DgDREB1A基因分离及对切花菊的遗传转化
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摘要
切花菊Cut Chrysanthemum(Dendranthema grandiflorum (Ramat.) Kitam)是世界4大切花之一,.具有色彩丰富、花型多样、瓶插寿命长等优点,是国际商品花卉总产值最高的花卉种类。在切花菊的栽培生产中,水资源的缺乏和冬季生产能耗成本高等因素严重影响着切花菊的周年生产规模与供应范围。为解决菊花冬季生产的能耗问题,采用现代分子生物学手段培育耐低温菊花新品种,完善菊花周年生产和稳定的供应技术体系意义重大。
采用PCR的方法,从菊花中克隆到了DgDREB1A基因,利用DNAMAN进行序列比对,所获序列与已知报道序列同源性达到98%,利用酶切、连接的方法,构建了转基因表达载体pBIG-DREBIA并通过冻融的方法转入了农杆菌LBA4404和EHA105中。
以切花菊‘神马’为试材,建立了无菌培养系,以无菌苗幼嫩叶片为外植体,优化了不定芽再生体系,叶片外植体在含有0.25mg/L2,4-D、1mg/LBA的MS培养基中诱导培养15d后,转入含有0.25mg/LNAA、1mg/LBA的分化培养基中培养3周,再生率达到100%。
在建立了切花菊高效再生体系的基础上,优化了遗传转化体系,并获得了转基因阳性植株,菊花叶片外植体预培养2d,用摇至OD600=0.5-0.7的农杆菌稀释10倍之后侵染10min.,黑暗条件下共培养2d,在5mg/L的kan.下连续筛选8周以上,将期间产生的抗性芽切下接种在含有7.5mg/L的生根培养中进行二次筛选,提取能够正常生根的抗性苗DNA,进行PCR检测,共获得PCR阳性植株8株,转化率达到1%。
本实验拟通过农杆菌介导的转基因育种手段,获得低温抗性良好的切花菊新品系,为切花菊冬季生产降低成本,为培育综合抗逆性良好的菊花新品种提供基础材料。
Cut chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitam) is one of the four main cut flower in the world,have the advantage of abundant colors、various flowertypes、and long vase life et.al.,and it is the highest value flower type in gross output value of international commodity flower. However,lack of water and high production cost seriously limit the scale of production and extent of supply. To solve the problem of enegy-consumable in the production of chrysanthemum in winter,It will be signaity to breed new cultivar that have low-temperature tolerance with modern molecular biology technology and perfect the system of all year production and sdeady supply technology.
With the method of PCR, we coloe DgDREB1A gene from chrysanthemum, sequence alignment with reported sequence,the homology reach to 98%, then we built transgene expression vector pBIG-DREB1A and transform it into Agrobacterium LBA4404 and EHA105.
Using cut chrysanthemum 'jinba' as the test material,we cultivated sterile culture system, using young leaves of the sterile plant as explant,we optimize the adventitious shoot regeneration system, the leaf explants were cultured 15 days in MS medium supplement with 0.25mg/L 2,4-D、1mg/L BA, then transferred onto development medium with 0.25mg/L NAA、1mg/L BA,after 3 weeks, the regeneration rate reach to 100%.
Based on the highly efficient regeneration,we optimized the genetic transformation system,and have got the positive lines,we found that 2 days preculture、OD600=0.05-0.07 engineer agrobacterium、10min.affection time and 2 days co-cultivation were necessary for the thansformation of chrysaanthemum,In our study,after 8 weeks selection under pressure,the resistent shoots were transferred to root medium which contain 7.5mg/L kan.to take the second selection,extract DNA of the rooted lines,PCR result showed that DgDREB1A was detected in 8 lines,the transformation ratio was 1%.
In this study, we use the method of genetic transformation mediated by agrobacterium tumefaciens,obtain new cut chrysanthemum lines that have good tolerance to low temperature,lower the cost for production in winter,supply the fundamental material for breeding new chrysanthemum cultivar that have good synthesized tolerance.
采用PCR的方法,从菊花中克隆到了DgDREB1A基因,利用DNAMAN进行序列比对,所获序列与已知报道序列同源性达到98%,利用酶切、连接的方法,构建了转基因表达载体pBIG-DREBIA并通过冻融的方法转入了农杆菌LBA4404和EHA105中。
以切花菊‘神马’为试材,建立了无菌培养系,以无菌苗幼嫩叶片为外植体,优化了不定芽再生体系,叶片外植体在含有0.25mg/L2,4-D、1mg/LBA的MS培养基中诱导培养15d后,转入含有0.25mg/LNAA、1mg/LBA的分化培养基中培养3周,再生率达到100%。
在建立了切花菊高效再生体系的基础上,优化了遗传转化体系,并获得了转基因阳性植株,菊花叶片外植体预培养2d,用摇至OD600=0.5-0.7的农杆菌稀释10倍之后侵染10min.,黑暗条件下共培养2d,在5mg/L的kan.下连续筛选8周以上,将期间产生的抗性芽切下接种在含有7.5mg/L的生根培养中进行二次筛选,提取能够正常生根的抗性苗DNA,进行PCR检测,共获得PCR阳性植株8株,转化率达到1%。
本实验拟通过农杆菌介导的转基因育种手段,获得低温抗性良好的切花菊新品系,为切花菊冬季生产降低成本,为培育综合抗逆性良好的菊花新品种提供基础材料。
Cut chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitam) is one of the four main cut flower in the world,have the advantage of abundant colors、various flowertypes、and long vase life et.al.,and it is the highest value flower type in gross output value of international commodity flower. However,lack of water and high production cost seriously limit the scale of production and extent of supply. To solve the problem of enegy-consumable in the production of chrysanthemum in winter,It will be signaity to breed new cultivar that have low-temperature tolerance with modern molecular biology technology and perfect the system of all year production and sdeady supply technology.
With the method of PCR, we coloe DgDREB1A gene from chrysanthemum, sequence alignment with reported sequence,the homology reach to 98%, then we built transgene expression vector pBIG-DREB1A and transform it into Agrobacterium LBA4404 and EHA105.
Using cut chrysanthemum 'jinba' as the test material,we cultivated sterile culture system, using young leaves of the sterile plant as explant,we optimize the adventitious shoot regeneration system, the leaf explants were cultured 15 days in MS medium supplement with 0.25mg/L 2,4-D、1mg/L BA, then transferred onto development medium with 0.25mg/L NAA、1mg/L BA,after 3 weeks, the regeneration rate reach to 100%.
Based on the highly efficient regeneration,we optimized the genetic transformation system,and have got the positive lines,we found that 2 days preculture、OD600=0.05-0.07 engineer agrobacterium、10min.affection time and 2 days co-cultivation were necessary for the thansformation of chrysaanthemum,In our study,after 8 weeks selection under pressure,the resistent shoots were transferred to root medium which contain 7.5mg/L kan.to take the second selection,extract DNA of the rooted lines,PCR result showed that DgDREB1A was detected in 8 lines,the transformation ratio was 1%.
In this study, we use the method of genetic transformation mediated by agrobacterium tumefaciens,obtain new cut chrysanthemum lines that have good tolerance to low temperature,lower the cost for production in winter,supply the fundamental material for breeding new chrysanthemum cultivar that have good synthesized tolerance.
引文
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