【摘要】：目的本研究以高粱HCN途徑的關(guān)鍵酶基因CYP71E1為目的基因,以玉米幼胚為外植體,通過根癌農(nóng)桿菌介導進行玉米遺傳轉(zhuǎn)化的研究。優(yōu)化A188玉米再生體系,分析不同外植體大小、農(nóng)桿菌濃度、侵染時間和熱處理時間對玉米遺傳轉(zhuǎn)化效率的影響,同時采用GUS組織化學染色和PCR技術(shù)對轉(zhuǎn)基因植株進行鑒定。利用高效率轉(zhuǎn)化技術(shù)解決因生物和非生物脅迫帶來玉米產(chǎn)量和品質(zhì)降低的瓶頸,為提供玉米新品種的轉(zhuǎn)化提供理論基礎(chǔ)。方法利用聚合酶鏈式反應(yīng)(PCR)從高粱中擴增出1854bp的CYP71E1基因全長。利用基于位點特異性重組的Gateway技術(shù),將CYP71E1基因重組到表達載體(p MDC141)中,將構(gòu)建成功的表達載體CYP71E1-p MDC141導入農(nóng)桿菌EHA105菌株。通過優(yōu)化外植體材料、誘導愈傷培養(yǎng)基、分化培養(yǎng)基、生根培養(yǎng)基,建立玉米再生體系。以攜帶CYP71E1-p MDC141(含GUS基因)質(zhì)粒的農(nóng)桿菌EHA105菌株侵染玉米優(yōu)良自交系A(chǔ)188的幼胚,通過對影響農(nóng)桿菌介導玉米幼胚的外植體大小、農(nóng)桿菌濃度、侵染時間、熱預處理時間進行優(yōu)化,提高GUS基因的平均轉(zhuǎn)化效率,同時對轉(zhuǎn)基因植株進行PCR和GUS檢測。結(jié)果1、利用基于位點特異性重組的Gateway技術(shù),通過TOPO克隆將高粱HCN途徑的關(guān)鍵酶基因CYP71E1克隆到入門載體p CRTM8/GW/TOPO?中,在LR克隆酶作用下將入門克隆重組到表達載體(p MDC141)中,成功構(gòu)建了p MDC141-CYP71E1植物表達載體,導入農(nóng)桿菌EHA105中。2、建立高效玉米再生體系,玉米幼胚大小為1.0~1.2mm時,愈傷誘導率最高,通過正交實驗的方法優(yōu)化誘導胚性愈傷培養(yǎng)基,由此得出誘導玉米A188愈傷組織的MS基礎(chǔ)培養(yǎng)基中添加L-pro、2,4-D、Ag NO3三因素的最優(yōu)組合為:L-pro700mg/L、2,4-D 2.0mg/L、Ag NO38 mg/L。分化培養(yǎng)基中玉米素濃度為5 mg/L時分化效率最高。生根培養(yǎng)基中IBA的最適濃度為2 mg/L。3、建立高效遺傳轉(zhuǎn)化體系,玉米幼胚大小為1.0~1.2mm,GUS的平均轉(zhuǎn)化效率為35.3%,農(nóng)桿菌菌液濃度為OD660nm為0.8,GUS的平均轉(zhuǎn)化效率為46.8%,侵染時間為5min和43℃熱預處理3 min,GUS的平均轉(zhuǎn)化效率分別為46.4%和45.4%。因此最佳轉(zhuǎn)化條件為:選擇幼胚大小為1.0~1.2mm、菌液濃度OD660nm=0.8、侵染時間5min、43℃熱預處理3 min。4、對獲得的轉(zhuǎn)基因植株進行PCR和GUS檢測,獲得12株轉(zhuǎn)基因苗。結(jié)論1、Gateway技術(shù)與傳統(tǒng)的克隆方法相比具有陽性克隆率高、快速、靈活等優(yōu)點,通過Gateway技術(shù)成功構(gòu)建p MDC141-CYP71E1表達載體。2、通過優(yōu)化外植體材料、愈傷誘導培養(yǎng)基、分化培養(yǎng)基、生根培養(yǎng)基,建立了A188玉米幼胚再生體系。3、通過對影響農(nóng)桿菌介導玉米幼胚的部分因素進行優(yōu)化,最佳轉(zhuǎn)化條件為:玉米幼胚大小為1.0~1.2mm、菌液濃度OD660nm=0.8、侵染時間5min、43℃熱預處理3min。4、獲得了高粱HCN途徑的關(guān)鍵酶基因CYP71E1陽性植株。
[Abstract]:Objective to study the genetic transformation of maize by Agrobacterium tumefaciens mediated by the key enzyme gene CYP71E1 of sorghum HCN pathway and maize embryo as explant. The regeneration system of A188 maize was optimized, and the effects of different explant size, Agrobacterium concentration, infection time and heat treatment time on the genetic transformation efficiency of maize were analyzed. The transgenic plants were identified by GUS histochemical staining and PCR technique. The high efficiency transformation technology was used to solve the bottleneck of maize yield and quality reduction caused by biological and abiotic stress, which provided the theoretical basis for the transformation of new maize varieties. Methods the CYP71E1 gene of 1854bp was amplified from sorghum by polymerase chain reaction (PCR). Using the Gateway technique based on site-specific recombination, the CYP71E1 gene was recombined into the expression vector (p MDC141), and the successfully constructed expression vector CYP71E1-p MDC141 was introduced into Agrobacterium tumefaciens EHA105 strain. Maize regeneration system was established by optimizing explant material, inducing callus medium, differentiation medium and rooting medium. Agrobacterium tumefaciens (EHA105) carrying CYP71E1-p MDC141 (containing GUS gene) plasmid was used to infect immature embryos of maize inbred line A188. The effects of Agrobacterium tumefaciens on the size of explant, concentration of Agrobacterium tumefaciens and infection time were studied. The thermal pretreatment time was optimized to improve the average transformation efficiency of GUS gene, and PCR and GUS were used to detect the transgenic plants. Results 1. Using Gateway technique based on site-specific recombination, the key enzyme gene CYP71E1 of sorghum HCN pathway was cloned into the entry vector p CRTM8/GW/TOPO? by TOPO cloning. Under the action of LR cloning enzyme, the primer clone was recombined into the expression vector (p MDC141), and the plant expression vector of p MDC141-CYP71E1 was successfully constructed, which was introduced into Agrobacterium tumefaciens EHA105. 2. To establish an efficient maize regeneration system. The callus induction rate of maize immature embryos was the highest when the size of immature embryos was 1.0~1.2mm. The medium of embryogenic callus induction was optimized by orthogonal experiment, and the MS basic medium for inducing callus of maize A188 was obtained by adding L-proz2O4-D into the basic medium of inducing callus of maize A188. The optimal combination of three factors of Ag NO3 is: L-pro700mg / L 24-D 2.0mg / L NO38 mg/L. When the concentration of zeatin in differentiation medium was 5 mg/L, the differentiation efficiency was the highest. The optimal concentration of IBA in rooting medium was 2 mg/L.3, to establish an efficient genetic transformation system. The average transformation efficiency of maize immature embryo was 1.0 ~ 1.2mmGUS and the average transformation efficiency of Gus was 35.3m. The concentration of Agrobacterium tumefaciens was 0.8, and that of Agrobacterium tumefaciens was 0.8. The average conversion efficiency of GUS was 46.8%, and the average transformation efficiency of 5min and thermal pretreatment at 43 鈩，

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