非组培遗传转化法将抗草甘膦基因导入蓖麻的研究
发布时间：2019-04-02 10:58
【摘要】：蓖麻是世界十大油料作物之一,是生物能源的首选作物。草害的发生是危害蓖麻生产的主要障碍之一。利用基因工程方法培育抗除草剂的作物品种可以有效的控制田间杂草。非组培的遗传转化法操作简单,成本低,用时短,避免了冗长繁琐的组织培养过程。本研究在蓖麻上首次采用农杆菌介导萌动种胚转化法,同时结合花粉管通道法开展蓖麻抗草甘膦遗传转化的研究。通过这两种方法分别摸索出了适合蓖麻的遗传转化条件,并获得了抗草甘膦蓖麻植株。主要结果如下:首次采用农杆菌介导萌动种胚转化法获得蓖麻抗草甘膦植株。结果表明,在生长点附近针刺三针,超声波强度800W,与农杆菌共培养16h为最佳转化条件。在草甘膦筛选前随机选取的800株转化处理植株中,检测到5株PCR阳性植株,平均转化率为6.3‰;喷施浓度为1.5‰草甘膦后,存活26株转化处理植株,经PCR和Southern blot检测,获得了 3株阳性抗草甘膦T0代植株。以草甘膦抗性植株为基数的转化率为11.5%。本研究证明,农杆菌介导萌动种胚转化法可以用于蓖麻的遗传转化,为蓖麻基因工程提供了新的思路和方法。采用花粉管通道法转化蓖麻,并对可能影响结实率转化率的三种因素DNA浓度、DNA溶剂、导入时间进行分析。结果表明,三种因素对结实率的影响差异不是特别显著,但是DNA浓度对转化率的影响却至关重要。综合考虑,DNA浓度以100ng/μL为佳。三种DNA溶剂以TE为优,导入时间为6-24h均可。通过对351株的T_1代植株PCR检测获得了 3株阳性植株。初步证明抗草甘膦EPSPS基因已成功转入蓖麻植株,为提高蓖麻花粉管通道法转化率提供了一定的理论依据。
[Abstract]:Castor is one of the top ten oil crops in the world and is the first choice for bioenergy. The occurrence of grass damage is one of the main obstacles to the production of castor. Using genetic engineering method to cultivate herbicide-resistant crop varieties can effectively control weeds in the field. The non-tissue culture genetic transformation method has the advantages of simple operation, low cost and short usage time, thus avoiding the tedious and tedious process of tissue culture. The genetic transformation of castor with glyphosate resistance by Agrobacterium tumefaciens-mediated embryo germination and pollen tube pathway was studied in this study for the first time. The genetic transformation conditions suitable for castor were found out by these two methods, and the plant resistant to glyphosate was obtained. The main results are as follows: for the first time, castor plant resistant to glyphosate was obtained by Agrobacterium-mediated germination embryo transformation. The results showed that the optimum transformation conditions were as follows: acupuncture three times near the growing point, ultrasonic intensity 800W, co-culture with Agrobacterium tumefaciens for 16h. Among the 800 transformed plants randomly selected before glyphosate screening, 5 PCR positive plants were detected with an average conversion rate of 6.3 鈥，
本文编号：2452502
[Abstract]:Castor is one of the top ten oil crops in the world and is the first choice for bioenergy. The occurrence of grass damage is one of the main obstacles to the production of castor. Using genetic engineering method to cultivate herbicide-resistant crop varieties can effectively control weeds in the field. The non-tissue culture genetic transformation method has the advantages of simple operation, low cost and short usage time, thus avoiding the tedious and tedious process of tissue culture. The genetic transformation of castor with glyphosate resistance by Agrobacterium tumefaciens-mediated embryo germination and pollen tube pathway was studied in this study for the first time. The genetic transformation conditions suitable for castor were found out by these two methods, and the plant resistant to glyphosate was obtained. The main results are as follows: for the first time, castor plant resistant to glyphosate was obtained by Agrobacterium-mediated germination embryo transformation. The results showed that the optimum transformation conditions were as follows: acupuncture three times near the growing point, ultrasonic intensity 800W, co-culture with Agrobacterium tumefaciens for 16h. Among the 800 transformed plants randomly selected before glyphosate screening, 5 PCR positive plants were detected with an average conversion rate of 6.3 鈥，
本文编号：2452502
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