农杆菌介导TLPD34基因的玉米遗传转化及检测
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摘要
以玉米优良自交系郑58、掖478为受体材料,以玉米茎尖生长点、幼穗(合子)为转化受体,采用农杆菌介导的转化技术,将抗真菌病害的水稻类甜蛋白(TLPD34)基因转入玉米,对后代进行除草剂筛选,分子检测及真菌接种试验,得到如下结论:
1.通过质粒pUbiTLPD34完整物理图谱,选择BamHI、HindIII两个酶切位点,对质粒进行单酶切和双酶切,得到与预期大小相同的各个片段,目的片段1100bp左右,质粒测序结果与NCBI提交的TLPD34基因序列达到97%的相似度。
2.Bar基因作为筛选标记基因,以草胺磷除草剂作为苗期筛选药物,在玉米3叶期通过不同浓度的涂抹试验发现,200 mg/L的草胺磷为转基因玉米适宜筛选浓度。
3.通过农杆菌介导的玉米茎尖转化,获得451株转化植株,经200 mg/L草胺磷有效质量浓度筛选,得到57株抗性苗,T0代检测后,获得13株阳性植株,转化率达2.9 %。对T0代PCR检测呈阳性的植株进行RT-PCR发现,7株能够进行正常转录。证明该方法可将外源基因TLPD34整合到玉米自交系郑58并进行正常转录。
4.用目的基因(TLPD34)的农杆菌重悬液(OD600值为0.6左右)侵染授粉24 h左右的郑58、掖478玉米自交系幼穗(合子转化),分别得到261粒、322粒种子,种子出苗率达到70%以上,在苗期进行除草剂筛选,分别得到了7.42 %、10.24 %的抗性苗率,PCR检测后得到0.99 %、1.18 %的转化率,说明掖478的合子转化效果优于郑58。
5.以郑58玉米茎尖和玉米合子为转化受体,用目的基因(TLPD34)的农杆菌重悬液(OD600值为0.6左右)进行侵染,在苗期进行除草剂筛选,分别得到12.64 %、7.42 %的抗性苗,PCR检测后分别得到2.9 %和0.99 %的阳性植株。说明茎尖优于合子转化转化效果。
6.在玉米苗期接种禾谷镰孢菌和串珠镰孢菌,拔节期前后玉米开始感病。结果表明转基因苗与非转基因苗均对禾谷镰孢菌均有明显的抗性,无明显差异,但转基因苗与非转基因苗相比,对串珠镰孢菌的抗性明显提高,非转基因苗接种串珠镰孢菌后叶片明显开始变黄,植株长势变弱,最终根部腐化,死亡。
7.在玉米拔节期用人工嵌入麦粒法进行立枯丝核菌接种试验,接菌后12h、24h、36h、48h、72h、84h、96h进行照相观察,发现非转基因苗与转基因苗相比,明显感病时间较晚,并且在病菌侵染后,对菌丝的进一步扩大有一定的抑制能力,耐病抗病性提高。
Excellent maize inbred line Zheng 58 and ye 478 as receptor material, the shoot apical point and young ears ( zygote ) of maize as transformation receptors, using the Agrobacterium-mediated transformation technology. The rice thaumatin-like protein (TLPD34) gene that antifungal diseases was transferred into corn, after selecting with glufosinate, molecule detection and Fungi inoculation test, we have conclusions as followed:
1. Through the plasmid pUbiTLPD34 complete physical map, choose enzymes cut plasmid sites, BamHI and HindIII, single and double digestion, get each segment with the same size as expected, the purpose clip is about 1100bp, the similarity is reached 97% after plasmid sequencing result compare with the sequencethat had submitted in NCBI.
2. Bar Gene as screening marker gene, choose glufosinate herbicide, configurate different mass concentration herbicides solution (glufosinate), smear non-Transformed plant of corn in 3 leaf stage, 200 mg/L glufosinate for genetically modified corn is best screening mass concentration.
3. Through Agrobacterium-mediated transformation, the gene was transferred into the shoot apical point of maize inbred line zheng 58, we have obtained 451 transgenic plants in all. After selecting with 200 mg/L effective mass concentration glufosinate, 57 transgenic plants were obtained, after T0 generation test, 13 positive transgenic plants were confirmed by PCR., the transformation frequency is about 2.9 %. 7 plants obtained after confirmed by RT-PCR amplification. The study showed that with this method the foreign gene TLPD34 has integrated into maize inbred line zheng 58 genome and transcription normally.
4. Cell suspension of Agrobacterium tumefaciens (OD=0.6), carrying TLPD34 genes, was applied onto the young ears of Zheng 58 and Ye 478, which had been previously pollinated for 8-12h with the pollen(Zygote Transformation). We obtained 261 and 322 seeds respectively, percentage of germination all reached more than 70%, after the same screening concentration with glufosinate, zheng 58 got 7.42 % resistant plantlets,ye 478 got 10.24 % resistant plantlets, after confirmed by PCR,the transformation frequency is about 0.99 % and 1.18 %, thus the effect is better for ye 478 compare to zheng 58 with this method.
5. Use the shoot apical point and young ears(zygote) of zheng 58 as transformation receptors, use the same cell suspension of Agrobacterium tumefaciens(OD=0.6), the same concentration of herbicides screening, get 12.64 % and 7.42% resistant seedlings respectively, after confirmed by PCR,the transformation frequency is about 2.9 % and 0.99 %. The study showed that the method with shoot apical point as transformation receptor is better for zheng 58.
6. The fungi inoculation test For transgenic and non-modified corn, corn started feeling sick at jointing stage. The transgenic seedlings have high resistance to Fusarium graminearum compared to non-modified seedlings, but transgenic seedlings resistance to Fusarium maniliforme increased significantly, non-modified seedlings leaf beginning to yellow, plant growing weaker, roots became corruption, and eventually death.
7. At jointing stage, several wheat seeds co-cultivate with Rhizoctonia solani were placed into the third sheath of the maize, photographic observation after 12h,24h,36h,48h,72h,84h and 96h , compared with transgenic seedlings, non-modified seedlings acquired susceptibility later, and after fungal infection, transgenic seedlings can inhibit the spread of the mycelium further expansion,the resistance to disease is improved.
1.通过质粒pUbiTLPD34完整物理图谱,选择BamHI、HindIII两个酶切位点,对质粒进行单酶切和双酶切,得到与预期大小相同的各个片段,目的片段1100bp左右,质粒测序结果与NCBI提交的TLPD34基因序列达到97%的相似度。
2.Bar基因作为筛选标记基因,以草胺磷除草剂作为苗期筛选药物,在玉米3叶期通过不同浓度的涂抹试验发现,200 mg/L的草胺磷为转基因玉米适宜筛选浓度。
3.通过农杆菌介导的玉米茎尖转化,获得451株转化植株,经200 mg/L草胺磷有效质量浓度筛选,得到57株抗性苗,T0代检测后,获得13株阳性植株,转化率达2.9 %。对T0代PCR检测呈阳性的植株进行RT-PCR发现,7株能够进行正常转录。证明该方法可将外源基因TLPD34整合到玉米自交系郑58并进行正常转录。
4.用目的基因(TLPD34)的农杆菌重悬液(OD600值为0.6左右)侵染授粉24 h左右的郑58、掖478玉米自交系幼穗(合子转化),分别得到261粒、322粒种子,种子出苗率达到70%以上,在苗期进行除草剂筛选,分别得到了7.42 %、10.24 %的抗性苗率,PCR检测后得到0.99 %、1.18 %的转化率,说明掖478的合子转化效果优于郑58。
5.以郑58玉米茎尖和玉米合子为转化受体,用目的基因(TLPD34)的农杆菌重悬液(OD600值为0.6左右)进行侵染,在苗期进行除草剂筛选,分别得到12.64 %、7.42 %的抗性苗,PCR检测后分别得到2.9 %和0.99 %的阳性植株。说明茎尖优于合子转化转化效果。
6.在玉米苗期接种禾谷镰孢菌和串珠镰孢菌,拔节期前后玉米开始感病。结果表明转基因苗与非转基因苗均对禾谷镰孢菌均有明显的抗性,无明显差异,但转基因苗与非转基因苗相比,对串珠镰孢菌的抗性明显提高,非转基因苗接种串珠镰孢菌后叶片明显开始变黄,植株长势变弱,最终根部腐化,死亡。
7.在玉米拔节期用人工嵌入麦粒法进行立枯丝核菌接种试验,接菌后12h、24h、36h、48h、72h、84h、96h进行照相观察,发现非转基因苗与转基因苗相比,明显感病时间较晚,并且在病菌侵染后,对菌丝的进一步扩大有一定的抑制能力,耐病抗病性提高。
Excellent maize inbred line Zheng 58 and ye 478 as receptor material, the shoot apical point and young ears ( zygote ) of maize as transformation receptors, using the Agrobacterium-mediated transformation technology. The rice thaumatin-like protein (TLPD34) gene that antifungal diseases was transferred into corn, after selecting with glufosinate, molecule detection and Fungi inoculation test, we have conclusions as followed:
1. Through the plasmid pUbiTLPD34 complete physical map, choose enzymes cut plasmid sites, BamHI and HindIII, single and double digestion, get each segment with the same size as expected, the purpose clip is about 1100bp, the similarity is reached 97% after plasmid sequencing result compare with the sequencethat had submitted in NCBI.
2. Bar Gene as screening marker gene, choose glufosinate herbicide, configurate different mass concentration herbicides solution (glufosinate), smear non-Transformed plant of corn in 3 leaf stage, 200 mg/L glufosinate for genetically modified corn is best screening mass concentration.
3. Through Agrobacterium-mediated transformation, the gene was transferred into the shoot apical point of maize inbred line zheng 58, we have obtained 451 transgenic plants in all. After selecting with 200 mg/L effective mass concentration glufosinate, 57 transgenic plants were obtained, after T0 generation test, 13 positive transgenic plants were confirmed by PCR., the transformation frequency is about 2.9 %. 7 plants obtained after confirmed by RT-PCR amplification. The study showed that with this method the foreign gene TLPD34 has integrated into maize inbred line zheng 58 genome and transcription normally.
4. Cell suspension of Agrobacterium tumefaciens (OD=0.6), carrying TLPD34 genes, was applied onto the young ears of Zheng 58 and Ye 478, which had been previously pollinated for 8-12h with the pollen(Zygote Transformation). We obtained 261 and 322 seeds respectively, percentage of germination all reached more than 70%, after the same screening concentration with glufosinate, zheng 58 got 7.42 % resistant plantlets,ye 478 got 10.24 % resistant plantlets, after confirmed by PCR,the transformation frequency is about 0.99 % and 1.18 %, thus the effect is better for ye 478 compare to zheng 58 with this method.
5. Use the shoot apical point and young ears(zygote) of zheng 58 as transformation receptors, use the same cell suspension of Agrobacterium tumefaciens(OD=0.6), the same concentration of herbicides screening, get 12.64 % and 7.42% resistant seedlings respectively, after confirmed by PCR,the transformation frequency is about 2.9 % and 0.99 %. The study showed that the method with shoot apical point as transformation receptor is better for zheng 58.
6. The fungi inoculation test For transgenic and non-modified corn, corn started feeling sick at jointing stage. The transgenic seedlings have high resistance to Fusarium graminearum compared to non-modified seedlings, but transgenic seedlings resistance to Fusarium maniliforme increased significantly, non-modified seedlings leaf beginning to yellow, plant growing weaker, roots became corruption, and eventually death.
7. At jointing stage, several wheat seeds co-cultivate with Rhizoctonia solani were placed into the third sheath of the maize, photographic observation after 12h,24h,36h,48h,72h,84h and 96h , compared with transgenic seedlings, non-modified seedlings acquired susceptibility later, and after fungal infection, transgenic seedlings can inhibit the spread of the mycelium further expansion,the resistance to disease is improved.
引文
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