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当前位置: 首页 > 文章 > 抗草甘膦与磷高效吸收基因双价表达载体的构建及对甘蓝型油菜的遗传转化 甘肃农业大学学报 2019 (3) 31-39,46
Position: Home > Articles > 抗草甘膦与磷高效吸收基因双价表达载体的构建及对甘蓝型油菜的遗传转化 Journal of Gansu Agricultural University 2019 (3) 31-39,46

抗草甘膦与磷高效吸收基因双价表达载体的构建及对甘蓝型油菜的遗传转化

作  者：
王朋宝;张晶晶;石菁;王威;曹智;张金文
单  位：
甘肃省作物遗传改良与种质创新重点实验室甘肃省干旱生境作物学重点实验室甘肃农业大学农学院
关键词：
甘蓝型油菜;植酸酶基因;遗传转化;特异性启动子;qRT-PCR
摘  要：
[目的]甘蓝型油菜是产油效率较高的油料作物,在其生长过程中往往伴随着土壤环境中磷素匮乏及杂草胁迫,严重影响着最终产量和品质.因此,同时解决有效磷匮乏及草害问题已经成为目前油菜研究的重要方向.[方法]采用PCR法克隆得到了与草甘膦抗性(eCTP和EPSPS)和磷高效吸收(Pht1;2和phyA)的相关目的基因,随后构建了双价植物表达载体,通过农杆菌介导法转化甘蓝型油菜,对转化植株进行PCR检测筛选,并通过qRT-PCR法对筛选的阳性植株内目的基因的表达进行定量分析.[结果]将PCR扩增得到的片段经琼脂糖凝胶电泳检测,结果符合预期.测序结果也与GenBank注册序列同源性高度一致,可用于后续试验.对转基因油菜进行草甘膦抗性筛选和PCR扩增检测,共获得5株转基因阳性植株.实时荧光定量结果发现转基因阳性植株内phyA和EPSPS基因的表达量均较对照植株有显著提高.[结论]本研究成功构建了抗草甘膦和磷高效吸收基因双价植物表达载体,获得的转基因阳性植株同时兼具草甘膦抗性及磷素高效吸收利用的特性,为培育甘蓝型油菜优良种质资源提供了理论依据.
作  者：
WANG Peng-bao;ZHANG Jing-jing;SHI Jing;WANG Wei;CAO Zhi;ZHANG Jin-wen;Gansu Provincial Key Lab of Crop Genetic Improvement and Germplasm Innovation,Gansu Provincial Key Lab of Aridland Crop Science,College of Agronomy,Gansu Agricultural University;College of Resources and Environmental Sciences,Gansu Agricultural University;
单  位：
WANG Peng-bao%ZHANG Jing-jing%SHI Jing%WANG Wei%CAO Zhi%ZHANG Jin-wen%Gansu Provincial Key Lab of Crop Genetic Improvement and Germplasm Innovation,Gansu Provincial Key Lab of Aridland Crop Science,College of Agronomy,Gansu Agricultural University%College of Resources and Environmental Sciences,Gansu Agricultural University
关键词：
Brassica napus;;phytase gene;;genetic transformation;;specific promoter;;qRT-PCR
摘  要：
【Objective】 Brassica napus is an oil-bearing crop with high oil production efficiency.The soil phosphorus lack and weeds stress are difficult problems which seriously affect the yield and quality.Therefore,it is an important field to find out the solutions to increase the available phosphorus deficiency and reduce the weeds damage at the same time.【Method】 The genes related to glyphosate resistance(eCTP and EPSPS) and phosphorus efficient uptake(Pht1;2 and phyA) were cloned by PCR.Subsequently,a bivalent plant expression vector was constructed and transformed into Brassica napus through agrobacterium-mediated transformation.Finally,the transformed plants were screened by PCR and the expression of the target genes in the positive plants was screened by qRT-PCR.【Result】 The fragments amplified by PCR were detected by agarose gel electrophoresis and the results were in line with expectations.The sequencing results were also highly homologous with the GenBank-registered sequences and could be used for subsequent experiments.Glyphosate resistance screening and PCR amplification tests were carried out on transgenic rape and 5 transgenic positive plants were obtained.Real-time fluorescence quantitative results showed that the expression levels of phyA and EPSPS genes in the transgenic positive plants were significantly higher than those in control plants.【Conclusion】 A bivalent plant expression vector for glyphosate resistance and phosphorus efficient absorption was successfully constructed.And the transgenic positive plants obtained have both glyphosate resistance and high-efficiency absorption and utilization of phosphorus.This will provide a theoretical basis for breeding excellent germplasm resources of Brassica napus.

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