红肉脐橙（Citrus sinensis [L.] Osbeck）番茄红素β-环化酶基因（Lcyb）功能的初步验证和分析
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摘要
红肉脐橙(Citrus sinensis[L.]Osbeck)是华盛顿脐(Citrus sinensis[L.]Osbeck)的红肉突变体,是果肉呈粉红的脐橙品种,其果肉中主要含有番茄红素和β-胡萝卜素,是研究柑橘果实类胡萝卜素代谢的良好素材。番茄红素β-环化酶(LCYB)在柑橘类胡萝卜素合成代谢途径中起关键作用,该酶能使反式番茄红素转化为β-胡萝卜素,是番茄红素向下游代谢物质转化的一个关键分支点。
目前,本课题组已经从红肉脐橙中克隆到番茄红素β-环化酶基因(Lcyb),为了对该基因的功能进行鉴定分析,本研究以红肉脐橙中克隆到的番茄红素β-环化酶基因(Lcyb)两个转录本的cDNA为目的片段,通过农杆菌介导的转基因手段,以番茄(Lycopersicon esculentum Mill.)为受体材料进行了遗传转化,通过抗性筛选,获得转基因植株群体,通过对转基因植株群体及其后代进行分子水平的检测和生理指标测定分析,表明红肉脐橙中克隆到的番茄红素β-环化酶基因(Lcyb)及其产物具有正常功能。主要结果如下:
1.利用已构建成功的含有红肉脐橙番茄红素β-环化酶基因(Lcyb)cDNA片段的载体,通过农杆菌介导的遗传转化方法将它们成功导入番茄品种中蔬5号中。获得卡那霉素抗性植株14棵,提取部分T_0代转化番茄PCR阳性植株的基因组DNA,经Southern杂交分析,证明载体的T-DNA区段已经整合到番茄基因组中,拷贝数1-3个不等。
2.T_0代转化番茄中有两棵植株的果实在成熟时果实色泽表现出明显的改变,成熟果实呈黄色,对照的非转化植株成熟果实则为红色。对T_0代番茄果实进行了色差值的测定,发现多数转基因果实相对于对照偏黄。通过HPLC(高效液相色谱)测定黄色转基因果实的色素成分和比例发现,其β-胡萝卜素的含量极显著的高于对照,而番茄红素的含量则显著低于对照。说明转基因植株中,外源β-环化酶基因(Lcyb)确实超量表达,致使番茄红素大量向β-胡萝卜素转化,导致果实积累β-胡萝卜素而呈现出黄色。
3.提取部分T_0代转化番茄PCR阳性植株的RNA,通过RT-PCR检测外源基因的相对表达量,14株转基因植株中有8株检测到目的基因的表达,表达量各有高低。通过Real-time PCR检测转基因植株果实中其他色素代谢相关基因的表达情况,发现在色素代谢途径中,位于Lcyb基因上游的基因在转基因植株中的表达量普遍降低,而位于Lcyb基因下游的基因在转基因植株中的表达量上升。
4.对Southern杂交检测的阳性植株,收获T_1代种子。对T_1世代植株PCR扩增分析鉴定,发现单拷贝的植株NPTⅡ基因分离符合1:3的比例。
'Cara Cara' navel orange(Citrus sinensis[L.]Osbeck) red-fleshed cultivar,is a bud sport of 'Washington' navel orange.The major pigments in the flesh of 'Cara Cara' navel orange are lycopene and beta-carotene.It is useful for the research of the carotenoid biosynthesis of the citrus fruits,lycopene beta-cyclase(LCYB) plays a very important role in the carotenoid biosynthesis of the citrus fruit.Lycopene is transformed into beta-carotene by the action of lycopene beta-cyclase(LCYB).It's a key point in the transformation from Lycopene to other pigments in the carotenoid metabolic pathway.
Based on the cloned lycopene beta-cyclase gene(Lcyb) from 'Cara Cara' navel orange, we transformed the cDNA fragment of this gene into tomato(Lycopersicon esculentum Mill.) plants 'Zhongshu No.5' using the Agrobacterium-mediated transformation to verify its function.We verified that the kanamycin-resistant gene had been integrated into the genome DNA after PCR and Southern blotting hybridization;and analyzed the physiological and biochemical characteristics of the transgenic plants.The main results were as follows:
1.Expression vectors which were constructed with cDNA fragments of the Lcyb gene were introduced into the tomato plants via Agrobacterium-mediated transformation. A total of 14 kanamycin-resistant plants were obtained.The PCR and Southern blot analysis of the putative transgenic plants showed that the T-DNA was integrated into the tomato genome with various copy numbers from 1-3.
2.The color of the fruits from two transgenic tomato plants were obvious yellow compared with the red fruits from the non-transgenic tomato plants.Measurements of the contents of lycopene and beta-carotene in the transgenic fruits by using high performance liquid chromatography(HPLC),showed that the contents of beta-carotene in yellow transgenic fruits were much higher than that in red fruits of wide-type plants.Oppositely, the contents of lycopene were very low in the yellow transgenic fruits.It's suggested that over-expression of lycopene beta-cyclase gene(Lcyb) in the transgenic plants resulted in accumulation of beta-carotene in the transgenic fruits which was absence in the wide-type plants.
3.RT-PCR analysis showed that the activities of target gene in 8 out of 14 different transgenic plants expressed,whereas the expression levels were different in individuals. Real-Time PCR analysis showed the expression of genes had involved in the carotenoid biosynthesis in the transgenic fruits.
4.The inheritance pattern of the transgenes in T_1 segregation population were analyzed based on PCR with NPTⅡprimers.And the results showed that NPTⅡgene inherited as a dominant genetic locus with the segregation ratio 3:1.
目前,本课题组已经从红肉脐橙中克隆到番茄红素β-环化酶基因(Lcyb),为了对该基因的功能进行鉴定分析,本研究以红肉脐橙中克隆到的番茄红素β-环化酶基因(Lcyb)两个转录本的cDNA为目的片段,通过农杆菌介导的转基因手段,以番茄(Lycopersicon esculentum Mill.)为受体材料进行了遗传转化,通过抗性筛选,获得转基因植株群体,通过对转基因植株群体及其后代进行分子水平的检测和生理指标测定分析,表明红肉脐橙中克隆到的番茄红素β-环化酶基因(Lcyb)及其产物具有正常功能。主要结果如下:
1.利用已构建成功的含有红肉脐橙番茄红素β-环化酶基因(Lcyb)cDNA片段的载体,通过农杆菌介导的遗传转化方法将它们成功导入番茄品种中蔬5号中。获得卡那霉素抗性植株14棵,提取部分T_0代转化番茄PCR阳性植株的基因组DNA,经Southern杂交分析,证明载体的T-DNA区段已经整合到番茄基因组中,拷贝数1-3个不等。
2.T_0代转化番茄中有两棵植株的果实在成熟时果实色泽表现出明显的改变,成熟果实呈黄色,对照的非转化植株成熟果实则为红色。对T_0代番茄果实进行了色差值的测定,发现多数转基因果实相对于对照偏黄。通过HPLC(高效液相色谱)测定黄色转基因果实的色素成分和比例发现,其β-胡萝卜素的含量极显著的高于对照,而番茄红素的含量则显著低于对照。说明转基因植株中,外源β-环化酶基因(Lcyb)确实超量表达,致使番茄红素大量向β-胡萝卜素转化,导致果实积累β-胡萝卜素而呈现出黄色。
3.提取部分T_0代转化番茄PCR阳性植株的RNA,通过RT-PCR检测外源基因的相对表达量,14株转基因植株中有8株检测到目的基因的表达,表达量各有高低。通过Real-time PCR检测转基因植株果实中其他色素代谢相关基因的表达情况,发现在色素代谢途径中,位于Lcyb基因上游的基因在转基因植株中的表达量普遍降低,而位于Lcyb基因下游的基因在转基因植株中的表达量上升。
4.对Southern杂交检测的阳性植株,收获T_1代种子。对T_1世代植株PCR扩增分析鉴定,发现单拷贝的植株NPTⅡ基因分离符合1:3的比例。
'Cara Cara' navel orange(Citrus sinensis[L.]Osbeck) red-fleshed cultivar,is a bud sport of 'Washington' navel orange.The major pigments in the flesh of 'Cara Cara' navel orange are lycopene and beta-carotene.It is useful for the research of the carotenoid biosynthesis of the citrus fruits,lycopene beta-cyclase(LCYB) plays a very important role in the carotenoid biosynthesis of the citrus fruit.Lycopene is transformed into beta-carotene by the action of lycopene beta-cyclase(LCYB).It's a key point in the transformation from Lycopene to other pigments in the carotenoid metabolic pathway.
Based on the cloned lycopene beta-cyclase gene(Lcyb) from 'Cara Cara' navel orange, we transformed the cDNA fragment of this gene into tomato(Lycopersicon esculentum Mill.) plants 'Zhongshu No.5' using the Agrobacterium-mediated transformation to verify its function.We verified that the kanamycin-resistant gene had been integrated into the genome DNA after PCR and Southern blotting hybridization;and analyzed the physiological and biochemical characteristics of the transgenic plants.The main results were as follows:
1.Expression vectors which were constructed with cDNA fragments of the Lcyb gene were introduced into the tomato plants via Agrobacterium-mediated transformation. A total of 14 kanamycin-resistant plants were obtained.The PCR and Southern blot analysis of the putative transgenic plants showed that the T-DNA was integrated into the tomato genome with various copy numbers from 1-3.
2.The color of the fruits from two transgenic tomato plants were obvious yellow compared with the red fruits from the non-transgenic tomato plants.Measurements of the contents of lycopene and beta-carotene in the transgenic fruits by using high performance liquid chromatography(HPLC),showed that the contents of beta-carotene in yellow transgenic fruits were much higher than that in red fruits of wide-type plants.Oppositely, the contents of lycopene were very low in the yellow transgenic fruits.It's suggested that over-expression of lycopene beta-cyclase gene(Lcyb) in the transgenic plants resulted in accumulation of beta-carotene in the transgenic fruits which was absence in the wide-type plants.
3.RT-PCR analysis showed that the activities of target gene in 8 out of 14 different transgenic plants expressed,whereas the expression levels were different in individuals. Real-Time PCR analysis showed the expression of genes had involved in the carotenoid biosynthesis in the transgenic fruits.
4.The inheritance pattern of the transgenes in T_1 segregation population were analyzed based on PCR with NPTⅡprimers.And the results showed that NPTⅡgene inherited as a dominant genetic locus with the segregation ratio 3:1.
引文
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