茎瘤固氮根瘤菌ORS 571与小麦互作机理的初步研究
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摘要
茎瘤固氮根瘤菌是一种内生固氮菌,它可以促进禾本科作物的生长。为了探讨茎瘤固氮根瘤菌与小麦互作的机理,本实验构建原核表达载体,为弄清根瘤菌的内生定殖规律奠定了基础;选取小麦不同生长时期的样品,对代表固氮水平和矿质元素吸收的硝酸还原酶,代表抗逆性的丙二醛含量和过氧化物酶活性进行了测定;利用实时定量PCR技术,对固氮菌与小麦互作初期防卫反应相关基因的表达情况进行了检测。结果表明:
(1)将质粒pEGFP-C1中EGFP基因克隆至原核表达载体pGEX-4T-1上,成功构建重组质粒pGEX-4T-EGFP。下一步计划将重组质粒转入茎瘤固氮根瘤菌中,通过激光共聚焦显微镜来观测被浸染的小麦组织,初步判断在小麦体内的定殖与分布;
(2)将茎瘤固氮根瘤菌制成的包衣包裹小麦种子,大田种植。硝酸还原酶活性在拔节期处理显著性高于对照,期间对小麦固氮相关基因的表达和矿质元素的吸收有一定的促进作用。丙二醛含量在灌浆期处理显著性高于对照,在小麦快成熟过程中对抗逆性有一定的提高。多酚氧化酶和过氧化物酶活性处理和对照各时期之间差异不明显;
(3)虽然茎瘤固氮根瘤菌是一种益生菌,但与小麦互作初期仍然会引发小麦的防卫反应。选取侵染初期1h,3h,6h,9h,12h,24h,48h的小麦根,用实时定量PCR检测pox,sams,sod等防卫基因的表达情况。pox分别在1h和9h显著性高于对照,而sams则在第24h才达到最大显著性差异。sod与pox和sams相比,处理反而在第1h小于对照,在第24h和48h达到最大显著性差异。
Azorhizobium caulinodans ORS 571 is an endogenous azotobacter, which can promote growth of cereal crops. To find out colonization rule between wheat interaction with Azorhizobium caulinodans, a recombinant plasmid was constructed. We mensurate enzymatic activity of nitratereductase of wheat which represent nitrogen-fixing related gene expression and mineral element assimilation. Make use of real-time PCR to test defense related gene expression in preliminary stage of wheat interaction with Azorhizobium caulinodans. The result showed:
(1) The EGFP gene was amplified by using PCR amplification methods and cloned to the prokaryotic expression vector pGEX-4T-1, a recombinant plasmid pGEX-4T-EGFP was successfully constructed. The next project is put the recombinant plasmid transformed into Azorhizobium caulinodans ORS 571 and infect wheat, then observe wheat organization use laser confocal miropsopy, preliminary assessment its colonization and distribution in wheat organization.
(2) Make the Azorhizobium caulinodans ORS 571 as coating to package wheat seed and planting in field. Nitratereductase activity is significantly high compared with CK in shooting period. Nitratereductase may improve wheat nitrogen-fixing gene expression and assimilate mineral element. MDA content is significantly high compared with CK in watery stage and it can enhance stress resistance in this period. Polyphenoloxidase and peroxidase activity have not obvious difference among groups.
(3)Although Azorhizobium caulinodans ORS 571 is a beneficial bacteria to wheat plant, it also can induce defense response. We choose wheat root simple of different time that 1h,3h,6h,9h,12h,24h and 48h, use real-time PCR amplification to check transcriptional level expression of defense-related gene, include pox,sams,and sod. The result show that pox transcriptional level expression is significantly high compared with CK at 1h and 9h, but the sams reach this difference at 24h. Compareing with pox and sams, sod transcriptional level expression below the CK at 1h and this tendency have significantly difference at 24h and 48h.
(1)将质粒pEGFP-C1中EGFP基因克隆至原核表达载体pGEX-4T-1上,成功构建重组质粒pGEX-4T-EGFP。下一步计划将重组质粒转入茎瘤固氮根瘤菌中,通过激光共聚焦显微镜来观测被浸染的小麦组织,初步判断在小麦体内的定殖与分布;
(2)将茎瘤固氮根瘤菌制成的包衣包裹小麦种子,大田种植。硝酸还原酶活性在拔节期处理显著性高于对照,期间对小麦固氮相关基因的表达和矿质元素的吸收有一定的促进作用。丙二醛含量在灌浆期处理显著性高于对照,在小麦快成熟过程中对抗逆性有一定的提高。多酚氧化酶和过氧化物酶活性处理和对照各时期之间差异不明显;
(3)虽然茎瘤固氮根瘤菌是一种益生菌,但与小麦互作初期仍然会引发小麦的防卫反应。选取侵染初期1h,3h,6h,9h,12h,24h,48h的小麦根,用实时定量PCR检测pox,sams,sod等防卫基因的表达情况。pox分别在1h和9h显著性高于对照,而sams则在第24h才达到最大显著性差异。sod与pox和sams相比,处理反而在第1h小于对照,在第24h和48h达到最大显著性差异。
Azorhizobium caulinodans ORS 571 is an endogenous azotobacter, which can promote growth of cereal crops. To find out colonization rule between wheat interaction with Azorhizobium caulinodans, a recombinant plasmid was constructed. We mensurate enzymatic activity of nitratereductase of wheat which represent nitrogen-fixing related gene expression and mineral element assimilation. Make use of real-time PCR to test defense related gene expression in preliminary stage of wheat interaction with Azorhizobium caulinodans. The result showed:
(1) The EGFP gene was amplified by using PCR amplification methods and cloned to the prokaryotic expression vector pGEX-4T-1, a recombinant plasmid pGEX-4T-EGFP was successfully constructed. The next project is put the recombinant plasmid transformed into Azorhizobium caulinodans ORS 571 and infect wheat, then observe wheat organization use laser confocal miropsopy, preliminary assessment its colonization and distribution in wheat organization.
(2) Make the Azorhizobium caulinodans ORS 571 as coating to package wheat seed and planting in field. Nitratereductase activity is significantly high compared with CK in shooting period. Nitratereductase may improve wheat nitrogen-fixing gene expression and assimilate mineral element. MDA content is significantly high compared with CK in watery stage and it can enhance stress resistance in this period. Polyphenoloxidase and peroxidase activity have not obvious difference among groups.
(3)Although Azorhizobium caulinodans ORS 571 is a beneficial bacteria to wheat plant, it also can induce defense response. We choose wheat root simple of different time that 1h,3h,6h,9h,12h,24h and 48h, use real-time PCR amplification to check transcriptional level expression of defense-related gene, include pox,sams,and sod. The result show that pox transcriptional level expression is significantly high compared with CK at 1h and 9h, but the sams reach this difference at 24h. Compareing with pox and sams, sod transcriptional level expression below the CK at 1h and this tendency have significantly difference at 24h and 48h.
引文
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房增国,赵秀芬,孙建好,张福锁,李隆. 2009.接种根瘤菌对蚕豆/玉米间作系统氮营养的影响.华北农学报, 24(4): 124~128
郝变青,马利平,乔雄梧. 2010. GFP标记的植物促生菌B96-Ⅱ-gfp的定殖能力研究.中国生态农业学报, 18(4): 861~865
何海健,陈婷飞. 2008.绿色荧光蛋白及其在分子生物学上的应用.金华职业技术学院学报, 8(4): 13~18
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