非組培遺傳轉(zhuǎn)化法將抗草甘膦基因?qū)氡吐榈难芯?/H1>
發(fā)布時(shí)間：2019-04-02 10:58
【摘要】：蓖麻是世界十大油料作物之一,是生物能源的首選作物。草害的發(fā)生是危害蓖麻生產(chǎn)的主要障礙之一。利用基因工程方法培育抗除草劑的作物品種可以有效的控制田間雜草。非組培的遺傳轉(zhuǎn)化法操作簡(jiǎn)單,成本低,用時(shí)短,避免了冗長(zhǎng)繁瑣的組織培養(yǎng)過程。本研究在蓖麻上首次采用農(nóng)桿菌介導(dǎo)萌動(dòng)種胚轉(zhuǎn)化法,同時(shí)結(jié)合花粉管通道法開展蓖麻抗草甘膦遺傳轉(zhuǎn)化的研究。通過這兩種方法分別摸索出了適合蓖麻的遺傳轉(zhuǎn)化條件,并獲得了抗草甘膦蓖麻植株。主要結(jié)果如下:首次采用農(nóng)桿菌介導(dǎo)萌動(dòng)種胚轉(zhuǎn)化法獲得蓖麻抗草甘膦植株。結(jié)果表明,在生長(zhǎng)點(diǎn)附近針刺三針,超聲波強(qiáng)度800W,與農(nóng)桿菌共培養(yǎng)16h為最佳轉(zhuǎn)化條件。在草甘膦篩選前隨機(jī)選取的800株轉(zhuǎn)化處理植株中,檢測(cè)到5株P(guān)CR陽性植株,平均轉(zhuǎn)化率為6.3‰;噴施濃度為1.5‰草甘膦后,存活26株轉(zhuǎn)化處理植株,經(jīng)PCR和Southern blot檢測(cè),獲得了 3株陽性抗草甘膦T0代植株。以草甘膦抗性植株為基數(shù)的轉(zhuǎn)化率為11.5%。本研究證明,農(nóng)桿菌介導(dǎo)萌動(dòng)種胚轉(zhuǎn)化法可以用于蓖麻的遺傳轉(zhuǎn)化,為蓖麻基因工程提供了新的思路和方法。采用花粉管通道法轉(zhuǎn)化蓖麻,并對(duì)可能影響結(jié)實(shí)率轉(zhuǎn)化率的三種因素DNA濃度、DNA溶劑、導(dǎo)入時(shí)間進(jìn)行分析。結(jié)果表明,三種因素對(duì)結(jié)實(shí)率的影響差異不是特別顯著,但是DNA濃度對(duì)轉(zhuǎn)化率的影響卻至關(guān)重要。綜合考慮,DNA濃度以100ng/μL為佳。三種DNA溶劑以TE為優(yōu),導(dǎo)入時(shí)間為6-24h均可。通過對(duì)351株的T_1代植株P(guān)CR檢測(cè)獲得了 3株陽性植株。初步證明抗草甘膦EPSPS基因已成功轉(zhuǎn)入蓖麻植株,為提高蓖麻花粉管通道法轉(zhuǎn)化率提供了一定的理論依據(jù)。
[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，
本文編號(hào)：2452502
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2452502.html
[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，
本文編號(hào)：2452502
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2452502.html
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