid B. napus varieties Zhongyou821, zero-erucic acid wild species Orychophragmus violaceus L.and Capsella bursapastrois L., wild species containing erucic acid Sinapis alba L., Sinapis arvensis L. and Isatis indigotica Fort. Comparison of FAE1sequences indicated that the FAE1gene of Zhongyou821showed more than85%identity with that of five wild species. Heterologous expression of the FAE1genes was performed in the yeast cell, all the FAE1genes from the wild cruciferous species can normally express in yeast cells. The yeast cells expressing FAE1genes from O. violaceus and C. buraspastroil showed no VLCFA formation, and those expressing FAE1genes from S. arvensis, S. alba, and I. indigotica produced trace amount VLCFA. Experimental results show that the low erucic acid trait of O. violaceus and C. buraspastroil comes from the inactivation of protein activity encoded by FAE1gene. The FAE1nucleotide sequences are highly conserved between high erucic acid species and zero erucic acid species.FAE1gene is the rate-limiting enzyme in the synthesis of erucic acid. The high erucic acid rapeseed varieties can be improved for low erucic acid rapeseed varieties through directed inhibition of FAE1gene function. The full-length sequence of FAE1gene was chosen to construct the FAE1RNAi vector. The FAE1sequences were cloned to downstream of the seed-specific Napin promoter as a head-to-head concatemer, and the inverted repeats of FAE1sequence were spaced apart by a histone H2A intron. The FAE1RNAi vector was transformed into high erucic acid B. napus varieties Zhongyou821by Agrobacterium-mediated method. After transformation,81transformants were obtained, in which,51positive seedlings were identified by PCR method. GC analysis of fatty acid composition of transgenic T1generation seeds revealed that the erucic acid content of transgenic rapeseed decreased in different degree compared with the wild type. The erucic acid content of recipient varieties Zhongyou821was43.35%, that of21transformants is less than35%, in which, the erucic acid content of two transformants decrease to10-20%. Near-infrared analysis of T2generation seeds showed that the erucic acid content of most lines displayed no significant difference compared with high erucic acid rapeseed. The erucic acid trait displayed separation ratio of1:3for the descendants of one line, the erucic acid content of low erucic acid transgenic plant was at the level of2%. The results revealed that the FAE1silence mediated by RNAi technology can effectively reduce the erucic acid content of transgenic plants, but can not reduce to zero erucic acid level. And low erucic acid transformants have obvious unintended effects, showing seed shrinkage, irregular shape, seed coat glossless and difficult to normally grow.On the basis of previous genomewalking and RACE experiment, we cloned17KCS gene members, including BnKCS1, BnKCS3, BnKCS4, BnKCS5, BnKCS6, BnKCS8, BnKCS9, BnKCS10, BnKCS11, BnKCS12, BnKCS13, BnKCS15, BnKCS16, BnKCS17, BnKCSl9, BnKCS20and BnKCS21. According to the pedigree analysis, BnKCS gene members can be divided into six sub-categories. The sequence homology ranges from45.6%to63.1%between the FAE1gene and other members of the KCS gene family. BnKCS4, BnKCS8, BnKCS9, BnKCS16, BnKCS17and FAE1genes belong to one sub-category, these genes show more than60%identity with FAE1gene. Expression profile analysis of BnKCS genes showed that there are9BnKCS genes expressing in the seeds, including BnKCS1, BnKCS4, BnKCSS, BnKCS6, BnKCS9, BnKCS10, BnKCS11, BnKCS19and BnKCS20. these genes exhibit greater than50%homology with FAE1gene. It was speculated that the FAE1full-length sequence was used to construct FAE1RNAi vector, which not only inhibit the expression of FAE1gene, but also synergisticlly inhibit the expression of homologous genes, thus, resulting in unintended effects. In the future, it is better to identify FAE1specific sequence for constructing FAE1RNAi or MicroRNAi vector on the basis of a comprehensive analysis of the sequences of the KCS gene family, to achieve the specific purpose of inhibiting the function of the FAE1gene.The conserved domain FAEl-CUT1-RPPA of KCS family was usend to search the KCS sequences across the whole genomic sequence of B. napus, and40KCS sequences were obtained. Wherereas, no homologous sequence of KCS3, KCS7, KCS8, KCS11, KCS12, KCS16and KCS21were retrieved. In this study, BnKCS3, BnKCS8, BnKCS11, Bn KCS12, BnKCS16and BnKCS21were isolated form B. napus. It is concluded that the KCS family contains46gene members at least in B, napus.The real-time PCR method was used to analyze the expression profile of BnKCS genes in different tissues of B. napus, including root, stem, leaf, bud, stigma, pollen,30d pod skin,40d pod skin,30d seeds and40d seed. The BnKCS genes have different expression pattern in different tissues or organs. The analysis results shows that18BnKCS genes are expressed in the floral organ of B. napus,10BnKCS genes expressed in the stem,13BnKCS genes expressed in leaves,9genes expressed in seed,8genes expressed in the pod skin.5genes (BnKCS2, BnKCS7, BnKCS8, BnKCS9and BnKCS21) are specifically expressed in floral organ, of which, BnKCS2and BnKCS9are specially expressed in the pollen, BnKCS21is specifically expressed in the petals.17BnKCS genes were cloned to downstream of the galactose-inducible promoter of the yeast expression vector pYES2/NT, to perform heterologous expression in yeast cells.15BnKCS gene can normally translate protein, and the BnKCS3and the BnKCS4fail to be expressed due to the codon preference. GC analysis of yeast fatty acid composition indicated that the VLCFAs were only observed in yeast expressing the BnKCS1and FAE1genes, and no VLCFA synthesis in yeast expressing other BnKCS genes. It was concluded that these BnKCS genes can not take advantage of the fatty acids in yeast as a catalytic substrate to synthesize VLCFA. In follow-up studies, VLCFAs should be added to the yeast medium in vitro for further studying of BnKCS gene function.To reveal the relationship between the KCS genes and wax biosynthesis, the homozygotes of7Arabidopsis Kcs mutant were identified, including kcs4, kcs7kcs9, kcsll kcs13, kcs16and kcs17. The KCS gene expression profile analysis showed that the target gene in the mutant was silenced due to the insertion of the T-DNA. The wax crystal density of the surface of Arabidopsis siliques and stems were scanned using a scanning electron microscope, and found that the wax crystal density of the mutant does not significantly change compared with that of the wild type. GC-MS was carried out to analyze the wax composition of Arabidopsis siliques, stems and leaves. Compared to the wild-type, part of the wax components were significantly reduced in stems, siliques and leaves for kcs mutants with except of the kcs4mutant. And the wax components with significant change have different chain length, including both the product of the acyl reduction pathway, such as primary alcohols, and that of decarbonylation pathway, such as alkanes, aldehydes and29C ketone. The kcs mutant did not appear serious deficiencies of surface wax, or excessive accumulation of a derivative of VLCFA and total loss of someone wax composition. This study revealed that the catalytic function was overlapping with other KCS gene for the KCS7, KCS8, KCS9, ACS11, KCS13, KCS16and KCS17genes, their catalytic product is not a VLCFA with specific chain length, involved in the metabolism of both acyl reduction pathway and decarbonylation pathway.