酵母耐铜基因CUP1改良荧光假单胞菌耐铜性研究初报

摘要/Abstract

摘要： 利用根际促生菌（PGPR）荧光假单胞菌（Pseudomonas fluorescens）等微生物的生物修复作用可治理土壤重金属污染。酿酒酵母耐铜基因CUP1编码铜金属硫蛋白（Cu-MT），对铜有很高的亲和性，可拮抗重金属铜毒性。为提高荧光假单胞菌的生物修复能力，将酿酒酵母耐铜基因CUP1克隆至荧光假单胞菌。通过引物设计，将SD序列添加至酵母耐铜基因CUP1编码序列前端，PCR扩增得到约200bp的CUP1片段，克隆至测序载体pUC19，经测序证明正确后，胶回收CUP1片段连接到具有广泛宿主范围的柯斯质粒pLAFR3上，经三亲接合转移至荧光假单胞菌AS1.1381，得到重组菌株AS1.1381/pL3CUP1。铜实验表明，重组菌株AS1.1381/pL3CUP1的抗铜能力（6mM Cu2+）显著高于AS1.1381/pLAFR3（4.5mM Cu2+），重组菌株抗铜性得到提高

关键词: 细胞内含物, 细胞内含物, 韧皮部, 原生质, 再分配, 物质运输

Abstract: Bioremediation with the microbe such as Pseudomonas fluorescens, a plant growth-promoting-rhizobacteria (PGPR), could be used to control heavy metal polluted soils. The copper-resistant gene CUP1 of bake yeast encodes copper metallothionein (Cu-MT) which possesses special affinity to copper ions. In this work, in order to improve the ability for bioremediation, the CUP1 gene was cloned into P. fluorescens. The SD sequence mediated by primer was added ahead of the CUP1 gene. The fragments of about 200 bp were amplified and cloned into the sequencing vector pUC19. After confirmed by sequencing, the fragments harboring CUP1 gene was gel-extracted and cloned into the wide-range-host cosmid pLAFR3, and then transformed into P. fluorescens AS1.1381 by tri-parents mating, generated the recombined strain AS1.1381/pL3CUP1. Copper resistance assay showed that the recombined strain AS1.1381/pL3CUP1 had higher copper resistance (6mM Cu2+) than the wide strain ASl.1381/pLAFR3 (4.5mM Cu2+).

中图分类号:

Q812
S476

姜伯乐,,吴圣进,,何勇强,. 酵母耐铜基因CUP1改良荧光假单胞菌耐铜性研究初报[J]. 中国农学通报, 2007, 23(9): 6-6.

Jiang Bole,, Wu Shengjin,, He Yongqiang,. Preliminary Report on Copper Resistance of Pseudomonas fluorescens Modified with Yeast CUP1 Gene[J]. Chinese Agricultural Science Bulletin, 2007, 23(9): 6-6.

参考文献

[1] 步雪琳.土壤污染防治刻不容缓.中国环境报,2004,4:22.
[2] 周东美,王慎强,陈怀满.土壤中有机污染物-重金属复合污染交互的作用.土壤与环境,2000,9(2):143-145.
[3] 赵学茂.土壤重金属污染的防治方法.甘肃农业,2006,235(2):228.
[4] Schippers B, Bakker A W, Bakker P A H M. Interactions of deleterious and beneficial rhizosphere microorganisms. Annu Rev Phytopathol,1987,25(1):339-358.
[5] Joshi-Tope G, Francis A J. Mechanisms of biodegradation of metal-citrate complexes by Pseudomonas fluorescens. J Bacteriol,1995,177(8):1989-1993.
[6] 朱荫湄,周启星.土壤污染与我国农业环境保护的现状、理论和展望.土壤通报,1999,30(3):132-135.
[7] Karin M, Najarian R, Haslinger A, et al. Primary structure and transcription of an amplified genetic locus: The CUP1 locus of yeast. Proc Natl Acad Sci USA,1984,81(2):337-341.
[8] 何勇强,陶勤南,唐纪良,等.酿酒酵母耐铜基因CUP1的克隆及其植物表达载体的构建.广西农业生物科学,2000,19(4):233-238.
[9] Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene,1985,33(1):103-119.
[10] Staskawicz B, Dahlbeck D, Keen N, et al. Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol,1987,169(12):5789-5794.
[11] Leong S A, Ditta G S, Helinski D R. Heme biosynthesis in Rhizobium. Identification of a cloned gene coding for delta-aminolevulinic acid synthetase from Rhizobium meliloti. J Biol Chem,1982,257(15):8724-8730.
[12] Turner P, Barber C, Danniels M. Behaviour of the transposons Tn5 and Tn7 in Xanthomonas campestris pv. campestris. Mol Gen Genet,1984,195:101-107.
[13] 郜宗智,郑梅.土壤污染现状及监测体系浅析.现代农业科技,2007,1:84-85.

[1]	杨颖, 康兰, 耿新, 陈玉珍, 卢存福. 植物原生质体再生细胞壁研究进展[J]. 中国农学通报, 2021, 37(4): 25-30.
[2]	王珺华,王凯琪,王凯,孙志宏,曹园,齐向英. 山丹丹原生质体的分离条件探究[J]. 中国农学通报, 2019, 35(34): 64-71.
[3]	牛瑜菲,彭建营. 酸枣花粉原生质体的分离条件研究[J]. 中国农学通报, 2019, 35(32): 53-56.
[4]	孙宇涵,王欢,李云. 浅谈林木体细胞融合技术[J]. 中国农学通报, 2016, 32(4): 136-143.
[5]	郝艳芳,王良群,刘勇,张微,杨伟,白鸿雁,武擘. 禾谷类作物原生质体培养研究进展[J]. 中国农学通报, 2016, 32(35): 19-23.
[6]	曹雪,戴忠良,秦文斌,潘跃平. 植物原生质体融合技术的研究进展[J]. 中国农学通报, 2016, 32(25): 84-90.
[7]	彭小群,唐然,解新明. 禾本科植物原生质体分离研究进展[J]. 中国农学通报, 2015, 31(1): 252-257.
[8]	欧阳泽怡周小玲罗佳田育新闫文德欧阳硕龙易浩宇. 杜仲人工林降水再分配特征研究[J]. 中国农学通报, 2014, 30(16): 41-47.
[9]	张振鲁杜茜周幸董宝池李启云王金刚. 不吸水链霉菌公主岭变种 769原生质体制备条件初探[J]. 中国农学通报, 2013, 29(9): 155-158.
[10]	张鹏1,2,3,龚玲凤3,4,庄卫东1,郑金水1,朱坚2,3. 杏鲍菇原生质体制备条件初探[J]. 中国农学通报, 2013, 29(31): 91-95.
[11]	常冬冬余志晟张洪勋. 产乙醇工程菌构建方法的研究进展[J]. 中国农学通报, 2013, 29(2): 120-124.
[12]	代海燕叶冬梅苏东玉那顺. 林业区划在森林火险天气等级预报中的应用[J]. 中国农学通报, 2013, 29(16): 41-44.
[13]	彭邵锋陆佳陈永忠王瑞陈隆升马力王湘南. 木本植物原生质体培养体系研究进展[J]. 中国农学通报, 2013, 29(1): 1-6.
[14]	蔡肖康向阳. 小青杨叶肉原生质体分离条件的研究[J]. 中国农学通报, 2011, 27(10): 18-22.
[15]	杨茹,刘世琦,张自坤,刘中良,张立伟,马琳. 大蒜原生质体游离和纯化的研究[J]. 中国农学通报, 2010, 26(4): 195-199.