摘要
花生(Arachis hypogaea L.)是世界上四大油料作物之一,在世界范围内的热带和亚热带地区都有种植。中国是世界上最大的花生生产国和消费国。维生素E是一种脂溶性的抗氧化剂,在人体、动物以及植物体内都有重要的生理作用。植物油是人类摄取维生素E的主要途径。植物包括花生在内具有高活性α-维生素E的含量非常低,为了提高花生中维生素E的含量,我们试图通过基因工程的办法改良现有的花生品种。本文报道维生素合成途径中的重要基因尿黑酸合成酶(VE_2)基因、2-甲基6-植基-苯醌甲基转移酶(VE_3)基因和γ-生育酚甲基转移酶(VE_4)基因的克隆,种子特异表达启动子引导的植物表达载体构建,以及这些载体对花生进行遗传转化的研究结果。
1、为了使维生素E相关的基因在花生胚中特异表达,通过PCR的方法在同源性分别为99.8%和100%的T载体中分离得到两个种子特异表达启动子。他们分别是大豆种子储藏蛋白启动子(7S)和油菜种子储藏蛋白启动子(2S)。这两个启动子的长度分别为795bp和1058bp。
2、提取拟南芥(Arabidopsis thaliana)叶子的总RNA,并根据GenBank所发表的VE_2、VE_3和VE_4序列设计引物,通过RT-PCR的方法分离得到这3个基因,他们和GenBank上所发表的序列同源性分别为98%、100%和100%。把这三个基因分别构建到中间表达载体pSPROK中,并且在VE2基因的上游插入7S,在VE3和VE4基因上游分别插入2S,来调控这3个基因在种仁特异表达。然后从这3个载体中酶切出分别带有启动子、基因和终止子的完整表达单元,构建到植物表达载体pCAMBIA1300中,所构建成的植物表达载体分别命名为pCAMBIA1300VE2·7S、pCAMBIA1300VE3·2S和pCAMBIA1300VE4·2S。测序分析显示3个基因的完整表达单元成功构建在植物表达载体pCAMBIA1300中。
3.为了更高效地改良花生中维生素E的合成效率,在单价载体的基础上构建了一个双价植物表达载体pCAMBIA1300 VE2·VE3,它含有VE_2和VE_3各自的启动子和终止子表达单元。为了同时改良花生中维生素E含量和油酸含量,亦构建得到双价植物表达载体pCAMBIA1300AT,它含有VE_4的完整表达单元和FAD2的反义RNA表达单元。
4、将pCAMBIA1300VE4·2S、pCAMBIA1300AT、pCAMBIA13002a等3个植物表达载体通过根癌农杆菌介导对花生进行遗传转化,提取抗性苗DNA进行PCR反应显示,他们的表达单元都已经插入到花生基因组中,各得到转基因苗3株、3株和6株,转化率分别为:2.00%、2.00%和3.85%。由于这些基因都是由种子特异表达启动子所引导,应待转基因苗种子收获后再作进一步检测。
综上所述,本研究构建了5个植物种子特异表达载体用来提高花生得维生素E含量,并将pCAMBIA1300VE4·2S、pCAMBIA1300AT、pCAMBIA13002a等3个载体对花生进行了转化,得到了转基因植株,为改良花生维生素E和油酸含量奠定了基础。
Peanut(Arachis hypogaea L.)is the world's fourth largest oilseed crop grown mainly in subtropical and tropical regions.China is the largest country of peanut production and consumption in the world.Vitamin E is an important class of lipid-soluble compounds with antioxidant activities that plays a very important role in plant,animal and human being.Plant oil is the main resource of the vitamin E in human nutritious.The content of activatedα-tocopherol is very low in plant,including peanut.To improve the rate of vitamin E component in peanut,we have tried to improve peanut vitamin E content by employing gene engineering.We reported here the cloning of homogentisic acid prenyltransferase(VE_2),2-methyl-6-phytylbenzoquinol(VE_3)andα-tocopherol methyltransferase(VE_4)genes,embryo-specific expression vectors construction and their transformation.
To make vitamin E genes express specifically in peanut embryo,two promoters already cloned in T-vectors were isolated with 99.8%and 100%identity to the sequences interested on GenBank,respectively.They are 7S seed storage protein gene promoter of soybean and 2S seed storage protein gene promoter of Brassica napus.The promoters region were 795bps and 1058bps in length.
Extracting total RNA from leaves of Arabidopsis thaliana and designing the primers according to the published sequence of VE_2,VE_3 and VE_4 genes on GenBank, we obtained those three genes,with 97%,100%and 100%identity to the conterpart sequences on GenBank,respectively,by RT-PCR.Those three genes were first constructed on intermediate expressing vector pSPROK with 7S promoter upstream to VE2 gene and 2S promoter upstream to VE3 and VE4 genes,respectively.Then the intact expression units with genes of interest flanked by specific promoters and nos terminals were cut and transferred to plant expressing vector pCAMBIA1300.Three plant expression vectors,named pCAMBIA1300VE2.7S,pCAMBIA1300VE3.2S and pCAMBIA1300VE4.2S,were obtained afterwards.Sequencing results showed that all the three genes were correctly inserted in plant expressing vector pCAMBIA1300.
To improve the tocopherol biosynthetic pathway and to increase the total amount of vitamine E,a plant bivalent expression vector,pCAMBIA1300 VE2.VE3,was constructed.It contains VE_2 and VE_3 genes,each has their own promoter and nos terminal.To improve both tocopherol and the rate of oleic acid component in peanut, another plant bivalent expression vector pCAMBIA1300AT was constructed.It has intact ultra-expression units of VE_4 and complete anti-RNA expression unit of FAD2.
Three vectors,pCAMBIA1300VE4.2S,pCAMBIA1300AT and pCAMBIA13002a, have been used to transform peanut variety Minghua 6,mediated by agrobacterium C58. Through many resistant transgenic seedling were obtained,only three,three and six seedlings with respective to genes of VE2,anti-RNA of FAD2 and VE4,and anti-FAD2 were shown to carry the genes of interest in the cells,after subjected to PCR assay,with transformation rate of 2.0%,2.0%and 3.85%.Further evaluation must be done after seed harvest,since those genes are all directed by seed specifically expressed prmoters.
In a word,we obtained five plant expressing vectors involved in increasing vatamine E contant.Plant expressing vectors pCAMBIA1300VE4-2S, pCAMBIA1300AT and pCAMBIA13002a were being used to transform peanut mediated by Agrobacterium and some transfomant seedlings were got,which provides basis in improving vitamin E and fatty acid composition in peanuts.
引文
[1]泽永等.GUS基因和NPTII基因在转基因花生后代的遗传研究[J],1999,花生科技(S1):241-245.
[2]白选杰等.论加快我国花生产业发展策略[J].中国工程科学,2007,(02):19-24.
[3]刘公社等.向日葵种质资源维生素E含量及相关变量的初步评价[J].植物遗传资源学报,2005,(02):178-181.
[4]胡英考.植物维生素E合成及其生物技术改良[J].中国生物工程杂志,2004,(01):32-35.
[5]李晓峰等.植物维生素E合成相关酶基因的克隆及其在体内功能研究进展[J].植物学通报,2006,(01):68-77.
[7]单世华等.花生子叶遗传转化再生体系影响因素的研究[J].花生学报,2007,(01):13-19.
[8]万勇善等.γ-维生素E甲基转移酶基因转化花生研究[J].中国粮油学报,2005,(01):61-64.
[9]刘风珍.Rs-αfP_1基因和γ-tmt基因转化花生及高效遗传转化体系的研究[D].山东农业大学博士论文,2004,5
[10]单世华,庄伟建等.以农杆菌为介导花生的遗传转化研究Ⅰ.质粒载体的分子鉴定及农杆菌菌株的转换[J].花生学报,2003,(02):9-13.
[11]宫旭洲,周垂钦.振兴花生产业,提高我国食用油自给率[J].中国油脂,2007,(01):9-12.
[12]欧阳青,樊春涛等.结球甘蓝γ-生育酚甲基转移酶cDNA的克隆、分析及其异源表达酶蛋白的功能研究[J].自然科学进展,2003,(07):709-715.
[13]欧阳青,蔡文启.天然维生素E的生物合成途径[J].植物生理学通讯,2003,(05):501-507.
[14]赵健,王斌,等.农杆菌介导的β-1,3-葡聚糖酶基因转化花生的研究[J].花生学报,2007,(01):20-23.
[15]黄智明,翁海波等.转入HPTl基因的油菜种子中维生素E含量的提高[J].植物生理学通讯,2006,(05):888-890.
[16]张吉民.中国花生产业现状与入世后发展对策[J].世界农业,2003,(01):25-27.
[17]张书标,庄伟建等.花生组织培养研究进展[J].福建农业大学学报,1999,(03):268-273.
[18]庄东红,邹湘辉等.农杆菌介导的花生遗传转化研究[J].中国油料作物学报,2003,(04):47-51.
[19]Anuradha,T.S.,S.K.Jami,et al.Genetic transformation of peanut(Arachis hypogaea L.) using cotyledonary node as explant and a promoterless gus::nptII fusion gene based vector[J]. J Biosci, 2006,31(2): 235-246.
[20] Bohles, H.. Antioxidative vitamins in prematurely and maturely born infants[J]. Int J Vitam Nutr Res , 1997,67(5): 321-328.
[21] Cheng, Z., S. Sattler, et al. Highly divergent methyltransferases catalyze a conserved reaction in tocopherol and plastoquinone synthesis in cyanobacteria and photosynthetic eukaryotes[J]. 2003,Plant Cell 15(10): 2343-2356.
[22] Collakova, E. and D. DellaPenna. Isolation and functional analysis of homogentisate phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis[J].Plant Physiol ,2001,127(3): 1113-1124.
[23] Collakova, E. and D. DellaPenna. Homogentisate phytyltransferase activity is limiting for tocopherol biosynthesis in Arabidopsis[J]. Plant Physiol,2003,131(2): 632-642. [24] Crowell, E.F., J.M. McGrath, and D.S. Douches, Accumulation of vitamin E in potato (Solanum tuberosum) tubers. Transgenic Res, 2007.
[25] DellaPenna, D., A decade of progress in understanding vitamin E synthesis in plants[J]. Journal of Plant Physiology 2005. 162: p. 729—737.
[26] DellaPenna, D., Progress in the dissection and manipulation of vitamin E synthesis[J]. Trends Plant Sci 2005.10(12): 574-579.
[27] Egnin, M., A. Mora, and C.S. Prakash, Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L. ) [J]. In Vitro Cell Dev Biol Plant, 1998. 34(4): p. 310-318
[28] DellaPenna, D., Biofortification of plant-based food: enhancing folate levels by metabolic engineering[J]. Proc Natl Acad Sci U S A, 2007. 104(10): p. 3675-6.
[29] Schultz, G. and J. Soil, [Biosynthesis of alpha-tocopherol (vitamin E), phylloquinone (2-methyl-3-phytylnaphtoquinone, vitamin K1) and other prenylquinones in plants. On the problem of inability of the biosynthesis in animals—a survey (author's transl)] [J]. Dtsch Tierarztl Wochenschr, 1980. 87(11): p. 410-2.
[30] Tian, L. and D. DellaPenna, Characterization of a second carotenoid beta-hydroxylase gene from Arabidopsis and its relationship to the LUT1 locus[J]. Plant Mol Biol, 2001. 47(3): p. 379-88.
[31] Bergmuller, E., S. Porfirova, and P. Dormann, Characterization of an Arabidopsis mutant deficient in gamma-tocopherol methyltransferase[J]. Plant Mol Biol, 2003. 52(6): p. 1181-90.
[32] Sattler, S.E., et al., Characterization of tocopherol cyclases from higher plants and cyanobacteria. Evolutionary implications for tocopherol synthesis and function[J]. Plant Physiol, 2003. 132(4): p. 2184-95.
[33] Rissler, H.M., et al., Chlorophyll biosynthesis. Expression of a second chl I gene of magnesium chelatase in Arabidopsis supports only limited chlorophyll synthesis[J]. Plant Physiol, 2002. 128(2): p. 770-9.
[34] Ajjawi, I. and D. Shintani, Engineered plants with elevated vitamin E: a nutraceutical success story. Trends Biotechnol, 2004. 22(3): p. 104-7.
[35] Rocheford, T.R., et al., Enhancement of vitamin E levels in corn[J]. J Am Coll Nutr, 2002. 21(3 Suppl):p. 191S-198S.
[36] Motohashi, R., et al., Functional analysis of the 37 kDa inner envelope membrane polypeptide in chloroplast biogenesis using a Ds-tagged Arabidopsis pale-green mutant[J]. Plant J, 2003. 34(5): p. 719-31.
[37] Gilliland, L.U., et al., Genetic basis for natural variation in seed vitamin E levels in Arabidopsis thaliana[J]. Proc Natl Acad Sci U S A, 2006. 103(49): p. 18834-41.
[38] Dahnhardt, D., et al, The hydroxyphenylpyruvate dioxygenase from Synechocystis sp. PCC 6803 is not required for plastoquinone biosynthesis[J]. FEBS Lett, 2002. 523(1-3): p. 177-81.
[39] Savidge, B., et al., Isolation and characterization of homogentisate phytyltransferase genes from Synechocystis sp. PCC 6803 and Arabidopsis [J]. Plant Physiol, 2002. 129(1): p. 321-32.
[40] DellaPenna, D., Plant metabolic engineering[J]. Plant Physiol, 2001. 125(1): p. 160-3. Maeda, H., et al., Tocopherols protect Synechocystis sp. strain PCC 6803 from lipid peroxidation[J]. Plant Physiol, 2005. 138(3): p. 1422-35.
[41] Havaux, M., et al., Vitamin E protects against photoinhibition and photooxidative stress in Arabidopsis thaliana[J]. Plant Cell, 2005. 17(12): p. 3451-69.
[42] Fujiwara, T., P. A. Lessard, et al. Seed-specific repression of GUS activity in tobacco plants by antisense RNA[J].Plant Mol Biol ,1992,20(6): 1059-69.
[43] Hofius, D. and U. Sonnewald . Vitamin E biosynthesis: biochemistry meets cell biology[J]. Trends Plant Sci ,2003,8(1): 6-8.
[44] Kanwischer, M., S. Porfirova, et al. Alterations in tocopherol cyclase activity in transgenic and mutant plants of Arabidopsis affect tocopherol content, tocopherol composition, and oxidative stress[J]. Plant Physiol,2005,137(2): 713-723.
[45] Lacorte C, Mansur E, Timmerman B,et al. Gene transfer into peanut(Arachis hypogaea L.) by Agrobacterium tumefaciens[J]. Plant Cell Reports, 1991,10:354-357
[46] Li Z, Jarret RL, Demski JW. Engineered resistance to tomato spotted wilt virus in transgenic peanut expressing the viral nucleocapsid gene[J]. Transgenic Research, 1997,6:297-305
[47] Little EL, Magbanua ZV, Parrott WA. A protocol for repetitive somatic embryogenesis from mature peanut epicotyls[J]. Plant Cell Reports, 2000, 19(4):351-357
[48] Livingstone DM, Birch RGPlant regeneration and microprojectile-mediated gene transfer in embryonic leaflets of peanut(Arachis hypogaea L.) [J]. Australian Journal of Plant Physiology, 1995, 22(4):585~591
[49] Livingstone DM, Birch RG. Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus
[50] produced from mature seeds[J]. Molecular Breeding, 1998, 10:1-9
[51] LANG Hui, WU Fang-Shen, WANG Dao-Wen,etc. Wheat Transformation by Electroporation with Ring Electrode [J]. Acta Genetica Sinica,2005,32(1):66~71
[52] Liu zhao-Hua, ZHANG zhen-shan, Guo Hong- Nian,etc. Expression of Two Plant Agglutinin Genes in Transgenic Tobacco Plants[J]. Acta Genetica Sinica,2005, 32(7):758~763
[53] McKently AH, Moore GA, Gardner FP. In vitro plant regeneration of peanut from seed explants[J].Crop Science, 1990,30(1): 192-196
[54] McKently AH,Moore GA,Doostar H,et al. Agrobacterium-mediated transformation of peanut(Arachis hypogaea L.) embryo axes and the development of transgenic plants [J].Plant Cell Reports, 1995,14:699-703
[55] McKently AH. Effect of genotype on somatic embryogenesis from axes of mature peanut embryos[J]. Plant Cell Tissue and Organ Culture, 1995,42(3): 251-254
[56] Ming C, His DCH, Phillips-GC. In vitro regeneration of valencia-type peanut (Arachis hypogaea L.) from cultured petiolules, epicotyl, sections and other seedling explants[J].Peanut Science, 1992, 19(2): 82-87
[57] Murashige,T.F. Skoog. A revised medium for rapid growth and bioassays with tobacco tissue cultures[J]. Physiol. Plant,1962,15: 473-494
[58] Murch SJ, Victor JMR, Krishnaraj S, et al. The role of proline in thidiazuron-induced somatic embryogenesis of peanut[J]. In Vitro Cellular and Developmental Biology Plant. 1999,35(1): 102-105
[59] Murthy BNS, Murch SJ, Saxena PK. Thidiazuron-induced somatic embryogenesis in intact seedlings of peanut (Arachis hypogaea L.): endogenous growth regulator levels and significance of cotyledons[J]. Physiology Plant, 1995,94(2): 268-276
[60] Murthy BNS, Saxena PK. Somatic embryogenesis in peanut(Arachis hypogaea L.):stimulation of direct differentiation of somatic embryos by forchlorfenuron (CPPU)[J].Plant Cell Reports, 1994,13(2-3): 145-150
[61] Mansur, E., C. Lacorte, et al. "Peanut transformation." Methods Mol Biol ,1995,44: 87-100.
[62] Norris, S. R., T. R. Barrette, et al. "Genetic dissection of carotenoid synthesis in arabidopsis defines plastoquinone as an essential component of phytoene desaturation." Plant Cell ,1995,7(12): 2139-2149.
[63] Norris, S. R., X. Shen, et al. Complementation of the Arabidopsis pds1 mutation with the gene encoding p-hydroxyphenylpyruvate dioxygenase[J]. Plant Physiol ,1998,117(4): 1317-23.
[64] Porfirova, S., E. Bergmuller, et al. Isolation of an Arabidopsis mutant lacking vitamin E and identification of a cyclase essential for all tocopherol biosynthesis[J]. Proc Natl Acad Sci U S A,2002,99(19): 12495-500.
[65] Rohini, V. K. and K. Sankara Rao. Transformation of peanut (Arachis hypogaea L.) with tobacco chitinase gene: variable response of transformants to leaf spot disease[J]. Plant Sci ,2001,160(5): 889-898.
[66] Sattler, S. E., Z. Cheng, et al. From Arabidopsis to agriculture: engineering improved Vitamin E content in soybean[J]. Trends Plant Sci ,2004,9(8): 365-7.
[67] Sattler, S. E., L. U. Gilliland, et al. Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination[J]. Plant Cell, 2004, 16(6): 1419-32.
[68] Sharma, K. K. and V. V. Anjaiah. An efficient method for the production of transgenic plants of peanut (Arachis hypogaea L.) through Agrobacterium tumefaciens-mediated genetic transformation[J]. Plant Science,2000,159(1): 7-19.
[69] Sharma, K. K. and P. Bhatnagar-Mathur (2006). Peanut (Arachis hypogaea L.) [J]. Methods Mol Biol 343: 347-358.
[70] Shintani, D. and D. DellaPenna. Elevating the vitamin E content of plants through metabolic engineering[J]. Science ,1998,282(5396): 2098-100.
[71] Shintani, D. K.Engineering plants for increased nutrition and antioxidant content through the manipulation of the vitamin E pathway[J]. Genet Eng (N Y) ,2006,27: 231-42.
[72] Shintani, D. K., Z. Cheng, et al. The role of 2-methyl-6-phytylbenzoquinone methyltransferase in determining tocopherol composition in Synechocystis sp. PCC6803[J]. FEBS Lett,2002,511(1-3): 1-5.
[73] Tanaka, J., H. Fujiwara, et al. Vitamin E and immune response. I. Enhancement of helper T cell activity by dietary supplementation of vitamin E in mice[J]. Immunology ,1979,38(4): 727-34.
[74] Tomlinson, K. L., S. McHugh, et al. Evidence that the hexose-to-sucrose ratio does not control the switch to storage product accumulation in oilseeds: analysis of tobacco seed development and effects of overexpressing apoplastic invertase[J]. J Exp Bot ,2004,55(406): 2291-303.
[75] Van Eenennaam, A. L., K. Lincoln, et al. Engineering vitamin E content: from Arabidopsis mutant to soy oil[J]. Plant Cell ,2003,15(12): 3007-19.
[76] Vidi, P. A., M. Kanwischer, et al. Tocopherol cyclase (VTE1) localization and vitamin E accumulation in chloroplast plastoglobule lipoprotein particles[J]. J Biol Chem ,2006,281(16): 11225-34.