ABSTRACT

Agrobacterium tumefaciens mediated transformation is a major tool in plant biotechnology. Catharanthus roseus suspension cultures were inoculated with A. tumefaciens strain carrying β-glucuronidase (gus) gene in presence of either Co²⁺ or cobalt nanoparticles (CoNPs) at 2.5, 5, 7.5, and 10 mg/L. Co²⁺ at 2.5 mg/L as well as CoNPs at 2.5, 5, and 7.5 mg/L enhanced expression of gus gene in plant cells. The enhancing effects can be attributed to the observed increase in virulence B1 (virB1) gene expression and potential subsequent improvement in transformation efficiency through the inhibitory effect of cobalt on ethylene biosynthesis in plant cells. Ethylene is a major hindrance against transformation success through suppression of vir gene expression. The Agrobacterium friend role of cobalt was lost upon a further increase in cobalt concentration due to abusing defense genes working against both heavy metal-induced oxidative stress and Agrobacterium-associated biotic stress. Results reflected the better performance of CoNPs, compared with Co²⁺ in the enhancement of expression of gus gene that can be attributed to lower sensitivity of plant defense system, represented with an expression of genes encoding phenylalanine ammonia-lyase (PAL), peroxidase (POD) and one of the class 1 pathogenesis-related proteins (CrPR1a), to cobalt nano form than ionic form. Such enhancements may suggest a potential role for CoNPs in enhancing the production of therapeutically important compounds employing transient expression of foreign genes in addition to improving Agrobacterium-mediated transformation that needs further investigation.

Abbreviations: The 2,4-D (2,4- dichlorophenoxyacetic acid), 5-bromo-4-choloro-3-indolyl-βglucuronic acid (X-gluc), aminoethoxyvinylglycine (AVG), cobalt nanoparticles (CoNPs), deionized water (DW), glucuronidase (GUS), glucuronidase gene (gus), hygromycin phosphotransferase gene (hpt), indole acetic acid (IAA), malondialdehyde (MDA), Murashige, and Skoog (MS), negative control (NC), pathogenesis-related (PR), phenylalanine ammonia-lyase (PAL), phenylalanine ammonia-lyase (PAL), peroxidase (POD), peroxidase gene (POD), polyvinylpyrrolidone (PVP), positive control (PC), standard deviation (SD), transmission electron microscopy (TEM), virulence genes (vir), YEP (yeast extract peptone)

摘要

根癌农杆菌介导的转化是植物生物技术中的主要工具。在具有2、5、7.5和10 mg / L的Co ²⁺或钴纳米颗粒（CoNPs）的情况下，用携带β-葡萄糖醛酸糖苷酶（gus）基因的根癌农杆菌接种长春花悬浮培养物。2.5 mg / L的Co ²⁺以及2.5、5和7.5 mg / L的CoNP增强了植物细胞中gus基因的表达。增强作用可归因于观察到的毒力B1（virB1）抑制钴对植物细胞中乙烯生物合成的影响，从而实现基因表达和转化效率的潜在提高。乙烯是通过抑制vir基因表达而阻碍转化成功的主要障碍。由于滥用针对重金属诱导的氧化应激和与农杆菌相关的生物胁迫的防御基因，钴的浓度进一步增加时，钴的农杆菌朋友角色就消失了。结果表明，与Co ²⁺相比，CoNPs在gus表达增强方面表现更好。可以归因于植物防御系统敏感性较低的基因，其表达形式为编码苯丙氨酸解氨酶（PAL），过氧化物酶（POD）和1类致病相关蛋白之一（CrPR1a）的基因对钴纳米形式的表达比离子形式。此类增强可能表明CoNP在利用外源基因的瞬时表达来增强治疗上重要的化合物的生产中可能具有潜在作用，此外还需要改进农杆菌介导的转化，这需要进一步研究。

缩写：2,4-D（2,4-二氯苯氧基乙酸），5-溴-4-胆甾-3-吲哚基-β-葡萄糖醛酸（X-gluc），氨基乙氧基乙烯基甘氨酸（AVG），钴纳米颗粒（CoNPs），去离子水（DW），葡萄糖醛酸酶（GUS），葡萄糖醛酸酶基因（gus），潮霉素磷酸转移酶基因（hpt），吲哚乙酸（IAA），丙二醛（MDA），Murashige和Skoog（MS），阴性对照（NC），发病机理相关（PR），苯丙氨酸解氨酶（PAL），苯丙氨酸解氨酶（PAL），过氧化物酶（POD），过氧化物酶基因（POD），聚乙烯吡咯烷酮（PVP），阳性对照（PC），标准差（SD），传输电子显微镜（TEM），毒力基因（vir），YEP（酵母提取蛋白p）