抗氧化蛋白SsPrxQ在向日葵中的表达研究
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摘要
逆境中盐碱胁迫是影响农作物生长、发育和产量的主要因素之一,近几年,随着分子生物学的迅速发展,转基因技术已逐渐成为耐盐碱领域研究的焦点。本研究通过农杆菌介导法将抗氧化蛋白基因(SsPrxQ)转入油葵中,以获得高耐盐碱的抗性植株。其中,目的基因SsPrxQ来源于耐盐碱植物碱蓬,并将其连入高效植物表达载体pBI121中,构建了植物表达载体pBI121-SsPrxQ,通过电击法将重组载体质粒导入农杆菌C58中,并进行油葵的侵染转化。转化受体是油料作物油葵,向日葵在再生体系与转基因方面已有相关报道,本研究在原有体系的基础上,进一步优化了向日葵的胚轴分化体系以及农杆菌遗传转化条件,获得胚轴分化培养基为MS+6-BA0.5mg/L+GA30.1mg/L,胚轴分化率达55%;生根培养基为MS+NAA0.6mg/L +6g/L agr,生根率达50%;最优的遗传转化条件为AS浓度100μmol/L,菌液浓度为OD600值0.6,预培养16小时,浸染10分钟,共培养3天,共培养温度24℃,Cef浓度250mg/L,Kan浓度40mg/L。通过Kan的逐步筛选最终得到5株抗性苗,用CTAB法提取转基因向日葵基因组DNA做PCR检测,检测出3株是阳性的,初步表明SsPrxQ基因已成功导入向日葵细胞中。同时提取转基因向日葵的总RNA做RT-PCR检测,结果显示3株均扩增出特异性目的片段,证明SsPrxQ基因已成功整合到转基因向日葵的基因组中,并实现转录。
In adverse circumstances, alkalic salt coerces is one of the major stresses effecting the crop plants growth, development and yield, the rapid advance in molecular biology promotes gene technology to become the focus in the field of salt-tolerant plants. In this study, to develop a salt-tolerant oil sunflower, a antioxidant protein gene SsPrxQ from Suaeda salsa. is transferred into oil sunflower, using an Agrobacterium-mediated method. Firstly,the functional gene is cloned and inserted into the plant vector pBI121, and a high efficient expression vector pBI121- SsPrxQ is obtained, and then transferred into Agrobacterium tumefaciens C58, and then oil sunflower was transformed by Agrobacterium tumefaciens infection. The acceptor system is oil crops sunflower. Its regeneration system and related gene transformation have appeared in some literature. This research optimizes the hypocotyl elongation and Agrobacterium transformation system on the base of previous results. The optimal hypocotyl elongation medium is MS+6-BA0.5mg/L+GA30.1mg/L, with the hypocotyl elongation rate of callus 55%; root regeneration medium is MS+NAA0.6mg/L +6g/L agr, with regenerating rate of root 50%. The best Agrobacterium transformation condition is AS concentration: 100μmol/L, A.tumefaciens concentration: OD600=0.6, pre-culture time 16 hours, inoculated time: 10min, co-culture time: 3 days, co-culture temperature: 24℃.concentration of Cef: 250mg/L,concentration of Kan: 40mg/L. 5 strains are gotten through the choice of Kan. We extracted genomic DNA by CTAB method from the leaves of transgenic sunflower plant for PCR analysis, result showed three strains were positive and it preliminarily showed that the SsPrxQ gene had been introduced into sunflower cells. And then we extracted genomic RNA from the leaves of transgenic sunflower plant for RT-PCR analysis and acquired a DNA fragment of correct size. It was confirmed that the SsPrxQ gene had integrated into the genome of the transgenic sunflower plants and realized transcription.
In adverse circumstances, alkalic salt coerces is one of the major stresses effecting the crop plants growth, development and yield, the rapid advance in molecular biology promotes gene technology to become the focus in the field of salt-tolerant plants. In this study, to develop a salt-tolerant oil sunflower, a antioxidant protein gene SsPrxQ from Suaeda salsa. is transferred into oil sunflower, using an Agrobacterium-mediated method. Firstly,the functional gene is cloned and inserted into the plant vector pBI121, and a high efficient expression vector pBI121- SsPrxQ is obtained, and then transferred into Agrobacterium tumefaciens C58, and then oil sunflower was transformed by Agrobacterium tumefaciens infection. The acceptor system is oil crops sunflower. Its regeneration system and related gene transformation have appeared in some literature. This research optimizes the hypocotyl elongation and Agrobacterium transformation system on the base of previous results. The optimal hypocotyl elongation medium is MS+6-BA0.5mg/L+GA30.1mg/L, with the hypocotyl elongation rate of callus 55%; root regeneration medium is MS+NAA0.6mg/L +6g/L agr, with regenerating rate of root 50%. The best Agrobacterium transformation condition is AS concentration: 100μmol/L, A.tumefaciens concentration: OD600=0.6, pre-culture time 16 hours, inoculated time: 10min, co-culture time: 3 days, co-culture temperature: 24℃.concentration of Cef: 250mg/L,concentration of Kan: 40mg/L. 5 strains are gotten through the choice of Kan. We extracted genomic DNA by CTAB method from the leaves of transgenic sunflower plant for PCR analysis, result showed three strains were positive and it preliminarily showed that the SsPrxQ gene had been introduced into sunflower cells. And then we extracted genomic RNA from the leaves of transgenic sunflower plant for RT-PCR analysis and acquired a DNA fragment of correct size. It was confirmed that the SsPrxQ gene had integrated into the genome of the transgenic sunflower plants and realized transcription.
引文
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