欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2012, Vol. 38 ›› Issue (12): 2185-2191.doi: 10.3724/SP.J.1006.2012.02185

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型黄籽油菜自交系转化体系的建立

林呐，刘列钊，殷家明，王瑞，柴友荣，李加纳*   

  1. 西南大学农学与生物科技学院 / 重庆市油菜工程技术研究中心 / 南方山地农业教育部工程研究中心，重庆 400716
  • 收稿日期:2011-12-01 修回日期:2012-07-05 出版日期:2012-12-12 网络出版日期:2012-10-08
  • 通讯作者: 李加纳, E-mail: ljn1950@swu.edu.cn
  • 基金资助:

    本研究由高等学校学科创新工程计划“111”项目(B12006)和国家自然科学基金项目(31071450)资助。

Establishment of a Transformation System Using Inbred Line of Yellow-Seeded Brassica napus

LIN Na,LIU Lie-Zhao,YIN Jia-Ming,WANG Rui,CHAI You-Rong,LI Jia-Na*   

  1. College of Agronomy and Biotechnology, Southwest University / Chongqing Rapeseed Engineering & Technology Research Center / Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400716, China
  • Received:2011-12-01 Revised:2012-07-05 Published:2012-12-12 Published online:2012-10-08
  • Contact: 李加纳, E-mail: ljn1950@swu.edu.cn

RichHTML

PDF

1294

被引次数

4

摘要：

利用黄籽甘蓝型油菜自交系建立和优化了遗传转化系统。首先构建了由质粒pCNR与Δ6脂肪酸脱氢酶基因插入到植物的高效表达载体pCAMBIA2301G。利用在Murashige和Skoog培养基(含有200 mmol L–1乙酰丁香酮)培养5~7 d的下胚轴外植体与农杆菌株LBA4404共培养63~69 h (pCNR)，再于芽诱导培养基上培养3个月诱导芽再生。在最佳条件下，平均转化效率约为1.3%。转化植株的GUS分析和PCR分析结果表明，外源基因成功导入甘蓝型油菜。Southern杂交表明，这些转化子含有目标基因1~2个拷贝。用气相色谱分析转基因植物种子的脂肪酸，γ-亚麻酸含量达8.2%。

关键词: 甘蓝型油菜, Δ6-脂肪酸饱和酶, 转化

Abstract:

In this research, we established a transformation system using an inbred line of yellow-seeded Brassica napus. Hypocotyl explants precultured for 5–7 d on Murashige and Skoog medium containing 200 μmol L–1 acetosyringone were cocultured with Agrobacterium tumefaciens strain LBA4404 (pCNR) for 63–69 hours.The plasmid pCNR was constructed by inserting Δ6-fatty acid desaturase gene from Rhizopusstolonifer into plant high-efficient expression vector pCAMBIA2301G. Kanamycin-tolerant shoots were regenerated on shoot induction medium for three months after Agrobacterium inoculation. The average transformation efficiency was about 1.3% under optimal conditions. Results from GUS assay and PCR analysis of transformed plants indicated that the introduced genewas integrated into B. napus genomes.The Southern blot revealed that those transformants carried one or two copies of the goal gene. The fatty acids of the transgenic plant seeds were analyzed by GC, and the γ-linolenic content was 8.2%.

Key words: Brassica napus, Δ6-fatty acid desaturase, Transformation

[1]Knutzon D S, Thompson G A, Radke S E, JohnsonW B, Knauf V C, Kridl J C. Modification of Brassica seed oil by antisense expression of a stearoly acyl carrier protein desaturase gene. Proc Natl Acad Sci USA, 1992, 89: 2624–2628



[2]Shi S-W(石淑稳), Zhou Y-M(周永明), Sun X-C(孙学成), Zhang X-L(张献龙). Transformation of Brassica napus with herbicide resistance gene. J Huazhong Agric Univ (华中农业大学学报), 1998, 17(3): 205–210 (in Chinese with English abstract)



[3]Xu B-B(许本波), Xie L-L(谢伶俐), Tian Z-H(田志宏), Yan H(严寒), He Y(何勇). Study on genetic transformation system of yellow-seed rapeseed (Brassica napus L.). Acta Agric Jiangxi (江西农业学报), 2007, 19 (8): 4–6 (in Chinese with English abstract)



[4]Poulsen G B. Genetic transformation of Brassica. Plant Breed, 1996, 115: 209–225



[5]Khan M R, Rashid H, Ansar M, Chaudry Z. High frequency shoot regeneration and Agrobacterium-mediated DNA transfer in Canola (Brassica napus). Plant Cell Tissue Organ Cult, 2003, 75: 223–231



[6]Leng H(冷虹), Li J N(李加纳), Lu H(陆合), Chai Y R(柴友荣), Yin J M(殷家明). Construction of seed-specific expression vector of Δ6-fatty acid desaturase gene. Chin Agric Sci Bull (中国农学通报), 2006, 22: 66–70 (in Chinese with English abstract)



[7]Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J, 1994, 6, 271–282



[8]Hood E E, Chilton W S, Chilton M D, Fraley R T. T-DNA and opine synthetic loci in tumors incited by Agrobacterium tumefaciens A281 on soybean and alfalfa plants. J Bacteriol, 1986, 168: 1283–1290



[9]Ohta S, Mita S, Hattori T, Nakamura K. Construction and expression in tobacco of a beta-glucoronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol, 1990, 31: 805–813



[10]An G, Ebert R R, Mitra A. Binary vectors. In: Gelvin S B, Schilperroort R A, eds. Plant Molecular Biology Manual. Dordrecht: Kluwer Academic Publishers, 1988. A3: pp 1–19



[11]Jefferson R A. Asaaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep, 1987, 5: 387–405



[12]Doyle J J, Doyle J L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull, 1987, 19: 11–15



[13]Rücker B, Röbbelen G. Impact of low linolenic acid content on seed yield of winter oilseed rape (Brassica napus L.). Plant Breed, 1996, 115: 226–230



[14]Tsukazaki H, Kuginuki Y, Aida R, Suzuki T. Agrobacterium-mediated transformation of a doubled haploid line of cabbage. Plant Cell Rep, 2002, 21: 257–262



[15]Godwin I, Todd G, Lloyd B F, Newbury H J. The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species. Plant Cell Rep, 1991, 9: 671–675



[16]Godwin A K, Testa J R, Handel L M, Liu Z, Vanderveer L A, Tracey P A, Hamilton T C. Spontaneous transformation of rat ovarian surface epithelial cells: association with cytogenetic changes and implications for repeated ovulation in the etiology of ovarian cancer. J Nat Cancer Inst, 1992, 84: 592–601



[17]Holford P, Hernandez N, Newbury H J. Factors influencing the efficiency of T-DNA transfer during co-cultivation of Antirrhinum majus with Agrobacterium bumefaciens. Plant Cell Rep, 1993, 11: 196–199



[18]Metz T D, Dixit R, Earle E D. Agrobacterium tumefaciens mediated transformation of broccoli (Brassica oleracea var. italica) and cabbage (B. oleracea var. capitata). Plant Cell Rep, 1995, 15: 287–292



[19]Takasaki T, Hatakeyama K, Ojima K, Watanabe M, Toriyama K, Hinata K. Factors influencing Agrobacterium-mediated transformation of Brassica rapa L. Breed Sci, 1997, 47: 127–136



[20]Henzi M X, Christey M C, McNeil D L. Factors that influence Agrobacterium rhizogenes-mediated transformation of broccoli (Brassica oleracea L. var. italica). Plant Cell Rep, 2000, 19: 994–999



[21]Deroles S C, Gardner R C. Analysis of T-DNA structure in a large number of transgenic petunias generated by Agrobacterium-mediated transformation. Plant Mol Biol, 1988, 11: 365–377



[22]Stoger E, Fink C, Pfosser M, Heberle B E. Plant transformation by particle bombardment of embryogenic pollen. Plant Cell Rep, 1995, 14: 273–278
[1] 陈松余, 丁一娟, 孙峻溟, 黄登文, 杨楠, 代雨涵, 万华方, 钱伟. 甘蓝型油菜BnCNGC基因家族鉴定及其在核盘菌侵染和PEG处理下的表达特性分析[J]. 作物学报, 2022, 48(6): 1357-1371.
[2] 秦璐, 韩配配, 常海滨, 顾炽明, 黄威, 李银水, 廖祥生, 谢立华, 廖星. 甘蓝型油菜耐低氮种质筛选及绿肥应用潜力评价[J]. 作物学报, 2022, 48(6): 1488-1501.
[3] 袁大双, 邓琬玉, 王珍, 彭茜, 张晓莉, 姚梦楠, 缪文杰, 朱冬鸣, 李加纳, 梁颖. 甘蓝型油菜BnMAPK2基因的克隆及功能分析[J]. 作物学报, 2022, 48(4): 840-850.
[4] 冯亚, 朱熙, 罗红玉, 李世贵, 张宁, 司怀军. 马铃薯StMAPK4响应低温胁迫的功能解析[J]. 作物学报, 2022, 48(4): 896-907.
[5] 黄成, 梁晓梅, 戴成, 文静, 易斌, 涂金星, 沈金雄, 傅廷栋, 马朝芝. 甘蓝型油菜BnAPs基因家族成员全基因组鉴定及分析[J]. 作物学报, 2022, 48(3): 597-607.
[6] 王瑞, 陈雪, 郭青青, 周蓉, 陈蕾, 李加纳. 甘蓝型油菜白花基因InDel连锁标记开发[J]. 作物学报, 2022, 48(3): 759-769.
[7] 王艳花, 刘景森, 李加纳. 整合GWAS和WGCNA筛选鉴定甘蓝型油菜生物产量候选基因[J]. 作物学报, 2021, 47(8): 1491-1510.
[8] 宋天晓, 刘意, 饶莉萍, Soviguidi Deka Reine Judesse, 朱国鹏, 杨新笋. 甘薯细胞壁蔗糖转化酶基因IbCWIN家族成员鉴定及表达分析[J]. 作物学报, 2021, 47(7): 1297-1308.
[9] 李杰华, 端群, 史明涛, 吴潞梅, 柳寒, 林拥军, 吴高兵, 范楚川, 周永明. 新型抗广谱性除草剂草甘膦转基因油菜的创制及其鉴定[J]. 作物学报, 2021, 47(5): 789-798.
[10] 唐鑫, 李圆圆, 陆俊杏, 张涛. 甘蓝型油菜温敏细胞核雄性不育系160S花药败育的形态学特征和细胞学研究[J]. 作物学报, 2021, 47(5): 983-990.
[11] 唐锐敏, 贾小云, 朱文娇, 印敬明, 杨清. 马铃薯热激转录因子HsfA3基因的克隆及其耐热性功能分析[J]. 作物学报, 2021, 47(4): 672-683.
[12] 周新桐, 郭青青, 陈雪, 李加纳, 王瑞. GBS高密度遗传连锁图谱定位甘蓝型油菜粉色花性状[J]. 作物学报, 2021, 47(4): 587-598.
[13] 李书宇, 黄杨, 熊洁, 丁戈, 陈伦林, 宋来强. 甘蓝型油菜早熟性状QTL定位及候选基因筛选[J]. 作物学报, 2021, 47(4): 626-637.
[14] 张春, 赵小珍, 庞承珂, 彭门路, 王晓东, 陈锋, 张维, 陈松, 彭琦, 易斌, 孙程明, 张洁夫, 傅廷栋. 甘蓝型油菜千粒重全基因组关联分析[J]. 作物学报, 2021, 47(4): 650-659.
[15] 唐婧泉, 王南, 高界, 刘婷婷, 文静, 易斌, 涂金星, 傅廷栋, 沈金雄. 甘蓝型油菜SnRK基因家族生物信息学分析及其与种子含油量的关系[J]. 作物学报, 2021, 47(3): 416-426.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2