Idenitfication And Functional Analysis Of Lipid Biosynthesis Related Genes In Peanut (Arachis Hypogaea L.)

Triacylglycerols （TAGs） are major source of essential polyunsaturated fatty acids in the humandiets and have a wide range of utilization in industry. They are increasingly used as renewable resourcesfor chemical industries and even biofuel. With the increasing demand of quality and quantity forvegetable oils, enhancing oil productivity and improving quality in oil crops are important objectives inplant breeding. Molecular breeding is one of the most important approaches to improve oil content. Abetter understanding of oil biosynthesis and its regulation mechanism in developing seeds is highlynecessary.Peanut （Arachis hypogaea）, with more than50%oil content in seed, is one of the most importantoil crops and serves as a good source of edible oil. In order to understand how different genes regulatefatty acid composition and TAG formation, we have constructed a seed full-length cDNA library with ahigh oil （56%） peanut line （06-4104）. Several ESTs related with biosynthesis and regulation of fattyacid and TAG were identified from the cDNA library. Based on the EST squences, we cloned theirfull-length sequences, analyzed the expression pattern of these genes, and elucidatdated their functionsand regulation mechanism in TAG biosynthesis via heterogeneous overexpression. The findings arehelpful to increase oil content and modify fatty acid composition through genetic engineering. The mainresults are as follows:1. Two full-length cDNAs encoding lysophosphatidic acid acyltransferase （LPAT） were clonedfrom peanut and designated as AhLPAT2（1753bp） and AhLPAT4（1631bp）. AhLPAT2and AhLPAT4encoded a putative membrane binding proteins with387and383amino acids, which were closelyrelated to AtLPAT2（Q8LG50） in Arabidopsis and GmLPAT4（XP₀03544428） in soybean, respectively.There were several copies of AhLPAT2in cultivated peanut genome. Both AhLPAT2and AhLPAT4wereubiquitously expressed in diverse organs. AhLPAT2was expressed predominately in developing seed,and its expression correlated well with the rate of oil accumulation.2. Seed-specific overexpression of AhLPAT2gene in Arabidopsis increased seed weight anddecreased protein content. Content of total fatty acid and unsaturated fatty acids significantly increasedcompared to the wild-type. In siliques of homozygous transgenic lines, the relative expression levels ofseveral genes （fatty acid and TAG biosynthesis, Suc metabolism and glycolytic pathway） were alsosignificantly increased.3. Four full-length cDNAs encoding transcription factors involved in embryonic development werecloned and characterized. Two types of transcription factors were obtained including AhLEC1-likeA andAhLEC1-likeB. The phylogenetic tree showed that AhLEC1-likeA and AhLEC1-likeB shared highidentity （>75%） with TcLEC1-like （CAM35799） in Theobroma cacao. It also shared a high amino acidsequence identity with the eukaryotic LEC1-type HAP3protein subunits from Arabidopsis thaliana andBrassica napus. AhLEC1-likeA and AhLEC1-likeB transcripts were both accumulated primarily indeveloping seed and consistent with the rate of oil accumulation. Transient expression analysis in onionepidermal cells indicated that AhLEC1-likeA and AhLEC1-likeB were specifically localized in nuclei.Yeast transactivation assay and truncated mutation analysis demonstrated that AhLEC1-likeA and AhLEC1-likeB were able to activate transcription in vitro and they contained more than one activationdomains.4. A constitutive over-expression vector of AhLEC1-likeA gene under the control of35S promoterand a seed-specific over-expression vector of AhLEC1-likeA under the control of napin promoter wereconstructed, respectively, and they were used to transform the wild-type Arabidopsis via Agrobacterium.There was no detrimental effect on embryogenesis and seed maturation in transgenic plants.Over-expression of AhLEC1-likeA enhanced fatty acid accumulation in seeds of both T2and T3generation transgenic plants. Generally, compared with that of wild-type seeds, the relative ratio ofC16:0and C18:2in total fatty acids were significantly decreased, whereas that of C20:1wassignificantly increased. The unsaturated fatty acid ratio was also slightly increased. The C18fatty acidsratio reduced, whereas the C20fatty acids increased more than15%compared with that of thenontransformed plants. In siliques of transgenic plants, the relative expression levels of genes in fattyacids biosynthesis genes were ubiquitously elevated. The most apparently up-regulated genes wereFAD2and KCS18. The relative expression of the genes encoding plastic enzymes involved in TAGassembly, Suc metabolism and glucolytic reaction, was also increased compared with control plants.Similarly, the expression of several additional transcription factors demonstrated previously to play arole in seed maturation and oil biosynthesis were also up-regulated. The results indicated thatAhLEC1-likeA may play an essential role in fatty acid biosynthesis and compostion modification.