| Lycopene content is an important indicator of tomato fruit quality. The higher content of lycopene in tomato fruit, the more colorful and the better quality they are. Medical researches show that lycopene has the health care functions of preventing and reducing the risk of many diseases.Although genetic engineering researches related to carotenoid synthesis has made significant progress, but it is not still clear which genes have a key role in the accumulation of lycopene in the fruit. Our previous studies have proved that fruit lycopene content could be significantly increased by importing the end fragments of chromosome 4 which contains the GGPS gene into nomal tomatoes.Further researches showed that there were 23 base differences between the GGPS genes cloned from Solanum habrochaites and from common tomato. Therefore, we speculate that the GGPS gene cloned from Solanum habrochaites may have an important role in increasing fruit lycopene content of tomato. Intensive study was carried on in this paper, GGPS genes cloned from Solanum habrochaites and common tomato were used as the target genes. Two plant expression vectors were constructed which were respectively named as PBI121-T-GGPS and PBI121-E-GGPS, and then transformed into Moneymaker via EHA105.By analysis of the relationship between lycopene content and gene expression in transgenic plants, it clarified the functional differences between two different sources of GGPS genes in regulating tomato lycopene accumulation.The major findings are as follows:1. An efficient regeneration transformation system of tomato was optimized and the positive transgenic plants were obtained by molecular biology methods.Using MS as the basal medium, the best hormone combination and concentration were obtained, of which Trans-zeatin was 2 mg?L-1+IAA0.2 mg?L-1, and kanamycin was 50 mg?L-1.Using leaf disc method, Moneymaker were transformated via EHA105, and the regeneration plants were obtained under the selection pressure of kanamycin. Through PCR detection, it was initially proved that the target gene had been imported into the genome; a part of transgenic plants of a single copy were selected by Southern blot; RT-PCR analysis showed that the GGPS gene imported in was transcribed normally in transgenic tomato. Results showed that GGPS gene had been inserted into the plant genome and it was normally transcribed and expressed. 15 positive plants with T-GGPS gene in T0 generation, 17 positive plants with E-GGPS gene and 4 positive plants transferred with empty vector were obtained in this study.2. We analyzed lycopene content and other physiological indexes in transgenic tomato of T1 generation. Measuring of lycopene content of T1 transgenic tomato by HPLC, results showed that lycopene contents in T-GGPS positive plants and the positive plants transferred with empty vector have significant differences. The lycopene content of the former varies from 70.8 mg?g-1 to 78 mg?g-1, the latter varies from mg?g-1 to 110.9 mg?g-1. Theβ-carotene content and chlorophyll content in transgenic plants have no difference compared with the control.3. By comparing T-GGPS transgenic plants with the negative control, it is proved that expression of GGPS gene was increased in different organs. Theβ-actin gene was chose as the standard gene. Semi-quantitative RT-PCR was used to analyze the expression level of GGPS gene in leaves, flowers, and four different stages of fruits (enlargement period, mature green, turning stage and mature) in transgenic tomato and wild tomato. The results showed that almost in the whole growing stages GGPS gene was expressed more in transgenic plants than in the wild tomato, but the fruit color changed stage is an exception. |
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