Cloning And Characterization Of Important Genes For Vitamin E Synthesis In Arachis Hypogaea

Vitamin E is an important antioxidative substance for human beings and animals, and human body cannot produce vitamin E itself, and mainly obtains it from vegetable oil, and other plant products. Vitamin E can eliminate oxygen free radicals and against lipid oxidation, and is considered to have function in reducing risk of cardiovascular diseases and cancer. In plants, vitamin E is thought to relate to stress tolerance and seed longevity, etc. As a lipid-soluble product, vitamin E exists in many parts of plants like leaf, flower and seeds, etc., mainly available in seed oil. There are two kinds of vitamin E, tocopherols and tocotrienols and each has alfa-, beta-, gamma-and sigma-four isoforms. The alfa type has the highest activity among the isoforms and human body is preferential to alfa-tocopherol during retained and distribution. Genes related to vitamin E bio-synthesis have been cloned from many photosynthetic organisms so far. These genes were being transformed into maize, soybean and rice to improve the quality of the crops through transgenic technology.Peanut （Arachis hypogasa） is an important oil and protein crop worldwide. Peanut seed contains as high as 50% of oil. To understand peanut vitamin E metabolism and to improve the vitamin E content of peanut in the end, we cloned several genes involved in the vitamin E synthesis by RACE including tocopherol cyclase gene （AhVTE1）, homogentisate phytyltransferase gene （AhVTE2-1 and AhVTE2-2） and tocopherol methyltransferase gene （AhVTE4/Ahy-TMT）. We also analyzed their structure feature, expression and evolutionary in the paper.The tocopherol cyclase gene was cloned by RACE based on the results of microarray and 454-sequencing. The gene was named AhVTE1 and its full length mRNA is 1622bp with an open reading frame （ORF） of 1368bp. Its genome DNA was also cloned consisting 9 introns. Multiple alignments showed that AhVTEl is similar to the counterpart of glycine_max （GeneBank Accession NO. XP₀03526485）. Phylogenetic tree construction demonstrated that AhVTE1 may derive from the wild type A. ipaensis. RT-PCR results showed that seed testa express the highest level of AhVTEl and in turn is root and leaf. During the process of embryo development (（20 DAP,40 DAP and 60 DAP）, the AhVTEl expression level gradually increase, with a significant rising in 60 DAP. Microarray analysis demonstrate that the AhVTEl did not react significantly to the treatment of salicylic acid （SA）, abscisic acid （ABA）, paclobutrazol （PAC） and jasmonic acid methyl ester （MeJ）, besides ethephon which decrease AhVTE1 expression by 22%. Low temperature decrease in AhVTE1 expression, while the drought stress upregulated its expression.Two homogentisate phytyltransferase genes （AhVTE2-1 and AhVTE2-2） were cloned by RACE and their full length are 1439bp and 1965bp, with an 1170bp ORF and a 1218bp ORF, respectively. Multiple alignments and Phylogenetic tree construction results showed that AhVTE2-1 and AhVTE2-2 belonged to different branches. The AhVTE2-1 location in the tree is far from other species VTE2-1 genes, while AhVTE2-2 is most similar to the glycine_max （GeneBank Accession NP₀01237900） VTE2-2. Evolution tress showed that the AhVTE2-2 is from the A. ipaensis. RT-PCR results showed that the AhVTE2-1 expression level in root is the highest and the AhVTE2-2 is in seed testa. And the AhVTE2-1 and AhVTE2-2 expression levels are gradually rising during the embryo development （20 DAP,40 DAP and 60 DAP）. Microarray data show that both the two genes decrease their expression level when suffered with low temperature and drought stress. The AhVTE2-1 expression levels after treated with the above five kinds of hormones are all increase and SA upregulated 6-folds of it. The AhVTE2-2 expression levels however, did not change much in expression by these hormones.By similarly method, tocopherol methyltransferase gene of peanut was got. The cDNA size is 1232bp with a 909 ORF. RT-PCR results show that the AhVTE4 expresses highest in the seed testa, and then in turn is pericarp, leaf, inflorescence, stem, root, peg and embryo. With the developing of embryo, the AhVTE4 expression level showed decrease. Microarray results show that AhVTE4 gene expression level was down regulated under PAC treatment, but SA, ABA and ETH did not affect the AhVTE4 gene expression level. AhVTE4 gene expression level decrease when suffer from drought stress, however, its expression level was not affect in low temperature.