Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2011, Vol. 31 Issue (04): 37-43    
研究报告     
香蕉内生克雷伯氏菌KKWB-5强启动子片段的分离及鉴定
夏启玉1, 孙建波1, 顾文亮2, 卢娟2, 卢雪花1, 王宇光1, 张欣3
1. 中国热带农业科学院生物技术研究所农业部热带作物生物技术重点开放实验室 海口 571101;
2. 海南大学农学院 海口 570228;
3. 中国热带农业科学院环境与植物保护研究所 儋州 571737
Isolation and Identification of Strong Promoter from Endophytic Klebsiella pneumoniae KKWB-5 of Banana
XIA Qi-yu1, SUN Jian-bo1, GU Wen-liang2, LU Juan2, LU Xue-hua1, WANG Yu-guang1, ZHANG Xin3
1. Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101,China;
2. College of Agriculture, Hainan University, Hai Kou 570228,China;
3. Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Dan Zhou 571737,China
 全文: PDF(1118 KB)   HTML
摘要：

目的:旨在获得香蕉内生克雷伯氏菌KKWB-5的强启动子片段,以应用于香蕉内生工程菌的构建。方法: 利用以kanr基因为报告基因的启动子探针载体pUCK在大肠杆菌Top10中克隆KKWB-5基因组DNA 的启动子片段;将筛选到的高抗Kan的质粒导入KKWB-5,分别于LB和香蕉杆浸汁培养基(BSM)平板上检测它们的抗Kan水平;选择在BSM上抗Kan水平最高的片段15,检测该片段的基因间隔区15P的启动子活性,最后以gfp为报告基因来验证片段15P的启动子活性。结果:有7个抗Kan 水平在2500μg/ml以上的Top10转化子;这7个质粒在导入KKWB-5后,它们在LB平板上的抗Kan 水平有不同程度的增加,但在BSM培养基上则大为减弱;片段15P具有启动kanr 基因的活性,且与原片段15的抗Kan 水平相同;重组质粒pUCK-6-15Pgfp,以Top10和KKWB-5为宿主菌,在LB培养基上培养时,在荧光显微镜下均能发出绿色荧光;以KKWB-5为宿主菌,在BSM培养基上培养时,在荧光显微镜下也能发出绿色荧光。 结论:片段15不仅在Top10中具有较强的启动子活性,而且在其供体菌KKWB-5中具有更强的启动子活性,其基因间隔区15P为主要的启动子区域,在BSM培养基上也具有较好的启动子活性,该启动子片段15P可以应用于KKWB-5内生工程菌的构建。
关键词： 肺炎克雷伯氏菌;  启动子;  分离;  鉴定;  gfp    
Abstract:

The aim is to obtain strong promoters from Endophytic Klebsiella pneumoniae KKWB-5 of Banana, which will be applied to construct an endophytic engineered bacterium of banana. Promoter probe plasmid pUCK whose report gene is kanamycin-resistance (Kanr) gene was used to screen promoter fragments from KKWB-5 DNA in E.coli Top10. There were 7 Top10 transformants whose resistant level to kanamycin were over 2500μg/ml. This 7 plasmids were transformed into KKWB-5, and KKWB-5 transformants were tested their resistant level to kanamycin on LB plate and banana stem medium (BSM) respectively. After the plasmids were transformed into KKWB-5, the resistant level to kanamycin of KKWB-5 transformants when cultured on LB showed various degree of increases, but when cultured on BSM it weakened to a great degree. Transformant K-pUCK-15 was the highest resistant clone to kanamycin on BSM, and then the intergenic spacer region 15P of inserted fragment 15 were tested its promoter activity by pUCK as Kanr gene for report gene and by pUCK-6 as green fluorescent protein gene for report gene. The results demonstrated that fragment 15P had the same resistant level to kanamycin with fragment 15, moreover, the Top10 and KKWB-5 transformants containing recombinant plasmid pUCK-6-15Pgfp could emit intense green fluorescence under UV excitation when cultured on LB, and KKWB-5 transformants could emit green fluorescence under UV excitation too when cultured on BSM. In conclusion, fragment 15 had strong promoter activity in Top 10, but had stronger promoter activity in KKWB-5. The intergenic spacer region 15P was the main promoter region of Fragment 15, and it had good promoter activity in KKWB-5 when cultured on BSM. Fragment 15P can be applied to construct an endophytic engineered bacterium of banana.
Key words: Klebsiella pneumoniae    Promoter    Isolation    Identification    gfp
收稿日期: 2010-11-15 出版日期: 2011-04-26
ZTFLH:  Q939  
基金资助:

中央级公益性科研院所基本科研业务费专项(ITBBZX2008-2-3)资助项目
通讯作者: 王宇光     E-mail: wygh28@263.net
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　

引用本文:

夏启玉, 孙建波, 顾文亮, 卢娟, 卢雪花, 王宇光, 张欣. 香蕉内生克雷伯氏菌KKWB-5强启动子片段的分离及鉴定[J]. 中国生物工程杂志, 2011, 31(04): 37-43.

XIA Qi-yu, SUN Jian-bo, GU Wen-liang, LU Juan, LU Xue-hua, WANG Yu-guang, ZHANG Xin. Isolation and Identification of Strong Promoter from Endophytic Klebsiella pneumoniae KKWB-5 of Banana. China Biotechnology, 2011, 31(04): 37-43.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I04/37


[1] Promputtha I, Lumyong S, Dhanasekaran V, et al. A phylogenetic evaluation of whether endophytes become saprotrophs at host senescence. Microbial Ecology, 2007, 53 (4): 579-590.

[2] Sturz A V, Christie B R, Nowak J. Bacterial endophytes: potential role in developing sustainable systems of crop production. Critical Reviews in Plant Science, 2000, 19(1): 1-30.

[3] 林玲, 乔勇升, 顾本康, 等. 植物内生细菌及其生物防治植物病害的研究进展. 江苏农业学报, 2008, 24(6): 969-974. Lin L, Qiao Y S, Gu B K, et al. Jiangsu J of Agr Sci, 2008, 24(6): 969-974.

[4] 胡萌. 植物内生细菌研究进展. 山东农业大学学报(自然科学版), 2008, 39(1):148-151. Hu M. Journal of Shandong Agricultural University (Natural Science), 2008, 39(1): 148-151.

[5] Lampel J S, Canter G L, Dimock M B, et al. Integrative cloning, expression, and stability of the cryIAc gene from Bacillus thuringiensis subsp.kurstaki in a recombinant strain of Clavibacter xyli subsp.cynodontis. Appl Environ Microbiol, 1994, 60(2): 501-508.

[6] Turner J, Lampel J S, Stearman R S, et al. Stability of the δ-endotoxin from Bacillus thuringiensis subsp.Kurstali in a recombinant strain of Clavibacter xyli subsp.Cynodontis. Appl Environ Microbiol, 1991, 57(12): 3522-3528.

[7] Downing K J, Thomson J A.Introduction of the Serratia marcescens chiA gene into an entophytic Pseudomonas fluorescens for the biocontrol of phytopathgenic fungi.Can J Microbiol, 2000, 46(4):363-369.

[8] 段灿星, 张青文, 徐静. 杀虫荧光假单胞工程菌对棉铃虫的离体及活体生物测定.中国生物防治, 2002, 18(2): 67-70. Duan C X, Zhang Q W, Xu J. Chinese Journal of Biological Control, 2002, 18(2):67-70.

[9] 张新建, 孙福在, 赵廷昌, 等. 促冻杀虫基因工程菌的构建. 中国农业科学, 2004, 37(2): 227-232. Zhang X J, Sun F Z, Zhao T C, et al. Scientia Agricultura Sinica, 2004, 37(2): 227-232.

[10] 夏启玉, 王宇光, 孙建波,等. 一株拮抗香蕉枯萎病的内生细菌的分离及其几丁质酶基因信号肽的分泌活性分析.中国生物工程杂志, 2010, 30(9): 24-30. Xia Q Y, Wang Y G, Sun J B, et al. China Biotechnology, 2010, 30 (9): 24-30.

[11] Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual. 3rded. New York: Cold Spring Harbor Laboratory Press, 2001.

[12] 潘皎,张义正. 枯草芽孢杆菌基因启动子的分离与鉴定. 微生物学报, 2004, 44(4): 457-460. Pan J, Zhang Y Z. Acta Microbiologica Sinica, 2004, 44(4): 457-460.

[13] 孟江萍, 尹一兵, 袁军,等. 肺炎链球菌体内启动子诱捕文库的构建与初步分析. 生物医学工程学杂志, 2007, 24 (1): 149-152. Meng J P, Yin Y B, Yuan J, et al. J Biomed Eng, 2007, 24 (1): 149-152.

[14] 魏云林, 季秀玲, 林连兵. 低温菌启动子分析及异源蛋白高效表达. 生物工程学报, 2008, 24(3): 415-422. Wei Y L, Ji X L, Lin L B. Chinese Journal of Biotechnology, 2008, 24(3): 415-422.
[1] 卜恺璇,周翠霞,路福平,朱传合. 细菌转录起始调控机制*[J]. 中国生物工程杂志, 2021, 41(11): 89-99.
[2] 朱亚鑫, 段艳婷, 高宇豪, 王籍阅, 张晓梅, 张晓娟, 徐国强, 史劲松, 许正宏. 不同D/L单体比γ-聚谷氨酸的合成与调控[J]. 中国生物工程杂志, 2021, 41(1): 1-11.
[3] 邱金戈,刘德武,孙宝丽,李耀坤,郭勇庆,邓铭,柳广斌. 动物外泌体分离方法的研究进展*[J]. 中国生物工程杂志, 2020, 40(9): 36-42.
[4] 杨娜,吴群,徐岩. 解淀粉芽孢杆菌合成surfactin的发酵策略优化 *[J]. 中国生物工程杂志, 2020, 40(7): 51-58.
[5] 玄美娟,张晓云,高莹,高丽影,吴佳婧,马梅,王艳梅,寇航,路福平,黎明. 大肠杆菌糖酵解途径和三羧酸循环启动子的表征及其应用 *[J]. 中国生物工程杂志, 2020, 40(6): 20-30.
[6] 胡益波,皮畅钰,张哲,向柏宇,夏立秋. 丝状真菌蛋白表达系统研究进展*[J]. 中国生物工程杂志, 2020, 40(5): 94-104.
[7] 潘炳菊,张宛怡,申会涛,刘婷婷,李中媛,罗学刚,宋亚囝. 甘露寡糖分离纯化研究进展*[J]. 中国生物工程杂志, 2020, 40(11): 90-95.
[8] 胡妍,李辉,何承文,朱婧,谢志平. 酵母亚细胞结构分离效率评估菌株的构建 *[J]. 中国生物工程杂志, 2020, 40(10): 10-23.
[9] 封金云,宿玲恰,吴敬. 多酶复配合成海藻糖及其分离提取的研究 *[J]. 中国生物工程杂志, 2019, 39(7): 65-70.
[10] 马占兵,党洁,杨继辉,霍正浩,徐广贤. 基于慢病毒系统的双荧光标记多功能自噬流监测系统建立与应用 *[J]. 中国生物工程杂志, 2019, 39(5): 88-95.
[11] 卢钟腾,呼高伟. 新型细胞穿膜肽的鉴定方法与其在抗肿瘤治疗中的应用[J]. 中国生物工程杂志, 2019, 39(12): 50-55.
[12] 黄宇,黄书婷,张夕梅,刘堰. 稀有鮈鲫HSP70基因启动子的克隆及功能分析[J]. 中国生物工程杂志, 2019, 39(10): 9-16.
[13] 安明晖,田文,韩晓旭,尚红. 表达HIV单链抗体的重组乳酸杆菌的构建及表型分析 *[J]. 中国生物工程杂志, 2019, 39(10): 1-8.
[14] 史超硕,李登科,曹雪,袁航,张钰文,于江悦,路福平,李玉. 两个不同启动子及其组合对碱性蛋白酶AprE异源表达的影响 *[J]. 中国生物工程杂志, 2019, 39(10): 17-23.
[15] 李亚芳,赵颖慧,刘赛宝,王伟,曾为俊,王金泉,陈洪岩,孟庆文. 鸡OV启动子表达HA对禽流感病毒攻击提供完全保护 *[J]. 中国生物工程杂志, 2018, 38(7): 67-74.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会