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在遗传转化获得抗性植株时,转化体的抗性筛选是遗传转化能否取得成功的关键步骤。通常在选择培养基中加入合适种类和浓度的筛选剂,使其产生一定的筛选压起到抗性筛选的作用。转化体内选择标记基因的表达产物可对特定筛选剂产生抗性,使转化受体材料继续保持正常的生长发育[1]。目前的研究中,卡那霉素、潮霉素等抗生素被普遍作为筛选剂使用[2-3]。但是由于水稻Oryza sativa胚性愈伤组织对抗生素具有生理抗性,以抗生素为选择标记进行抗性筛选,不能起到很好的筛选效果,且经抗生素筛选后的转化体在分化和再生阶段易受抑制或产生白化苗[4-6]。以草甘膦作为筛选剂可以提高选择的灵敏度,消除转化体生理抗性对筛选结果的影响,克服了以往研究中抗生素筛选的局限性。可遗传的草甘膦抗性基因突变率低,并可在后代中稳定表达,因此进行抗草甘膦作物的培育是可行的[7]。籼稻Oryza sativa subsp. indica和粳稻Oryza sativa subsp. japonica是栽培稻的2个亚种,随着水稻遗传转化技术的发展,大部分粳稻品种已经建立了成熟的遗传转化体系,并成功引入抗虫、抗病、生长发育调控等诸多有利基因[8]。而大多数籼稻品种组培特性不佳,愈伤组织诱导率低,继代过程易褐化且分化再生频率低,导致籼稻的遗传转化效率低,有的品种甚至难以转化。尤其是对生产上广泛推广、农艺性状优良的重要品种而言,其改良与育种进程受到严重限制[9]。CHAN等[10]于1992年尝试利用农杆菌Agrobacterium tumefaciens介导法转化籼稻幼根愈伤组织,对转化体进行Southern印记杂交,结果表明:目的基因片段已成功转入转化体细胞中。后经酶活性检测,目的基因可在转化体中稳定表达。1994年,HIEI等[11]为建立高效稳定的农杆菌遗传转化体系,采用了“双超元”载体,并通过在菌液添加乙酰丁香酮(As)活化Vir基因提高转化效率等方法,推进了遗传转化技术在籼稻中的研究应用。目前,虽然已有转抗草甘膦基因的籼稻遗传转化体系的报道,但是转化效率低,还未建立一个高效的转化体系[12]。基于此,本研究选取具有成功再生体系的籼稻‘中恢161’ Oryza sativa subsp. indica ‘Zhonghui 161’为材料,利用农杆菌介导法,转入草甘膦抗性基因(CP4),探索适合的草甘膦质量浓度用于抗性筛选,并对农杆菌介导的转化过程进行了合理优化,建立‘中恢161’农杆菌介导的转化体系。
Agrobacterium-mediated transformation of CP4 gene into indica rice
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摘要:
目的 建立籼稻‘中恢161’Oryza sativa subsp. indica ‘Zhonghui 161’农杆菌Agrobacterium tumefaciens介导的转化体系。 方法 以籼稻‘中恢161’的成熟胚为材料,设置了5个草甘膦质量浓度(100、200、300、400和500 mg·L−1)进行胚性愈伤组织的草甘膦敏感性试验。利用农杆菌介导法,将草甘膦抗性基因CP4-EPSPS导入‘中恢161’的胚性愈伤组织中,转化后的胚性愈伤组织分别在含有300、350和400 mg·L−1草甘膦的选择培养基上进行抗性筛选。抗性愈伤组织进一步分化、成苗。 结果 草甘膦质量浓度为300~400 mg·L−1时,愈伤组织褐化率约50%,具有很好的选择效果。经统计,300、350和400 mg·L−1草甘膦抗性筛选后,愈伤组织阳性率分别为40.16%、61.72%和84.04%,抗性愈伤组织的分化率为46.43%,成苗率为32.84%。共获得67株再生小苗,经PCR检测,43株成功转入CP4基因,再生植株阳性率为64.18%。 结论 建立了‘中恢161’农杆菌介导的转化体系。图5参20 Abstract:Objective The objective was to establish the Agrobacterium-mediated transformation system of Oryza sativa subsp. indica ‘Zhonghui 161’. Method 5 groups of glyphosate concentrations (100, 200, 300, 400 and 500 mg·L−1) were used to test the sensitivity of embryogenic callus to glyphosate. The glyphosate-resistant gene (CP4-EPSPS) was introduced into the embryogenic callus of ‘Zhonghui 161’ by Agrobacterium-mediated method. The transformed embryogenic callus was screened for glyphosate resistance on the selective medium containing 300, 350 and 400 mg·L−1 glyphosate. The resistant callus was further differentiated and seeded. Result When the concentration of glyphosate was 300−400 mg·L−1, the browning rate of callus was about 50%, showing a good selection effect. The positive rates of callus on 300, 350 and 400 mg·L−1 glyphosate were 40.16%, 61.72% and 84.04%, respectively. The further differentiation rate was 46.43%, and the seedling rate was 32.84%. A total of 67 regenerated plantlets were obtained, and 43 of them were successfully transformed into CP4 gene by PCR detection. The positive rate of regenerated plantlets was 64.18%. Conclusion Agrobacterium-mediated transformation system of ‘Zhonghui 161’ was established. [Ch, 5 fig. 20 ref.] -
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https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.20200436