农杆菌介导法将PSY基因转入玉米自交系的研究
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摘要
维生素A缺乏被世界卫生组织列为世界四大营养缺乏病之一。玉米作为世界上分布面积最广泛的粮食作物之一,却不含维生素A,以其为主食远远不能满足人体对维生素A的需要。因此玉米中维生素A的提高,将极大的改善贫困地区人群营养状况。通过转基因方法提高玉米中维生素A的含量是一条经济而切实可行的方法。
本论文以类胡萝卜素合成途径中的重要关键酶八氢番茄红素合成酶(PSY,phytoene synthase)为目的基因,旨在通过农杆菌介导法将PSY基因转入玉米优良自交系,以获得高维生素A原含量的转基因玉米新品质。目的基因PSY来源于草原龙胆,将其插入带有35S启动子和bar基因筛选标记的表达载体pCAMBIA2300中,构建了植物表达载体,并转入农杆菌C58中获得了工程农杆菌。实验中以优良玉米自交系7922,238,265,p138等的幼胚为材料,接种在诱导培养基上进行培养以获得愈伤组织。结果发现,幼胚大小在1~2mm,幼胚接种时盾片向上放置,愈伤诱导率较高。此外,愈伤的诱导受基因型和培养基成分的显著影响。7922,238和265的二型愈伤可长期继代并再生出植株,培养基中添加半胱氨酸和硝酸银能显著降低愈伤的褐化率。用农杆菌C58介导法转化7922,238等的二型愈伤,在转化前对愈伤的抗性进行了研究,发现草丁膦(L-phosphiothricin,PPT)浓度在5mg/L时,愈伤有80%死亡,所以将筛选浓度定为5mg/L。对影响农杆菌转化效率的因素进行研究,发现侵染时间在15min,共培培养基中添加葡萄糖和MES时,抗性愈伤率最高。通过PPT筛选获得17株抗性植株,经过PCR分子检测初步证明,有3株植株中整合有PSY外源基因。
The vitamin A dificiency is listed as one of the world’s four major nutrirional dificiency diseases by the World Health Organization.However,as one of the most widely distributed food crops in the world, maize dose not contain vitamin A, which is bound to lead to severe lack of vitamin A in some less developed area where maize is still the stable food. Therefore, the improvement of vitamin A in maize will greatly improve the nutritional status of people in poor areas .Transgenic approach to improve vitamin A content of maize is an economical and practical way.
In this thesis,we used Agrobacterium-mediated approach to transform the key enzyme of the carotenoid biosynehesis called PSY(phytoene synthase) into the maize,and we hope to obtain the transgenic maize high of vitamin A. The target gene PSY comes from Eustoma grandiflorum,and was inserted into the plant expression vector pCAMBIA2300 with the 35S promoter and the bar selection marker.The vector was then transformed into Agrobacterium tumefaciens C58.We induced embryonic callus from immature embryos of different inbred lines of maize such as 7922,238,265,p138,etc.,and studied the factors affecting callus induction rates.The results show that when the size of immature embryo is in 1~2mm,and the inoculated immature embryos placed scutellum up,we could obtain a higher callus induction rate.The genotype and medium compositon can influnce callus indution rate significantly.The second type callus of 7922,238 and 265 can be long-term subcultured and regenerate into plants.Medium containing cysteine and silver nitrate can significantly reduced the browning rate. Before the transformation,we studied the resistance of the callus and found that when PPT(L-phosphiothricin) concentration was 5mg/L,80% of the callus died,so the screening concentration is 5mg/L.We also studied the factors affecting Agrobacterium-mediated transformation,and found that when the infection time was 15min,and the co-culture medium was added with glucose and MES,we could get the highest rate of resistant callus.We obtained 17 resistant plants through the selection of PPT,the result of PCR molecular detection indicated that PSY has been inserted into three plants.
本论文以类胡萝卜素合成途径中的重要关键酶八氢番茄红素合成酶(PSY,phytoene synthase)为目的基因,旨在通过农杆菌介导法将PSY基因转入玉米优良自交系,以获得高维生素A原含量的转基因玉米新品质。目的基因PSY来源于草原龙胆,将其插入带有35S启动子和bar基因筛选标记的表达载体pCAMBIA2300中,构建了植物表达载体,并转入农杆菌C58中获得了工程农杆菌。实验中以优良玉米自交系7922,238,265,p138等的幼胚为材料,接种在诱导培养基上进行培养以获得愈伤组织。结果发现,幼胚大小在1~2mm,幼胚接种时盾片向上放置,愈伤诱导率较高。此外,愈伤的诱导受基因型和培养基成分的显著影响。7922,238和265的二型愈伤可长期继代并再生出植株,培养基中添加半胱氨酸和硝酸银能显著降低愈伤的褐化率。用农杆菌C58介导法转化7922,238等的二型愈伤,在转化前对愈伤的抗性进行了研究,发现草丁膦(L-phosphiothricin,PPT)浓度在5mg/L时,愈伤有80%死亡,所以将筛选浓度定为5mg/L。对影响农杆菌转化效率的因素进行研究,发现侵染时间在15min,共培培养基中添加葡萄糖和MES时,抗性愈伤率最高。通过PPT筛选获得17株抗性植株,经过PCR分子检测初步证明,有3株植株中整合有PSY外源基因。
The vitamin A dificiency is listed as one of the world’s four major nutrirional dificiency diseases by the World Health Organization.However,as one of the most widely distributed food crops in the world, maize dose not contain vitamin A, which is bound to lead to severe lack of vitamin A in some less developed area where maize is still the stable food. Therefore, the improvement of vitamin A in maize will greatly improve the nutritional status of people in poor areas .Transgenic approach to improve vitamin A content of maize is an economical and practical way.
In this thesis,we used Agrobacterium-mediated approach to transform the key enzyme of the carotenoid biosynehesis called PSY(phytoene synthase) into the maize,and we hope to obtain the transgenic maize high of vitamin A. The target gene PSY comes from Eustoma grandiflorum,and was inserted into the plant expression vector pCAMBIA2300 with the 35S promoter and the bar selection marker.The vector was then transformed into Agrobacterium tumefaciens C58.We induced embryonic callus from immature embryos of different inbred lines of maize such as 7922,238,265,p138,etc.,and studied the factors affecting callus induction rates.The results show that when the size of immature embryo is in 1~2mm,and the inoculated immature embryos placed scutellum up,we could obtain a higher callus induction rate.The genotype and medium compositon can influnce callus indution rate significantly.The second type callus of 7922,238 and 265 can be long-term subcultured and regenerate into plants.Medium containing cysteine and silver nitrate can significantly reduced the browning rate. Before the transformation,we studied the resistance of the callus and found that when PPT(L-phosphiothricin) concentration was 5mg/L,80% of the callus died,so the screening concentration is 5mg/L.We also studied the factors affecting Agrobacterium-mediated transformation,and found that when the infection time was 15min,and the co-culture medium was added with glucose and MES,we could get the highest rate of resistant callus.We obtained 17 resistant plants through the selection of PPT,the result of PCR molecular detection indicated that PSY has been inserted into three plants.
引文
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[5]段智利等,营养富集型高维生素A原玉米研究进展,云南农业科技,2007.51~53
[6]Eugenia M.A,Enfissi,et al,Genetic engineering of carotenoid formation in tamato,Phytochem Rev.2006,59~65
[7]朱长甫,植物类胡萝卜素生物合成及其相关基因在基因工程中的应用,植物生理与分子生物学学报,2004,30(6):609~618
[8]Paul D.Fraser,Peter M.Bramley,The biosynthesis and nutritional uses of carotenoids,Progress in Lipid Research ,2004. 228~265
[9]王玉萍,植物类胡萝卜素生物合成相关基因的表达调控及其在植物基因工程中的应用,分子植物育种,2006,4(1):103~110
[10]Guan-Nan Liu, Yue-Hui Zhu,Jiao-Guo Jiang, The metabolomics of carotenoids in engineered cell factory,Appl Microbiol Biotechnol.(2009)83:989~999
[11]Zhu C,Yamamura S,Nishihara M,cDNAs for the synthesis of cyclic carotenoids in petals of Genitana lutea and their regulation during flower development.Biochim Biophys Aeta.2003,1625(3):305~308
[12]Armstrong GA,Alberiti M,Lwach F.Nucleotide sequence,organization and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus.Mol Gen Genet,216(3):254~268
[13]Misawa N,Nakagawa M,Kobayashi K,Elucidation of the Erwinia uredovora carotenoid biosynthesis pathway by functional analysis of gene products expresses in Escherichia coli.Jbacterial,1990,172(12):6704~6712
[14]Bird CR,Ray JA,Fletcher JD,Boniwell JM,Bird AS,Teulieres C,Blain I,Bramley PM,Schuch W.Using antisense RNA to study gene function:inhibition of carotenoid biosynthesis in transgenic tomatoes.Bio Teechnol,19919(7):635~639
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[17]Moehs CP,Tian L,Osteryoung KW.DellaPenna D.Analysis of carotenoid biosynthetic gene expression during marigold petal development.Plant Mol Biol,2001,45(3):281~293
[18]陈大明,博士后学位论文,中国科学院植物研究所,2001
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