根癌农杆菌（Agrobacterium tumefaciens）介导的多年生黑麦草（Lolium perenne L.）遗传转化
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摘要
多年生黑麦草(Lolium perenne L．)是优良的牧草和草坪草，是畜牧业和草坪业重要的产业支柱。由于自交不亲和，传统遗传改良困难而复杂。生物技术尤其是转基因技术对其遗传改良带来希望。本研究通过根癌农杆菌(Agrobacterium tumefaciens)介导，对多年生黑麦草进行遗传转化，成功地将外源报告基因或目的基因导入至多年生黑麦草中，获得转基因植株。
研究首先对多年生黑麦草的转化条件进行了优化，建立起根癌农杆菌介导的遗传转化方法。在选择的9种多年生黑麦草成熟胚诱导的愈伤组织中，经用根癌农杆菌侵染、百龙霉素选择，从4种多年生黑麦草材料(金牌美达丽、神枪手、多福和利美斯)中获得了抗性愈伤组织和抗性植株。
在利用报告基因进行转化中，金牌美达丽、神枪手、多福的抗性植株是由携质粒pCAMBIA2301的农杆菌EHA105转化后获得的。该质粒含有选择基因npt Ⅱ和报告基因gus，均由CaMV35S启动子驱动。经PCR、GUS组织染色验证结果表明，绝大多数抗性植株是转npt Ⅱ和gus基因的植株。其中金牌美达丽从1次转化中获得9株转基因植株：神枪手从2次转化中分别获得4和12苗；从3次转化中分别获得转基因多福植株17、18和15苗。最高转化效率可以达到4.3％。烟草汁在一定程度上有提高转化效率的作用。以质粒pCAMBIA2301为模板进行探针PCR地高辛标记，Southern bloting分析金牌美达丽9株转基因植株npt Ⅱ基因，结果表明，转基因植株外源基因拷贝数低，其中6株为单拷贝，
Perennial ryegrass (Lolium perenne L.) (2n=2x=14), a graminaceous crop, is one of the most widely distributed grass in the temperate regions for forage and turf. Because of the highly self-incompatible, the conventional plant breeding is difficult and selection schemes are complex. Biotechnological approaches, especially genetic transformation for the direct introduction of agronomically useful genes, hold great promise.
A method Agrobacterium tumefaciens transformation for perennial ryegrass was developed using the calli derived from mature embryos, and the transgenic plants were successfully obtained.
In delivering report genes, nine ryegrass varieties were employed and their calli were inoculated with a disarmed Agrobacterium tumefaciens strain EHA105 harboring binary vector pCAMBIA2301 or p132. Transgenic plants were obtained from 4 varieties of perennial ryegrass ( Gold Medalist, Top Gun, Tove and Limes) by selection with paromomycin.
The transgenic seedlings of Gold Medalist, Top Gun, and Tove were produced by using the plasmid pCAMBIA2301. The transgenic calli and plants were identified by PCR and GUS histochemical assay, which showed that the foreign genes were presented in host genome and expressed. Overall, 9 transgenic seedlings were obtained from Gold Medalist in one transformation event; 4 and 12 ones of Top Gun were gained from 2 events separatively; and 17, 18 and 15 transgenic seedlings were produced from Tove in 3 independent events, respectively. Dig-labelled Southern-bloting analysis for the 9 transgenic seedlings of Gold
研究首先对多年生黑麦草的转化条件进行了优化，建立起根癌农杆菌介导的遗传转化方法。在选择的9种多年生黑麦草成熟胚诱导的愈伤组织中，经用根癌农杆菌侵染、百龙霉素选择，从4种多年生黑麦草材料(金牌美达丽、神枪手、多福和利美斯)中获得了抗性愈伤组织和抗性植株。
在利用报告基因进行转化中，金牌美达丽、神枪手、多福的抗性植株是由携质粒pCAMBIA2301的农杆菌EHA105转化后获得的。该质粒含有选择基因npt Ⅱ和报告基因gus，均由CaMV35S启动子驱动。经PCR、GUS组织染色验证结果表明，绝大多数抗性植株是转npt Ⅱ和gus基因的植株。其中金牌美达丽从1次转化中获得9株转基因植株：神枪手从2次转化中分别获得4和12苗；从3次转化中分别获得转基因多福植株17、18和15苗。最高转化效率可以达到4.3％。烟草汁在一定程度上有提高转化效率的作用。以质粒pCAMBIA2301为模板进行探针PCR地高辛标记，Southern bloting分析金牌美达丽9株转基因植株npt Ⅱ基因，结果表明，转基因植株外源基因拷贝数低，其中6株为单拷贝，
Perennial ryegrass (Lolium perenne L.) (2n=2x=14), a graminaceous crop, is one of the most widely distributed grass in the temperate regions for forage and turf. Because of the highly self-incompatible, the conventional plant breeding is difficult and selection schemes are complex. Biotechnological approaches, especially genetic transformation for the direct introduction of agronomically useful genes, hold great promise.
A method Agrobacterium tumefaciens transformation for perennial ryegrass was developed using the calli derived from mature embryos, and the transgenic plants were successfully obtained.
In delivering report genes, nine ryegrass varieties were employed and their calli were inoculated with a disarmed Agrobacterium tumefaciens strain EHA105 harboring binary vector pCAMBIA2301 or p132. Transgenic plants were obtained from 4 varieties of perennial ryegrass ( Gold Medalist, Top Gun, Tove and Limes) by selection with paromomycin.
The transgenic seedlings of Gold Medalist, Top Gun, and Tove were produced by using the plasmid pCAMBIA2301. The transgenic calli and plants were identified by PCR and GUS histochemical assay, which showed that the foreign genes were presented in host genome and expressed. Overall, 9 transgenic seedlings were obtained from Gold Medalist in one transformation event; 4 and 12 ones of Top Gun were gained from 2 events separatively; and 17, 18 and 15 transgenic seedlings were produced from Tove in 3 independent events, respectively. Dig-labelled Southern-bloting analysis for the 9 transgenic seedlings of Gold
引文
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