农杆菌介导的Lyz-GFP基因对匍匐翦股颖Penn A-1转化和表达的研究

草业学报 ›› 2012, Vol. 21 ›› Issue (2): 141-148.

农杆菌介导的Lyz-GFP基因对匍匐翦股颖Penn A-1转化和表达的研究

安惠惠^1,2,3,马晖玲^1,2,3*,李坚⁴,白生军^1,3,马祥^1,3

1.草业生态系统教育部重点实验室甘肃农业大学,甘肃兰州 730070;
2.甘肃省草业工程实验室,甘肃兰州 730070;
3.中-美草地畜牧业可持续发展研究中心,甘肃兰州 730070;
4. 陆地水循环及地表过程重点实验室中国科学院地理科学与资源研究所,北京 100101

收稿日期:2010-12-01 出版日期:2012-02-25 发布日期:2012-04-20
通讯作者: E-mail:mahl@gsau.edu.cn
作者简介:安惠惠(1983-),女,甘肃天水人,硕士。E-mail:anhh2004@163.com
基金资助:
草业生态系统教育部重点实验室(甘肃农业大学)开放课题(CYZS-2011-002)和甘肃省作物遗传改良与种质创新重点实验室项目-匍匐翦股颖抗病转基因植株的选育(033160)资助。

A study on transformation and expression of the Lyz-GFP genes mediated by agrobacteria in creeping bentgrass variety Penn A-1

AN Hui-hui^1,2,3, MA Hui-ling^1,2,3, LI Jian⁴, BAI Sheng-jun^1,3, MA Xiang^1,3

1.Key Laboratory of Grassland Ecosystem, Gansu Agricultural University, Lanzhou 730070, China;
2.Ministry of Education,　Pratacultural Engineering Laboratory of Gansu Province, Lanzhou 730070, China;
3.Sino-U.S. Centers for Grazingland Ecosystem Sustainability, Lanzhou 730070, China;
4.Key Laboratory of Water Cycle and Related Land Surface Processes, Institute of Geographic Sciences and Natural Resources Research, CAS, Beijing 100101, China

Received:2010-12-01 Online:2012-02-25 Published:2012-04-20

摘要/Abstract

摘要： 以匍匐翦股颖Penn A-1成熟胚为供试材料,建立了其植株高效再生体系。利用农杆菌介导法将溶菌酶与绿色荧光蛋白Lyz-GFP双元基因转入匍匐翦股颖Penn A-1胚性愈伤组织中, 经培养获得抗病转基因植株。并对Lyz-GFP双元基因转化Penn A-1的适宜条件进行了研究。研究结果表明,在2.0 mg/L 2, 4-D+0.1 mg/L 6-BA的MS培养基上Penn A-1愈伤组织诱导率最高,可达36%,且质量最好。愈伤组织在MSO+0.5 mg/L NAA上分化率最高,达42.5%;浓度为300 mg/L的头孢霉素(Cef)可抑制农杆菌LBA4404的生长;Penn A-1胚性愈伤组织经携有pBI121-Lyz-GFP的农杆菌LBA4404 (OD₆₀₀值0.3~0.5)侵染10~15 min,共培养3 d后,转化愈伤组织生长状况良好,转化率达12.5%,且在后期的生长和转化苗的再生中有良好的表现, 转化苗再生率为27.5%; 转化植株有较强的荧光表达量,并经PCR检测,获得的2株匍匐翦股颖Penn A-1转化植株中均扩增出750 bp的目标基因片断。

Abstract: Mature embryos of bentgrass (Agrostis stolonifera) Penn A-1 were used to establish a highly efficient regeneration system. The dual genes Lyz-GFP (Lysozyme gene and green fluorescence protein gene) were transformed into embryonic calli of creeping bentgrass by an agrobacterium-mediation method to obtain transgenic plants with disease resistance capability. The best conditions for transformation of the Lyz-GFP genes in Penn A-1 were studied. The callus induction ratio of Penn A-1 was highest at 36% on MS medium supplemented with 2.0 mg/L 2, 4-D and 0.1 mg/L 6-BA, and the calli had the optimum growth status. The highest differentiation ratio was 42.5% on MSO medium supplemented with 0.5 mg/L NAA. The study also showed that growth of agrobacterium LBA4404 was restrained by 300 mg/L cefotaxime (Cef). Transformed calli performed well in later growth periods as well as in the regeneration of transgenic plants after infection with agrobacterium LBA4404 (OD₆₀₀: 0.3-0.5) carried with pBI121-Lyz-GFP for 10-15 min, and co-cultured for 3 days. The transformation ratio of calli was 12.5%; the regeneration ratio of transgenic plants was 27.5%; the transgenic plants strongly expressed fluorescence, and the 750 bp target fragments of the GFP gene were amplified by PCR from 2 acquired transgenic plants with expression of Penn A-1 fluorescence.

中图分类号:

安惠惠,马晖玲,李坚,白生军,马祥. 农杆菌介导的Lyz-GFP基因对匍匐翦股颖Penn A-1转化和表达的研究[J]. 草业学报, 2012, 21(2): 141-148.

AN Hui-hui, MA Hui-ling, LI Jian, BAI Sheng-jun, MA Xiang. A study on transformation and expression of the Lyz-GFP genes mediated by agrobacteria in creeping bentgrass variety Penn A-1[J]. Acta Prataculturae Sinica, 2012, 21(2): 141-148.

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