Cloning And Transformation Of Lettuce Vitamin E Biosynthesis Genes LSHPPD And LSHPT

Vitamin E is a generic term for a group of lipid soluble antioxidants, which can be separated into two groups, tocopherols and tocotrienols. Vitamin E plays a crucial role in human health and nutrition. It can only be synthesized by photosynthetic organisms, including higher plants and cyanobacteria. The dietary intake of vitamin E of human body is usually derived from plant materials or drugs. Lactuca sativa L. (lettuce) is a leafy vegetable which is commonly used in various dishes. Thus it is quite meaningful to use genetic engineering strategy to improve vitamin E pathway for increasing vitamin E content in lettuce. The biological function of three enzymes, including 4-hydroxyphenylpyruvate dioxygenase (HPPD), homogentisate phytyltransferase (HPT) and?-tocopherol methyltransferase (?-TMT), was studied in this paper. All of the three enzymes were involved in the vitamin E biosynthetic pathway, and their impact on the pathway was investigated in lettuce (Lactuca sativa L. var. longifolia (romaine lettuce)).1. Molecular cloning and characterization of LsHPPD and LsHPT genes from L. sativa. Genes encoding HPPD and HPT, designated as LsHPPD and LsHPT, were isolated from L. sativa. Sequence data of LsHPPD and LsHPT had been deposited at GenBank under accession numbers of FJ194493 and FJ194492, respectively. The full-length cDNA sequence of LsHPPD contained 1,743 base pairs (bp). It was predicted to encode a protein of 446 amino acids. Multiple alignment of amino acid sequences indicated that LsHPPD shared 77% sequence identity with the HPPD from Medicago truncatula L. It had been reported that the enzyme HPPD contains a Fe²⁺ as an essential cofactor for catalytic activity. Based on the results of multiple alignment and three-dimensional modeling, two histidine residues (His-222, His-304) and one glutamic acid residue (Glu-390) in LsHPPD enzyme were presumed to be involved in the interaction with Fe²⁺. The full-length cDNA of LsHPT contained 1,670 bp, and it was predicted to encode a protein of 395 amino acids. Multiple alignment indicated that LsHPT shared 68% sequence identity with the HPT from Arabidopsis thaliana L. It had been reported in A. thaliana and spinach (Spinacia oleracea L.) that HPT is located in the chloroplast envelope. The LsHPT was a highly hydrophobic protein, and the first 75 N-teriminal amino acids of it exhibited features of a chloroplast targeting sequence. Thus it was presumed that LsHPT might be located in the chloroplast envelope as well. Southern blot analysis suggested that LsHPPD and LsHPT genes were present at low copy number in the lettuce genome. The expression profile showed that the transcriptional level of these two genes was the highest in seeds, followed by that in the fifth to tenth leaves, while in the first to second leaves, roots, cotyledons and stems, the transcriptional level was relatively lower. Generally, the total tocopherol content was relatively higher in the tissues wherein the gene transcriptional level was higher. The expression level of LsHPPD and LsHPT genes was significantly increased accompanied with accumulation of tocopherol content in lettuce plants subjected to stress treatments (high light, drought, and low temperature) and plant hormones (methyl jasmonate and abscisic acid). These results indicated that LsHPPD and LsHPT genes might play an important role in lettuce tocopherol biosynthesis in different tissues and under various inducing conditions.2. Transient expression of LsHPPD and LsHPT genes in L. sativa. In order to further assess the impact of LsHPPD and LsHPT genes on the lettuce vitamin E biosynthesis, transient expression of these two genes via agroinfiltration was utilized. Transcriptional level of LsHPPD and LsHPT genes was rapidly increased in a short time. Real-time fluorescent quantitative RT-PCR analysis showed that the expression level (2-??Ct values) of LsHPPD and LsHPT genes were increased to 13.18 folds and 12.47 folds, respectively, compared with control. High performance liquid chromatography (HPLC) results suggested that the?- tocopherol content in leaves transiently expressing LsHPPD was 25.57±1.37?g/g FW, which were 5.37 folds of the control. The?-tocopherol content in leaves transiently expressing LsHPT was 88.44±3.15?g/g FW, which were 18.58 folds of the control. Transient expression analysis indicated that enhancing the expression level of LsHPPD and LsHPT genes was of benefit to tocopherol biosynthesis, while LsHPT showed a better effect on the tocopherol biosynthesis compared with LsHPPD.3. Stable expression of LsHPPD and LsHPT genes in A. thaliana and L. sativa. Based on the results of transient expression, LsHPPD and LsHPT were utilized in the genetic transformation of A. thaliana to study their impact on the vitamin E biosynthetic pathway. The total content of?- and?-tocopherol in transgenic Arabidopsis expressing LsHPPD and LsHPT genes was increased to 1.51 folds and 2.09 folds, respectively. The results suggested that stable expression of LsHPPD and LsHPT genes could enhance the vitamin E biosynthesis in plants. In order to increase the tocopherol content in lettuce and improve its nutritional quality, LsHPPD and LsHPT genes were introduced into L. sativa by means of genetic transformation. The total content of?- and ?-tocopherol in lettuce overexpressing LsHPPD was 11.57±0.39?g/g FW, which were 1.70 folds of wild-type plants. The total content of tocopherol in lettuce overexpressing LsHPT was 17.77±0.41?g/g FW, which were 2.61 folds of the control. The T1 generation maintained a relatively high level of gene expression and tocopherol content, indicating the genetic stability in the transgenic lines.4. Co-transformation of L. sativa with AtHPT and AtTMT genes from A. thaliana. Overexpression of LsHPPD and LsHPT genes, especially LsHPT, could enhance the tocopherol content in lettuce. However, these two genes were incapable of changing the tocopherol composition. AtHPT and AtTMT genes were constructed into a dual-gene co-transformation vector and transformed into lettuce. The total tocopherol content in transgenic lettuce was increased to 13.62±0.26?g/g FW and the?-tocopherol:?-tocopherol ratio was increased to 4.24. The total vitamin E activity was increased to 3.51 folds compared with control.The results suggested that simultaneous overexpression of AtHPT and AtTMT genes could enhance both the tocopherol content and the?-tocopherol percentage, leading to a significant increase in the total vitamin E acitvity. In this paper, two genes, LsHPPD and LsHPT were isolated from L. sativa. Transgenic plants (A. thaliana and L. sativa) with elevated vitamin E content and activity were obtained through Agrobacterium-mediated genetic transformation. Our work has laid a necessary foundation for the further improvement of the vitamin E biosynthetic pathway in lettuce.