多毛番茄GGPS基因对提高番茄中番茄红素含量的功能研究
摘要
番茄红素(Lycopene)含量是评价番茄果实品质的重要指标。番茄红素含量越高,果实越鲜艳,品质越好。医学研究表明:番茄红素具有预防并降低人类患多种疾病的保健功能。
虽然类胡萝卜素合成的基因工程研究已经取得了显著的进展,但是哪些基因对于果实中的番茄红素积累具有关键作用尚不是很清楚。本课题组在前期的研究中发现,将载有GGPS基因的多毛番茄第4条染色体片段导入到普通番茄中,能显著提高果实中番茄红素含量。进一步的研究表明,多毛番茄中的GGPS基因与普通番茄中的GGPS基因存在23个碱基差异。因此,推测多毛番茄的GGPS基因对于提高番茄果实中番茄红素含量可能具有重要作用。
本试验拟在此研究基础上,以多毛番茄和普通番茄的GGPS基因为目的基因,构建植物表达载体(分别命名为PBI121-T-GGPS和PBI121-E-GGPS),通过根瘤农杆菌EHA105介导转入到番茄红素含量一般的番茄材料Moneymaker中。通过分析转基因植株中目的基因表达与番茄红素含量的关系,明确两种不同来源的GGPS基因对于番茄果实中番茄红素含量积累的调控功能上的差异。主要研究结果如下:
1、优化了番茄高效转化再生体系并利用分子生物学方法分析并获得了转基因阳性植株。以MS为基本培养基,筛选并获得了最佳的激素组合和浓度配比:ZT 2 mg·L-1+IAA0.2 mg·L-1;确定卡那霉素筛选阳性植株的最合适浓度为50 mg·L-1。采用叶盘法,通过根瘤农杆菌EHA105介导番茄转化Moneymaker,在卡那霉素的选择压力下筛选并获得再生植株。PCR检测初步证明目的基因已插入到受体材料基因组内;Southern印迹杂交从阳性转基因株系中筛选出一部分单拷贝的转基因植株;RT-PCR分析表明插入的GGPS基因已在转基因番茄体内正常转录表达。上述分子生物学方法检测结果表明:GGPS基因已经插入到植物基因组中并且能够转录及表达;T0代获得了转T-GGPS基因的阳性植株15株,转E-GGPS基因的阳性植株17株,转空载体的阳性植株4株。
2、分析了T1代转基因植株番茄红素含量等生理指标。利用高效液相色谱法(HPLC)测定T1代转基因番茄果实中番茄红素含量:转T-GGPS基因的阳性植株与转E-GGPS基因的阳性植株相比,番茄红素含量存在极显著差异,前者番茄红素含量在104.1-110.9 mg?Kg-1之间,后者番茄红素含量在70.8-78.0 mg?Kg-1之间。转基因植株与对照相比,β-胡萝卜素含量和叶绿素含量没有差异性。
3、转T-GGPS基因植株与阴性对照相比,GGPS基因在不同器官中的表达量增加。以β-actin基因为内标基因,对转T-GGPS基因植株和野生番茄植株的叶、花以及四个不同时期(膨大期、绿熟期、转色期和完熟期)的果实中所含的GGPS基因进行半定量研究。结果表明:转基因植株与阴性对照相比,除转色期的果实中变化不明显外,在不同器官中GGPS基因的表达量在转录水平上显著增加。
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.
虽然类胡萝卜素合成的基因工程研究已经取得了显著的进展,但是哪些基因对于果实中的番茄红素积累具有关键作用尚不是很清楚。本课题组在前期的研究中发现,将载有GGPS基因的多毛番茄第4条染色体片段导入到普通番茄中,能显著提高果实中番茄红素含量。进一步的研究表明,多毛番茄中的GGPS基因与普通番茄中的GGPS基因存在23个碱基差异。因此,推测多毛番茄的GGPS基因对于提高番茄果实中番茄红素含量可能具有重要作用。
本试验拟在此研究基础上,以多毛番茄和普通番茄的GGPS基因为目的基因,构建植物表达载体(分别命名为PBI121-T-GGPS和PBI121-E-GGPS),通过根瘤农杆菌EHA105介导转入到番茄红素含量一般的番茄材料Moneymaker中。通过分析转基因植株中目的基因表达与番茄红素含量的关系,明确两种不同来源的GGPS基因对于番茄果实中番茄红素含量积累的调控功能上的差异。主要研究结果如下:
1、优化了番茄高效转化再生体系并利用分子生物学方法分析并获得了转基因阳性植株。以MS为基本培养基,筛选并获得了最佳的激素组合和浓度配比:ZT 2 mg·L-1+IAA0.2 mg·L-1;确定卡那霉素筛选阳性植株的最合适浓度为50 mg·L-1。采用叶盘法,通过根瘤农杆菌EHA105介导番茄转化Moneymaker,在卡那霉素的选择压力下筛选并获得再生植株。PCR检测初步证明目的基因已插入到受体材料基因组内;Southern印迹杂交从阳性转基因株系中筛选出一部分单拷贝的转基因植株;RT-PCR分析表明插入的GGPS基因已在转基因番茄体内正常转录表达。上述分子生物学方法检测结果表明:GGPS基因已经插入到植物基因组中并且能够转录及表达;T0代获得了转T-GGPS基因的阳性植株15株,转E-GGPS基因的阳性植株17株,转空载体的阳性植株4株。
2、分析了T1代转基因植株番茄红素含量等生理指标。利用高效液相色谱法(HPLC)测定T1代转基因番茄果实中番茄红素含量:转T-GGPS基因的阳性植株与转E-GGPS基因的阳性植株相比,番茄红素含量存在极显著差异,前者番茄红素含量在104.1-110.9 mg?Kg-1之间,后者番茄红素含量在70.8-78.0 mg?Kg-1之间。转基因植株与对照相比,β-胡萝卜素含量和叶绿素含量没有差异性。
3、转T-GGPS基因植株与阴性对照相比,GGPS基因在不同器官中的表达量增加。以β-actin基因为内标基因,对转T-GGPS基因植株和野生番茄植株的叶、花以及四个不同时期(膨大期、绿熟期、转色期和完熟期)的果实中所含的GGPS基因进行半定量研究。结果表明:转基因植株与阴性对照相比,除转色期的果实中变化不明显外,在不同器官中GGPS基因的表达量在转录水平上显著增加。
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.
引文
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