【作者】 王伟；

【导师】 王义强；

【作者基本信息】 中南林业科技大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 CaNZ是CaN(Calcineurin)基因的正义表达基因,是在真核生物中存在的一个Ca2+及CaM依赖的Ser/Thr蛋白磷酸酶,它包括一个催化亚基calcineurin A和一个Ca2+结合亚基calcineurin B,其催化亚基calcineurin A活性受CaM调节。本实验在筛选烟草高效再生体系基础上,利用带有信号肽和CaM结合肽的载体,以农杆菌为媒介将CaN基因导入烟草中,预期过表达的融合蛋白将会被分泌到细胞外并与质外体CaM相结合,抑制质外体CaM的功能,从而构建出质外体CaM的反义植株,观察质外体CaM反义植株的表型改变,为进一步开展CaM在植物生长发育过程中的功能研究提供基础。研究结果如下：(1)以14-16d烟草叶片为外植体,以MS为基本培养基,通过附加激素配方对比,筛选出适合材料再生的最佳培养基配方：MS+IAA 0.5mg·L-1+6-BA 1.5mg·L-1为芽分化的培养基,1/2MS+NAA0.1mg·L-1为生根培养基。(2)用Kan进行烟草叶片抗性筛选。当Kan浓度小于50mg·L-1时,叶片能正常分化出芽；当Kan浓度为75mg·L-1时,叶片大多数白化；当Kan浓度超过100mg·L-1时,芽分化停止。所以,以Kan浓度100mg·L-1为烟草叶片分化芽抗性筛选的临界浓度；而培养物根对抗生素较为敏感,Kan 50mg·L-1为筛选临界值。通过对烟草叶片转化过程中各种条件的比较试验,获得其转化最佳条件为：叶片预培养3d后,在10倍稀释(OD600=0.8)的根癌农杆菌菌液中浸染6-8min,然后放置于铺有一层无菌滤纸的分化培养基上共培养3d。(3)以抗性植株的总DNA为模板,以质粒DNA为阳性对照,以为转化植株为阴性对照,利用外援基因的特异引物进行PCR扩增,结果表明,37株转化苗中有21棵检测结果为阳性,转化效率为56.7%。初步证明CaNZ基因已经转入烟草基因组中。(4)大部分PCR检测为阳性的转化植株与对照植株相比,生长缓慢,株型矮小,种子成熟期推迟。更多还原

【Abstract】 CaNZ is the CaN plus-sense gene. CaN is a Ca2+/CaM-depended Ser/Thr proteinphosphotase existing in the eucaryons, which includes a catalytic subunit—calcineurin A and a Ca2+ combined’subunit—calcineurin B. The activity of calcineurin A is regulated by CaM. According to the researching methods for animals, through the purpose gene transfered by Agrobacteritunm, fusion protein of expected expression is excreted out of cell and combin with CaM with the signal peptide and bearer protein of CaM combined peptide. So the function of CaM would be restrained, thereby constructing the CaM’s antisense plants. By observing the phenotype change of CaM’s antisense plants, the function of CaM on the plants’growth and development was suggested.(1) First, the regeneration system of somatic embryogenesis is established for alfalfa genetic transformation. The 14-16 days old tabaco leaf with high regenerability was selected as the plant material. Based on the MS medium, a series of medium were devised and selected by effect. The medium of inducing differentiation of explants is MS+IAA 0.5mg·L-1+6-BA 1.5mg·L-1,the medium of including rootage of sprouts is 1/2MS+NAA0.1mg/L.(2) Kanamycin is used to select transigenic explants. When Kanamycin concentration is below 50mg/L, the tobacco explants grow well;when Kanamycin concentration is 75mg/L, a majority of explant became white; when concentration is 100mg/L,all explant become white,so Kanamycin concentration of 100mg/L could be regarded as the selection-pressure. The rootage of sprouts is sensitive to antibiotic. So it is enough to select explant,when Kanamycin concentration is 50mg/L. The factors influencing Agrobacterium-mediated tabacco transformation were studied in systematism, When three days pre-cultured, Agrobacteritunm in the phrase of loizarithlm Growth was deluted to 1/10 of original concentration, explants were mixed with cell suspension of Agrobacterium in 6-8 minutes, three days col-cultivated on the axenic filter paper.(3) General DNA of transgenic plants was taken as template, the DNA of plasmid as was taken masculine contrast, the DNA of common green tabacco plants was taken as negative contrast, then carried through PCR amplification. PCR analysis showed that 21 plants were masculine plants. The percent of transformation frequency is 56.7%. we elementarily confirmed that CaNZ gene existed in the DNA of plants.(4) A majority of plants analysed masculine were short and grow slowly,compare to the contrast.Certainly,there were morphological characters. We need more evidences if want to confirm the effect of gene transformed on calcineurin signalling mechanisms.更多还原