【作者】 刘艺平；

【导师】 何松林；

【作者基本信息】 河南农业大学 ， 森林培育， 2015， 博士

【摘要】 牡丹(Paeonia suffruticosa),为世界著名的观赏花卉,其花朵硕大、色彩艳丽、芳香四溢,备受人们喜爱。本研究以牡丹叶柄诱导的愈伤组织为材料,进行了牡丹胚性愈伤组织的诱导;克隆牡丹体细胞胚相关SERK基因,并采用实时荧光量PCR技术进行了SERK基因的表达研究;采用农杆菌介导转化法构建了适用于牡丹愈伤组织的遗传转化体系。主要研究结果如下:1.牡丹胚性愈伤组织的诱导本试验选择牡丹‘凤丹白’和‘乌龙捧盛’叶柄诱导的愈伤组织为材料,利用CaCl2、H2O2和硝普纳(SNP,NO供体)进行牡丹胚性愈伤组织的诱导,研究这3种细胞信号转导因子对牡丹愈伤组织生长、分化的影响。通过形态学和显微切片观察,从总体表现来看,‘乌龙捧盛’在愈伤组织生长过程中的褐化程度比‘凤丹白’严重得多,已经影响到愈伤的正常生长和增殖。在不同浓度处理下培养30 d后,愈伤组织的形态上出现了结构变疏松,质地变软,有雪花状和凸起状愈伤组织的形成,这些变化和胚性愈伤组织的形成有关。在这3种因子中,以SNP处理下的变化最显著。通过不同时期内酶活性和内源激素变化的研究发现,CaCl2诱导牡丹胚性愈伤中应保持较高浓度1.6 mmol·L-1以上,诱导时间约在10-20 d;H2O2在牡丹胚性愈伤组织诱导中的作用并不显著;SNP在较低的浓度水平50μmol·L-1以下就可以促进牡丹胚性愈伤的形成,并可能促进体细胞胚的形成。2.牡丹体细胞胚发生相关SERK基因的克隆与表达分析本研究应用同源克隆结合RACE技术首次从牡丹?凤丹白‘叶柄诱导的愈伤组织中成功克隆得到了SERK基因的cDNA全长序列,命名为PsSERK,在GenBank中的注册号为KF876175。该基因的cDNA全长2362 bp,包括299 bp的5非编码区、179 bp的3′非编码区和1884 bp的开放阅读框共编码627个氨基酸。在GenBank中检索发现牡丹PsSERK基因核苷酸序列与其它植物的SERK基因序列高度同源,相似性均在82%以上。对牡丹PsSERK基因的生物信息学分析结果表明,基因编码627个氨基酸,属于亲水性蛋白,定位在细胞膜中,具备跨膜结构,4个保守结构域,24个磷酸化位点,具有信号肽序列,二级结构以α螺旋结构和无规则卷曲结构为主。与不同植物来源的SERK的氨基酸同源性为91%-95%。本研究采用实时荧光定量PCR技术,以Actin作为内参基因分析了在牡丹的叶子、花和胚性愈伤组织中PsSERK基因的转录水平表达变化。结果显示该基因在花中表达量最低,叶子中稍有表达,而在胚性愈伤组织中表达非常高。这表明PsSERK基因的表达与牡丹胚性愈伤的形成密切相关。3.农杆菌介导的牡丹愈伤组织遗传转化体系构建本试验通过农杆菌介导的方法,对牡丹愈伤组织遗传转化体系的优化进行初步的研究。对遗传转化过程中菌株的选择、影响转化率的各项影响因子(预培养时间、菌液浓度、侵染时间、共培养时间等)进行了初步筛选,初步建立植物组织培养液侵染法的牡丹愈伤组织遗传转化体系。潮霉素浓度3 mg·L-1为临界筛选浓度;头孢霉素初始抑菌浓度为200 mg·L-1,在继代培养时降低至100 mg·L-1。不经过预培养,直接用OD600=0.6的菌液侵染20 min,在22℃黑暗环境下共培养3 d,用200 mg·L-1头孢霉素水涮洗浸泡愈伤30 min后,接入含有100mg·L-1头孢霉素的液体增殖培养基,振荡培养24 h后,接至含有100 mg·L-1头孢霉素和3mg·L-1潮霉素的固体筛选培养基中,20 d转接一次。牡丹愈伤组织转化后GUS染色温度为24℃,染色后均能成功检测到GUS瞬时表达活性。更多还原

【Abstract】 Tree peonies（Paeonia suffruticosa）are famous ornamental flowers frequently seen in gardens nearly all over the world.People enjoy their large,showy,colorful and fragrant flowers.In this study peony callus in vitro from petioles was used as the material for induction of embryogenic callus;cloning of SERK gene from callus,expression analysis of SERK gene by real-time quantitative PCR technology in P.suffruticosa.genetic transformation system of P.suffruticosa callus was constructed in this study.The main results are as follows:1.Induction of peony embryogenic callusIn this study the callus inducted by petioles of P.suffruticosa ‘Feng dan bai’ and ‘Wu long peng sheng’ were as explants to induce embryonic callus.Experiments were executed to evaluate the effect of different concentrations of Ca Cl₂,H₂O₂ and SNP（NO donor）on peony callus growth and differentiation.The results showed that the degree of browning in ‘Wu long peng sheng’ was much more serious than in ‘Feng dan bai’ through morphology and microscopic observation,the growth and multiplication of the callus were influenced in ‘Wu long peng sheng’.Under the different concentration of treatments a consistently occurring phase of response,within 30 d of culture,was the appearance of clear,loose,soft,raised and “snow-flake” callus tissue on the surface of the explants.These changes were related to the formation of the embryonic callus.In these three factors,the effects of SNP treatment were the most significant.The studies about the changes of antioxidase activity and endogenous phytohormones in P.suffruticosa callus during the different periods showed that embryonic callus was induced under CaCl₂ at above 1.6 mmol·L-1 after 10-20 d,H₂O₂ was not significant in the peony embryonic callus induction.SNP at low concentration level belowμmol·L-1 promoted the formation of the peony embryonic callus and may be conducive to the peony somatic embryogenesis.2.Cloning and expression of SERK gene in P.suffruticosaIn this study,the full-length cDNA of SERK gene was cloned by homology cloning combined with RACE technology from the callus inducted by petioles of P.suffruticosa ’Feng dan bai’,and named as Ps SERK（Gen Bank accession number KF876175）.Sequencing of the full-length cDNA got 2362 bp,contained 299 bp 5′ untranslated region,179 bp 3′untranslated region and 1884 bp open reading frame,and encoding 627 amino acids.Nucleotide sequence alignment showed that the Ps SERK align had high homology more than 82% with of SERK genes from other species in Gen Bank.The results of bioinformatics analysis of Ps SERK showed that Ps SERK gene encoded 627 amino acids and was a kind of hydrophilic protein,mainly located in the plasma membrane,contained transmembrane domains,4 conserved structure domains,24 phosphorylation sites and a signal peptide sequence.The secondary structure of Ps SERK was made up mostly of alpha helix and random coil.When compared the Ps SERK with the other plant amino acid sequences,the identity percentage varied from 91% to 95%.Actin gene was used as the reference gene for Ps SERK gene expression analysis by real-time quantitative PCR technology in the leaves,petals and embryonic calluses of P.suffruticosa.The results showed that transcriptional expression levels of Ps SERK gene at different tissues were expressed at different degree,the expression level was the lowest at the petals;the next was at the leaves,and the highest was at the embryonic calluses,which suggested that Ps SERK gene expression played an important role during embryonic calluses morphogenesis in P.suffruticosa.3.Constructing an Agrobacterium-mediated genetic transformation system of P.suffruticosa callusThe optimization of Agrobacterium mediated genetic transformation system was studied in the peony callus.During the genetic transformation the strains were selected and at the same time the study optimized the various impact factors,the pre-cultured time,concentration of bacterium,infection time,co-cultured time.And Agrobacterium mediated genetic transformation system of peony callus had been established.The minimum lethal homomycin concentration of peony callus was 3 mg·L-1 and the minimum lethal cephalosporin concentration for growth of agrobacterium in the initial select medium was 200 mg·L-1,and that for the continuous medium was 100 mg·L-1.The system was no pre-culture,numerical OD600 0.6,20 minutes for infection,3 days for co-culture in darkness at 22℃.And then the callus was rinsed and immersed using 200 mg·L-1 cephalosporin for 30 minutes.The callus treated was transferred to the liquid culture medium added 200 mg·L-1 cephalosporin for by shaking culture for 24 hours and then was transferred to the screening solid medium added 200 mg·L-1 cephalosporin and 3 mg·L-1 homomycin,and was done once 20 days.The peony callus after transformation was successfully tested by GUS expression at the staining temperature of 24℃.更多还原