Construction of a cDNA library of Ipomoea pes-caprae L.and screening for cadmium tolerant genes
-
摘要: 厚藤（Ipomoea pes-caprae（L.） Sweet）是一种具有重要生态、观赏及药用价值的沙滩植物，对重金属镉（Cd）具有一定的富集能力，可作为Cd污染滨海地区的修复植物进行引种栽植和利用。本研究通过Gateway技术构建厚藤的cDNA文库，将该文库质粒转化酵母对Cd敏感的突变株ycf1△，采用全长cDNA过表达基因捕获系统（FOX）筛选厚藤重金属Cd胁迫耐受相关基因，并采用酵母互补实验进行基因的功能验证。本研究获得了2个能够恢复ycf1△对Cd敏感表型的重组质粒，经测序分析，该重组质粒包含的cDNA全长序列分别对应厚藤植物螯合肽合成酶基因（phytochelatin synthase）和金属硫蛋白基因（metallothionein），分别将其命名为IpPCS和IpMT，通过功能分析，初步认定该基因为编码Cd耐受和解毒相关蛋白的候选基因。Abstract: Ipomoea pes-caprae(L.) Sweet,as a coastal beach halophyte with great ecological,ornamental,and medicinal value,has the capacity to enrich heavy metal cadmium and can be used in phytoremediation.We constructed a cDNA library for expression in yeast with tissues of Ipomoea pes-caprae by gateway technology,and then introduced the library plasmids into cadmium-sensitive yeast mutant strain,ycf1△,and screened the library through the FOX gene hunting system based on functional screening in yeast.We obtained two full-length cDNAs of Ipomoea pes-caprae,which could enhance the Cd tolerance of ycf1△.Sequence analyses indicated that these two cDNAs encoded phytochelatin synthase (IpPCS) and metallothionein (IpMT).This research lays a good foundation for further clarifying the molecular mechanism of plants responding to cadmium,and provides a reference cDNA library for functional screening to clone other stress-related genes in Ipomoea pes-caprae.
-
Keywords:
- cDNA library /
- Cadmium /
- Ipomoea pes-caprae(L.) Sweet
-
-
[1] 申旭红, 肖飞鹏, 肖进中. 中国三大海域沉积物中重金属的分布特征及其生态危害[J]. 安徽农业科学, 2012, 40(24):12199-12201. Shen XH, Xiao FP, Xiao JZ. Distribution of heavy metals in the sediments of the polluted coastal regions in China and its ecological danger[J]. Journal of Anhui Agricultural Sciences, 2012, 40(24):12199-12201.
[2] 梁开, 黄向清, 郑志昌. 我国南方沿海海域有害重金属污染之现状调查[C]//2007年全国铅污染监测与控制治理技术交流研讨会论文集. 北京:中国环境科学学会, 2010:329-336. [3] 马玉, 李团结, 王迪, 彭艳超, 蔡钰灿, 王爱军. 珠江口滨海湿地沉积物重金属污染现状及潜在生态危害[J]. 热带地理, 2011, 31(4):353-356. Ma Y, Li TJ, Wang D, Peng YC, Cai YC, Wang AJ. Status and potential ecological hazards of heavy metal pollution in sediments of coastal wetlands in the Pearl River Estuary[J]. Tropical Geography, 2011, 31(4):353-356.
[4] 欧阳蒲月, 刘楠, 张伟伟, 王俊, 简曙光. 海滩植物厚藤(Ipomoea pescaprae)的生物学及生理生态特性[J]. 湖南科技大学学报:自然科学版, 2011, 26(4):117-121. Ou Yang PY, Liu N, Zhang WW, Wang J, Jian SG. Biological and eco-physiological characteristics of a beach plant Ipomoea pescaprae[J]. Journal of Hunan University of Science and Technology:Natural Science Edition, 2011, 26(4):117-121.
[5] 杜月青, 刘建强, 陈立潮, 金水虎. 鳝藤等4种藤本植物耐盐性试验[J]. 浙江林业科技, 2011, 31(4):51-57. Du YQ, Liu JQ, Chen LC, Jin SH. Salt stress on four species of climbing plant[J]. Journal of Zhejiang for Science and Technology, 2011, 31(4):51-57.
[6] Manigauha A, Kharya MD, Ganesh N. In vivo antitumor potential of Ipomoea pes-caprae on melanoma cancer[J]. Pharmacogn Mag, 2015, 11(42):426-433.
[7] 陆彬斌, 孟柱, 赵志忠, 袁建平. 海南昌化铅锌矿区土壤重金属含量及优势植物富集特征[J]. 河南科技,2013, 1:184-189. Lu BB, Meng Z, Zhao ZZ, Yuan JP. Heavy metal content and dominant plant enrichment characteristics of soils in Changhua lead-zinc mine area, Hainan province[J]. Journal of Henan for Science and Technology, 2013, 1:184-189.
[8] Wang HL, Tian CY, Jiang L, Wang L. Remediation of heavy metals contaminated saline soils:a halophyte choice[J]. Environ Sci Technol, 2013, 48(1):21-22.
[9] 杨亚洲, 张春华, 郑青松, 张春银, 葛滢. 碱蓬和滨藜对镉和钠吸收、转运及亚细胞分布特性的比较研究[J]. 农业环境科学学报, 2015, 34(4):619-626. Yang YZ, Zhang CH, Zheng QS, Zhang CY, Ge Y. Comparative study on cadmium and sodium absorption, transport and subcellular distribution in Suaeda salsa and Atriplex triangularis[J]. Journal of Agro-Environment Science, 2015, 34(4):619-626.
[10] Li ZS, Szczypka M, Lu YP, Thiele DJ, Rea PA. The yeast cadmium factor protein(YCF1) is a vacuolar glutathione S-conjugate pump[J]. J Biol Chem, 1996, 271(11):6509-6517.
[11] Ichikawa T, Nakazawa M, Kawashima M, Iizumi H, Kuroda H, et al. The FOX hunting system:an alternative gain-of-function gene hunting technique[J]. Plant J, 2006, 48(6):974-985.
[12] 张伟, 郭钦, 阮辉, 何国庆. β-1,3-1,[BFY]4-葡聚糖酶在工业酿酒酵母表面的诱导展示表达研究[J]. 中国食品学报, 2010, 10(5):131-136. Zhang W, Guo Q, Ruan H, He GQ. Studies on inducible surface-display expression of β-1,3-1,[BFY]4-Glucanase on industrial Sacchraomyces Cerevisiae surface[J]. Journal of Chinese Institute of Food Science and Technology, 2010, 10(5):131-136.
[13] 张灿, 陈虹, 余忆玄, 王立军, 韩建波, 陶平. 我国沿海地区城镇污水处理厂污泥重金属污染状况及其处置分析[J]. 环境科学, 2013, 34(4):1345-1350. Zhang C, Chen H, Yu YX, Wang LJ, Han JB, Tao P. Pollution characteristics of heavy metals in sludge from wastewater treatment plants and sludge disposal in Chinese coastal areas[J]. Environmental Science, 2013, 34(4):1345-1350.
[14] 丁自立, 李书谦, 周旭, 曹凑贵. 植物修复土壤重金属污染机制与应用研究[J]. 湖北农业科学, 2014, 53(23):5617-5623. Ding ZL, Li SQ, Zhou X, Cao CG. Mechanisms and applications of phytoremediation of heavy metal contamination in soils[J]. Hubei Agricultural Sciences, 2014, 53(23):5617-5623.
[15] 倪晓鹏, 高志红. 园艺作物基因组测序研究进展[J]. 江苏农业科学, 2016, 44(2):9-13. Ni XP, Gao ZH. Advances in genome sequencing of horticultural crops[J]. Jiangsu Agricultural Sciences, 2016, 44(2):9-13.
[16] 吴倩, 张磊, 黄志平, 王大刚, 胡国玉. 转录组测序及其在野生大豆基因资源发掘中的应用[J]. 大豆科学, 2013, 32(6):845-851. Wu Q, Zhang L, Huang ZP, Wang DG, Hu GY, Transcription sequencing and its application on discovering the gene resources of wild soybean[J]. Soybean Science, 2013, 32(6):845-851.
[17] 贾昌路,张瑶,朱玲,张锐. 转录组测序技术在生物测序中的应用研究进展[J]. 分子植物育种, 2015, 13(10):2388-2394. Jia CL, Zhang Y, Zhu L, Zhang R. Application of transcriptional sequencing technology in biological sequencing[J]. Molecular Plant Breeding, 2015, 13(10):2388-2394.
[18] Higuchi M, Kondou Y, Ichikawa T, Matsui M. Full-length cDNA overexpressor gene hunting system (FOX hunting system)[J]. Methods Mol Biol, 2011, 678:77-89.
[19] Asano T, Hakata M, Nakamura H, Aoki N, Komatsu S, et al. Functional characterization of OsCPK21, a calcium-dependent protein kinase that confers salt tolerance in rice[J]. Plant Mol Biol, 2011, 75(1-2):179-191.
[20] Higuchi-Takeuchi M, Matsui M. Screening for gene function using the FOX (Full-length cDNA over expressor gene) hunting system[J]. Methods Mol Biol, 2014, 1056:201-210.
[21] Song WY, Martinoia E, Lee J, Kim D, Kim DY, et al. A novel family of cys-rich membrane proteins mediates cadmium resistance in Arabidopsis[J]. Plant Physiol, 2004, 135(2):1027-1039.
[22] Shim D, Hwang JU, Lee J, Lee S, Choi Y, et al. Orthologs of the class A4 heat shock transcription factor HsfA4a confer cadmium tolerance in wheat and rice[J]. Plant Cell, 2009, 21(12):4031-4043.
[23] Blanvillain R, Kim JH, Wu S, Lima A, Ow DW. OXIDATIVE STRESS 3 is a chromatin-associated factor involved in tolerance to heavy metals and oxidative stress[J]. Plant J, 2009, 57(4):654-665.
[24] Kunihiro S, Saito T, Matsuda T, Inoue M, Kuramata M, et al. Rice DEP1, encoding a highly cysteine-rich G protein γ subunit, confers cadmium tolerance on yeast cells and plants[J]. J Exp Bot, 2013, 64(14):4517-4527.
[25] Kuramata M, Masuya S, Takahashi Y, Kitagawa E, Inoue C, et al. Novel cysteine-rich peptides from Digitaria ciliaris and Oryza sativa enhance tolerance to cadmium by limiting its cellular accumulation[J]. Plant Cell Physiol, 2009, 50(1):106-117.
[26] Shitan N, Horiuchi K, Sato F, Yazaki K. Bowman-Birk proteinase inhibitor confers heavy metal and multiple drug tolerance in yeast[J]. Plant Cell Physiol, 2007, 48(1):193-197.
[27] 张妍茹, 佟少明, 侯和胜. 植物螯合肽合成酶的催化机制研究进展[J]. 天津农业科学, 2016, 22(2):27-30. Zhang YR, Dong SM, Hou HS. Research progress of catalytic mechanism of phytochelatin synthase[J]. Tianjin Agricultural Science, 2016, 22(2):27-30.
[28] Rea PA. Phytochelatin synthase:of a protease a peptide polymerase made[J]. Physiol Plant, 2012, 145(1):154-164.
[29] Zanella L, Fattorini L, Brunetti P, Roccotiello E, Cornara L, et al. Over expression of AtPCS1 in tobacco increases arsenic and arsenic plus cadmium accumulation and detoxification[J]. Planta, 2016, 243(3):605-622.
[30] 孟红恩, 刘忠渊. 植物金属硫蛋白研究进展[J]. 广东农业科学, 2014, 15:133-138. Meng HE, Liu ZY. Research advances of plant metallothioneins[J]. Guangdong Agricultural Sciences, 2014, 15:133-138.
[31] Leszczyszyn OI, Imam HT, Blindauer CA. Diversity and distribution of plant metallothioneins:a review of structure,properties and functions[J]. Metallomics, 2013, 5(9):1146-1169.
[32] Hassinen VH, Tervahauta AI, Schat H, Kärenlampi SO. Plant metallothioneins-metal chelators with ROS scavenging activity?[J]. Plant Biol (Stuttg), 2011, 13(2):225-232.
[33] 张伟, 郭钦, 阮辉, 何国庆. β-1,3-1,[BFY]4-葡聚糖酶在工业酿酒酵母表面的诱导展示表达研究[J]. 中国食品学报, 2010, 10(5):131-136. Zhang W, Guo Q, Ruan H, He GQ. Studies on inducible surface-display expression of β-1,3-1,[BFY]4-Glucanase on industrial Sacchraomyces cerevisiae surface[J]. Journal of Chinese Institute of Food Science and Technology, 2010, 10(5):131-136.
计量
- 文章访问数: 866
- HTML全文浏览量: 3
- PDF下载量: 1402
下载:
