【作者】 周延清；

【导师】 贾敬芬；

【作者基本信息】 西北大学 ， 植物细胞工程， 2005， 博士

【摘要】 本文旨在:Ⅰ.建立河南三种主栽经济植物地黄、山药和大豆种质遗传多样性的ISSR和RAPD标记分析体系,为利用DNA分子标记技术合理利用、保护、鉴定和改良这三种植物品种提供理论和技术依据。Ⅱ.建立发根农杆菌对怀地黄的转化及毛状根的植株再生体系。Ⅲ.分离克隆大豆油酰基-△12-去饱和酶基因fad 2-1和构建反义基因表达载体,为利用农杆菌介导的遗传转化技术培育大豆和怀地黄新品种提供理论和技术依据。 Ⅰ.利用ISSR和RAPD标记技术对这三种经济植物种质遗传多样性进行了检测。其中,ISSR标记技术首次用于这一研究。所取得的主要进展如下:（1）.用CTAB法提取了10个地黄品种、28个山药品种和10个大豆品种以及16个怀地黄单株的基因组DNA,建立了适用于山药基因组DNA提取的改良CTAB方法。（2）.以怀地黄基因组DNA为模板,优化出了适宜于地黄ISSR分析的合适的退火温度（53-55℃）和扩增体系:25μL PCR反应体积,包含1×Taq DNA酶缓冲液（10mmol/L Tris-HC l,50mmol/L KCl,0.1%Trion X-100,pH9.0）,2.5 mmol/L MgCl₂,1.0-1.5U Taq酶,60ng模板DNA,0.4μmmol/L引物,dATP、dGTP、dCTP和dTTP各0.4 mmol/L。（3）.在此基础上,从44个ISSR引物中分别筛选出了适合于地黄、山药和大豆ISSR标记分析的引物10条、7条和8条;从80条RAPD引物中分别筛选出了适合于地黄和山药RAPD标记分析的引物17条和2条。（4）.10条ISSR引物对10个地黄品种（系）扩增出110条带,多态条带比率（PPB）为71.82%,平均多样性指数（Ⅰ）为0.3577,遗传相似系数（GS）在0.557～0.979,平均GS为0.665;17条RAPD引物对10个地黄品种（系）扩增出177条带,多态条带比率（PPB）为61.58%,平均多样性指数（Ⅰ）为0.3135,遗传相似系数（GS）在0.63～0.93,平均GS为0.7545。两种分子标记的分析结果呈极显著正相关（r=0.649）;利用2条ISSR引物对16个怀地黄单株扩增出17条带,多态条带比率（PPB）为64.71%。使用3条RAPD引物对16个怀地黄单株扩增出7条带,多态条带比率（PPB）为57.14%。（5）.7条ISSR引物对28个山药品种扩增出65条带,多态条带比率（PPB）为83.01%,平均多样性指数（Ⅰ）为0.4379,遗传相似系数（GS）在0.33～0.96,平均GS为0.6246。2条RAPD引物对28个山药品种扩增出23条带,多态条带比率（PPB）,为82.6%。（6）.8个ISSR引物对10个大豆品种扩增出89条带,多态条更多还原

【Abstract】 The objectives of this dissertation are: Ⅰ .to establish an efficient ISSR and RAPD-based genetic diversity detection system of three economic plants (Rehmannia glutinosa Libosch, Dioscorea opposita Thunb and Glycine max Merr); Ⅱ .to establish an appropriate transformation and regeneration protocol for Rehmannia glutinosa Libosch .f. hueichingensis(Chao et schih) Hsiao; Ⅲ.to clone the Oleoyl- △12-desaturase gene from Glycine max Merr and to construct its antisense expression vector in order to lay the theoretical and technological foundation for the DNA marker-based cultivar identification and improvement in three plants as well as the genetic breeding via Agrobacterium-mediated transformation in Glycine max Merr and Rehmannia glutinosa Libosch .f. hueichingensis(Chao et schih) Hsiao.Ⅰ .RAPD and ISSR markers were used to investigate the genetic diversity of three economical plants, ISSR marker of which was used for the first time.The main progresses obtained are as follows: (1).By means of CTAB method, the DNAs were extracted from 10 cultivars (lines) in Rehmannia glutinosa Libosch, 28 cultivars in Dioscorea opposita Thunb, 10 cultivars in Glycine max Merr and 16 individuals in Rehmannia glutinosa Libosch .f. hueichingensis(Chao et schih) Hsiao, respectively. CTAB method for the DNA extraction from Dioscorea opposita Thunb was improved. (2).The genomic DNA, from a young regenerated plant of Rehmannia glutinosa Libosch .f. hueichingensis (Chao et schih) Hsiao, was used as a template to optimize ISSR-PCR amplification conditions for Rehmannia glutinosa Libosch as follows: proper annealing temperatures from 53℃ to 55 ℃ and a PCR reaction volume of 25μL, including 1.0-1.5 U Taq DNA polymerase, 3.0 mmol/L MgCl2, 1 x Taq DNA polymerase buffer (10mmol/L Tris-HCl, 50mmol/L KCl, 0.1% Trion X-100, pH9.0 ), 60ng template DNA, 0.4μm mol/L primer, 0.4 m mol/L each of dATP、 dGTP、 dCTP and dTTP. (3). Based on the optimized conditions, among 44 ISSR primers and 80 RAPD primers tested, the 10,7 and 8 ISSR primers were selected for the ISSR analyses in Rehmannia glutinosa Libosch, Dioscorea opposita Thunb and Glycine max Merr, respectively; 17 and 2 RAPD primers were done for the RAPD analyses in Rehmanniaglutinosa Libosch and Dioscorea opposita Thunb. (4).Among the 10 cultivars in Rehmannia glutinosa Libosch, the 10 ISSR primers amplified 110 bands with a percentage of polymorphic bands (PPB) of 71.82% and 17 RAPD primers amplified 177 bands with one (PPB) of 61.58%. The genetic diversity, estimated by Shannon’s index, was 0.3577 by ISSR markers and 0.3135 by RAPD markers. Genetic similarity (GS) value was from 0.557 to 0.979, whose mean value was 0.665 by ISSR markers. GS value range was from 0.63 to 0.93, whose mean value was 0.7545 by RAPD markers. Furthermore, the correlation coefficient of 0.649 between RAPD and ISSR markers indicated that both markers were significantly correlated. Among the 16 individuals, the 2 ISSR primers amplified 17 bands with one (PPB) of 64.71% and 3 RAPD primers amplified 7 bands with one (PPB) of 57.14%. (5). Among the 28 cultivars, the 7 ISSR ISSR primers amplified 65 bands with one (PPB) of 83.01% and 2 RAPD primers amplified 23 bands with one (PPB) of 82. 6%. The genetic diversity, estimated by Shannon’s index, was 0.4379 by ISSR markers. GS value range was from 0.33 to 0.96, whose mean value was 0.6246 by ISSR markers. (6). Among the 10 cultivars in Glycine max Merr, the 8 ISSR primers amplified 89 bands with one (PPB) of 62.5%. The genetic diversity, estimated by Shannon’s index, was 0.2865. GS value range was from 0.60 to 0.75, whose mean value was 0.6912 by ISSR markers. (7). 10 Rehmannia cultivars (lines) could be divided into two similar groups by the dendrograms performed by both markers; 28 yam cultivars 10 soybean cultivars could be divided into four groups and 10 soybean cultivars could be done into two groups by the dendrograms performed by ISSR markers. (8). Primary content analysis was employed to evaluated the resolving power of the markers to differentiate between the cultivars.,whose results were very consistent to that of the above dendrograms.The results revealed that the quality of the extracted DNAs reached to the standads of both markers .the optimized reaction system was appropriate for ISSR analysis; Both markers could not only detect a quite high PPB and Shannon’index (I) among different cultivars in each of these three plants but among different individuals in Rehmannia glutinosa Libosch .f. hueichingensis (Chao et schih) Hsiao. However, different plants had different PPBs, Is,GS value ranges and mean GS values.The genetic diversity(PPB and I) among different cultivars in Rehmannia glutinosa Libosch was higher that among different individuals in Rehmannia glutinosa Libosch .f. hueichingensis (Chao et schih) Hsiao. Among the three plants’ PPBs and Is by ISSR markers, Yam’s PPB and I were the biggest,and Rehmannia’s PPB and I were gigger than soybean’s; Yam’s PPB and I were higher than Rehmannia’s by RAPD markers. While the result "yam GS value range > Rehmannia GS one>soybean GS one" was revealed , the result "yam mean GS value range < Rehmannia mean GS one<soybean mean GS one" was found. Rehmannia GS value range by ISSR markers was bigger than its GS one by RAPD markers.However, Rehmannia mean GS value by ISSR markers was smaller than its mean GS one by RAPD markers.Therefore,RAPD and ISSR markers were suitable for the detection of germplasm genetic diversity in three plants.Ⅱ. The genetic transformation in Rehmannia glutinosa Libosch .f. hueichingensis (Chao et schih) Hsiao was carried out for the first time using Agrobacterium rhizogenes-mediated transformation. The hairy roots were induced from the young leaf, stem and petiole explants by infection with Agrobacterium rhizogenes strainA4, 15834 and LBA9402.Transformed hairy roots could grow rapidly on solid or liquid 1/2 MS medium without any phytohonnones. Their clones were established on the above solid 1/2 MS medium. The effect of different Agrobacterium rhizogenes strains, explants, media, culture methods and sucrose concentration on hairy root growth was studied. The effect of sucrose concentration on the catalpol content in transformed hairy roots was analyzed The plant regeneration from the transformed hairy roots was investigated. The results showed that (1). transformed hairy roots could be induced from leaf explants at a higher frequency of 32.6+1.76% as compared to stem and petiole ones; (2).hairy roots could grow well on phytohormone-free solid or liquid 1/2 MS medium(30g/L sucrose) ,but the hairy roots could grow better in the latter. After a culture of 35d, the fresh weight of liquid-cultured hairy roots increased by twice more than that of solid-cultured hairy roots , which did by 8. 91 ±0.51 times. The growth curve of hairy roots on phytohormone-free solid 1/2 MS medium was similar to that of them on phytohormone-free liquid 1/2 MS medium, and their growth peaks appeared after they were inoculated for about two weeks. (3).At the range of sucrose concentrations from15 to 75 mg/L, 30mg/L was favorable to the catalpol production in hairy roots. When hairy roots were cultured in liquid 1/2 MS medium for 35 days , a catalpol content of 0.557mg/g.FW in them was obtained, which was higher than that (0 mg/g.FW) in non-transformed roots,48.5% that in fresh Rehmannia root and 18% that in dried Rehmannia root, respectively.(4). A good hormone combination of 3.0m g / L 6-BA and 0.2 m g /L KT was selected. After hairy root segments were cultured for 40d on solid 1/2 MS medium plus the hormone combination, calluses were induced from them at a percentage of 100%, from which shoots were re-differentiated at one of 51.49%. (5).The shoots could take root on a solid 1/2 MS medium at a frequency of 100%. (6).Transgenic plantlets showed such phenotypic alterations as winkled leaves, shortened internodes, dwarfing of plant height, numerous adventitious roots and fewer stomata and dispersed chloroplasts in the leaf lower epidermis in comparison to the corresponding non-trangenic plants. The observed differences may reflect the modification of morphological root characters by integration of rol genes. Survival rate of the transplanted plantlets was up to 82.4%. The stable introduction of rol genes into this kind of medical plant and its transformed roots was confirmed by opine detection and PCR analysis.Ⅲ.One of key ways to improve soybean fatty quality is the increasement of oleic acid content by antisense gene technology. Oleoyl- A 12-desaturase plays an important role in the control of the production of polyenoic acids from oleic acid in soybean, one of whose two genes is named as fad2-1genz. Firstly, genomic DNA of the soybean cultivar Jidou6 was extracted by CTAB method.Then, the fad2-1gene fragment was amplified with polymerase chain reaction (PCR) with primers designed according to the retrieved soybean fad2-1gene sequence from NCBI. It was sequenced after cloned into the BamHI and SalI sites of the pMD18-T vector and transferred to E.coli JM109. The result of DNA sequence analysis indicated that the cloned fragment was approximately 660 base pairs (bp) in size, having a complete base identity to its original gene. In order to down-regulate the expression of soybean fad2-1gene, the cloned fragment was inserted in an antisense orientation into the BamHI and SalI sites between the CaMV35S promoter and the Nos terminator of binary vector pbt .The antisense fad2-1genefragment was transferred into Agrobacterium tumefaciens strain LBA4404. PCR detection and double digestion test showed that the gene fragment had been in E.coli JM109 and the strain LBA4404.The positive LBA4404 transformants are now being used in soybean transformation. To our knowledge, this is the first report in soybean Oleoyl- A 12-desaturase gene cloning in our country.更多还原