[1]李琳,付晓佳,杨燕燕,等.农杆菌介导菜豆几丁质酶基因转化洋桔梗[J].江苏农业学报,2015,(03):512-519.[doi:10.3969/j.issn.1000-4440.2015.03.008]
　LI Lin,FU Xiao-jia,YANG Yan-yan,et al.Agrobacterium-mediated transformation of a bean chitinase gene into Eustoma grandiflorum[J].,2015,(03):512-519.[doi:10.3969/j.issn.1000-4440.2015.03.008]
点击复制

农杆菌介导菜豆几丁质酶基因转化洋桔梗()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年03期
页码:
512-519
栏目:
遗传育种·生理生化
出版日期:
2015-06-30

文章信息/Info

Title:
Agrobacterium-mediated transformation of a bean chitinase gene into Eustoma grandiflorum
作者:
李琳; 付晓佳; 杨燕燕; 韦银凤; 陈崇顺
（南京师范大学生命科学学院，江苏省生物多样性与生物技术重点实验室，江苏南京210023）
Author(s):
LI Lin; FU Xiao-jia; YANG Yan-yan; WEI Yin-feng; CHEN Chong-shun
(Jiangsu Key Laboratory of Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China)
关键词:
洋桔梗; 几丁质酶基因; 遗传转化; 真菌病害
Keywords:
lisianthus; chitinase gene; transformation; fungal disease
分类号:
Q943.2
DOI:
10.3969/j.issn.1000-4440.2015.03.008
文献标志码:
A
摘要:
通过农杆菌介导法将抗真菌的几丁质酶基因转入洋桔梗，以提高其对真菌病害的抗性。结果表明，成功克隆了抗真菌活性较强的菜豆几丁质酶基因，构建了植物表达载体，并对洋桔梗Double Mariachi Pink叶块进行转化，获得了卡那霉素抗性植株,对抗性植株进行了2次PCR检测（nptⅡ基因），第1次PCR检测获得了13个阳性株系,培养60 d后再进行第2次PCR检测，获得11个阳性株系,再对生长良好的5个转基因株系进行RT-PCR检测，获得了3个阳性株系。3个阳性株系的几丁质酶平均活性（0.215 U、0.225 U和0.286 U）均显著高于未转化植株（0.133 U），转基因株系几丁质酶粗提液对大豆链霉菌5A、5E和灰葡萄孢菌3种指示真菌的抑菌圈直径均显著大于未转化植株，其中抑菌效果最好的株系对上述3种指示真菌的抑菌圈直径与对照相比，分别增大了106.00%、90.91%和71.53%。
Abstract:
An antifungal chitinase gene was transferred into the leaf segments of lisianthus [Eustoma grandiflorum (Raf.) Shinn.] Double Mariachi Pink to enhance the resistance to fungal diseases by the Agrobacterium-mediated method. Thirteen kanamycin-resistant lines were obtained and analyzed by PCR, among which, three transgenic lines were confirmed nptII gene positive by RT-PCR. The chitinase activities of the three transgenic lines were 0.215 U, 0.225 U and 0.286 U, significantly higher than the non-transformed line (0.133 U). The inhibitory effects (in terms of the diameter of inhibition zone) on three fungi (Streptomyce 5A, 5E and boytrytis cinerea) were significantly greater than the non-transformed line; The strongest inhibitory transgenic line showed larger diameters of inhibition zone by 106.00%, 90.91% and 71.53%. 

参考文献/References:


[1]OHKI T, KAJIHARA H, MAOKA T. First of Moroccan pepper virus on lisianthus (Eustoma grandiflorum) in Japan [J]. J Gen Plant Pathol, 2014, 80: 90-93.
[2]KAWABATA S, YOKOO M, NII K. Quantitative analysis of corolla shapes and petal contours in single-flower cultivars of lisianthus [J]. Sci Hort, 2009, 121: 206-212.
[3]THIRUVENGADAM M, YANG C H. Ectopic expression of two MADS box genes from orchid (Oncidium Gower Ramsey) and lily (Lilium longifiorum) alters fiower transition and formation in Eustoma grandifiorum [J]. Plant Cell Rep, 2009, 28:1463-1473.
[4]KIL E J, BYUN H S, HWANG H. First report of tomato yellow leaf curl virus infecting Eustoma (Eustoma grandiflorum) in Korea [J]. Plant Dis, 2014, 98(8): 1163.
[5]TRUTER M, WEHNER F C. Crown and root infection of Lisianthus caused by Fusarium solani in South Africa [J]. Plant Dis, 2004, 88(5): 573. 
[6]PAOLETTI M G, PIRMENTEL D. Genetic engineering in agriculture and the environment [J]. BioScience, 1996, 46(9): 665-673. 
[7]BOLLER T, GEHRI A, MAUCH F, et al. Chitinase in bean leaves: induction by ethylene, purification, properties, and possible function [J]. Planta, 1983, 157: 22-31.
[8]HAMMAM I, SIALA R, JRIDI M, et al. Partial purification and characterization of chiIO8, a novel antifungal chitinase produced by Bacillus cereus IO8 [J]. J Appl Microbiol, 2013, 115(2): 358-366. 
[9]曲敏,王全伟,李新玲. 菜豆几丁质酶基因的克隆、序列分析及其表达 [J]. 大豆科学, 2007, 26(2): 121-126.
[10]SCHLUMBAUM A, MAUCH F, VGELI U, et al. Plant chitinase are potent inhibitors of fungal growth [J]. Nature, 1986, 324: 355-356.
[11]BROGLIE K E, CHET I, HOLLLIDAY M, et al. Transgenic plant with enchanced resistance to the fungal pathogen Rhizoctonia solani [J]. Science, 1991, 254(5035): 1194-1197.
[12]刘伟华,李文雄,胡尚莲,等. 基因枪法向小麦导入几丁质酶基因的研究 [J]. 西北植物学报, 2003, 23(1): 54-59.
[13]王晶晶,屈淑平,崔崇士. 农杆菌介导几丁质酶基因转化南瓜的研究 [J]. 北方园艺, 2007 (10): 184-185.
[14]萨姆布鲁克. 分子克隆实验指南 [M]. 3版. 金冬雁，译. 北京: 科学出版社, 2002.
[15]杨燕燕，陈崇顺，瞿大枞,等. 洋桔梗高频再生系统的建立及其卡那霉素敏感性测定 [J]. 江苏农业科学, 2007(2): 98-100.
[16]王转梅. 烟草碱性β-1,3-葡聚糖酶基因转化Double Mariachi Pink洋桔梗的研究[D]. 南京: 南京师范大学, 2009.
[17]KATAPODIS P, CHRISTAKO P V, KEKOS D, et al. Optimization of xylanase production by Chaetomium thermophilum in wheat straw using response surface methodology [J]. Biochem Eng J, 2007, 35: 136-141.
[18]SAQIB A A N, WHITNEY P J. Differential behaviour of the dinitrosalicylic acid (DNS) reagent towards mono- and di-saccharide sugars [J]. Biomass Bioenerg, 2011, 35(11): 4748-4750.
[19]吴子健,王新字,石晓旭,等. TUV26菌株几丁质酶酶解所产几丁寡糖产物的分析 [J]. 食品研究与开发, 2010, 31(10): 178-181.
[20]陈崇顺,徐风彩,李明启. 21科41种(变种)植物叶片几丁质酶系的研究 [J]. 植物资源与环境学报, 1993, 2(4): 28-33.
[21]林慧珍,苟萍. 灰葡萄孢菌的诱变及其致病性研究 [J]. 新疆农业科学, 2011, 48(5): 877-881.
[22]陈崇顺,朱雪峰,郁志芳. 豇豆几丁质酶纯酶液对不同真菌的作用 [J]. 植物保护学报, 2000, 27(4): 375-376. 
[23]VAN DER KROL A R, MUR L A, BELD M, et al. Flavonoid genes in petunia: addition of a limited marker of gene copies may lead to a suppression of gene expression [J]. Plant Cell, 1999, 2: 291-299.
[24]贾庆利,龚振辉,李大伟. 转基因植物的筛选 [J]. 陕西农业科学, 2004(5): 53-54.
[25]王关林，方宏筠. 植物基因工程 [M]. 2版. 北京: 科学出版社, 2002: 188,395,666-668.
[26]DEAN J D, GOODWIN P H, HSIANG T. Comparison of relative RT-PCR and northern blot analyses to measure expression of β-1,3-glucanase in Nicotiana benthamiana infected with Colltotrichum destructivum [J]. Plant Mol Biol Rep, 2002, 20: 347-356.
[27]GUO H M, ZHANG Y Q, WAN F H, et al. Agrobacterium-mediated transformation of Eupatorium adenophorum [J]. Plant Cell Tiss Org Cult, 2010, 103:417-422.
[28]JIA Z C, SUN Y M, YUAN L, et al. The chitinase gene (Bbchit1) from Beauveria bassiana enhances resistance to Cytospora chrysosperma in Populus tomentosa Carr [J]. Biotechnol Lett, 2010, 32: 1325-1332.
[29]JAYARAJ J, PUNJA Z K. Combined expression of chitinase and lipid transfer protein genes in transgenic carrot plants enhances resistance to foliar fungal pathogens [J]. Plant Cell Rep, 2007, 26:1539-1546. 
[30]CELIKKOL A U, MAHMOUDIAN M, KAMCI H, et al. Agrobacterium tumefaciens-mediated genetic transformation of a recalcitrant grain legume, lentil (Lens culinaris Medik) [J], Plant Cell Rep, 2009, 28: 407-417.
[31]LEE S N, CHOI S H, RYU K B, et al. Molecular and cytogenetic assessment of transgenic hot peppers resistant to Cucumber Mosaic Virus [J]. Hort Environ Biotechnol, 2011, 52(2): 211-217.
[32]TOHIDFAR M, MOHAMMADI M, GHAREYAZIE B. Agrobacterium-mediated transformation of cotton (Gossypium hirsutum) using a heterologous bean chitinase gene [J]. Plant Cell Tiss Org Cult, 2005, 83(1): 83-96.
[33]TOHIDFAR M, RASSOULI H, NAGHNAZAI A, et a1. Evaluation of stability chitinase gene in transgenic offspring of cotton (Gossypium hirsutum) [J]. Iran J Biotechnol, 2009, 7(1):45-49.
[34]张志忠,吴菁华,吕柳新,等. 植物几丁质酶及其应用研究进展 [J]. 福建农林大学学报: 自然科学版, 2005, 34(4): 494-499.〖ZK）〗〖FL）〗

相似文献/References:

[1]张倩,孙晶,廖静,等.RNA干扰技术抑制洋桔梗ACC合酶基因的表达[J].江苏农业学报,2017,(02):267.[doi:doi:10.3969/j.issn.1000-4440.2017.02.005]
　ZHANG Qian,SUN Jing,LIAO Jing,et al.Inhibition of ACC synthase gene expression in Eustoma grandiflorum using RNA interference technology[J].,2017,(03):267.[doi:doi:10.3969/j.issn.1000-4440.2017.02.005]
[2]付晓佳,张倩,李琳,等.农杆菌介导烟草抗真菌β-１，３-葡聚糖酶基因转化洋桔梗[J].江苏农业学报,2016,(01):44.[doi:10.3969/j.issn.1000-4440.2016.01.007]
　FU Xiao-jia,ZHANG Qian,LI Lin,et al.Agrobacterium-mediated transformation of tobacco β-1,3-glucanase gene into Eustoma grandiflorum (Raf.) Shinn.[J].,2016,(03):44.[doi:10.3969/j.issn.1000-4440.2016.01.007]

备注/Memo

备注/Memo:
收稿日期：2014-12-11 作者简介：李琳（1989-）女，安徽宣城人，硕士研究生，主要从事植物生物技术与分子生物学研究。（E-mail）lilin675910@163.com 通讯作者：陈崇顺，（E-mail）chenchongshun@njnu.edu.cn
更新日期/Last Update: 2015-06-30