【作者】 李俊；

【导师】 尹伟伦； 夏新莉；

【作者基本信息】 北京林业大学 ， 植物学， 2008， 博士

【摘要】 中国沙棘（Hippophae rhamnoides L.subsp.sinensis Rousi）是广泛分布在欧亚大陆上,具有生态、医用、食用价值的多功能经济乔灌木;其优良苗木的繁殖因各种限制因素,难以满足市场的大量需求。近年来,又面临大面积死亡的现象,抗逆性不够被认为是根本原因,抗逆基因的转入可能是解决问题的最佳途径。中国沙棘体细胞胚胎发生体系的建立,既能提供大量优良苗木,又能为其转基因研究奠定基础。又由于中国沙棘被应用在食品、药品、饲料等生产领域中,抗逆性基因BADH作为目的基因与标记基因转入植物体内,可达到提高转化株的抗逆性及消除抗性标记的目的,意义重大。因此,本论文在前人有关研究的基础之上,通过2年的试验,成功建立了以中国沙棘以茎尖为材料通过间接途径诱导体细胞胚胎发生的再生体系,并对体细胞胚发生过程中的相关影响因素进行了探讨分析。在BADH遗传转化拟南芥（Arabidopsisthahana（Linn.）Heynh.）试验中,进行了其作为标记基因判断阳性植株的相关表型筛选依据的探索研究,为以后相关工作的开展提供坚实的背景资料。主要研究结果如下:1.以中国沙棘组培苗的茎尖为外植体,进行愈伤组织及胚性愈伤组织诱导试验,结果发现在1/4MS培养基上的愈伤组织发生率最高,对愈伤组织的诱导具有关键作用;而无论单独使用NAA还是2,4-D都不利于胚性愈伤组织的发生,所以在胚性愈伤组织诱导中,生长素与细胞分裂素的结合是必须的;同时得出结论:NAA与KT的结合能更有效的诱导胚性愈伤组织的发生。2.在中国沙棘愈伤组织及胚性愈伤组织诱导试验基础之上,深入探讨诱导中国沙棘胚性愈伤组织发生的影响因素。试验结果表明,以1/4MS+KT1.0mg/L+NAA0.3mg/L+琼脂粉6 5g/L+白砂糖30g/L为最适配方,愈伤组织诱导率达到90.10%,胚性愈伤组织诱导率达到77.67%,平均体细胞胚的发生数达到15.48。3.通过对中国沙棘子叶和下胚轴在诱导培养基上不同放置方式的研究发现,接种方式不同导致愈伤组织及胚性愈伤组织的诱导效果不同。子叶的愈伤组织和胚性愈伤组织的诱导率由高到低依次为:子叶切口向下>远轴面（背面）向下>近轴面（正面）向下;而下胚轴极性下端倾斜接触培养基比水平接触培养基更有利于愈伤组织和胚性愈伤组织的诱导。4.通过将中国沙棘茎尖、子叶、下胚轴分别接种在诱导培养基上的试验发现:茎尖的诱导效果远远高于子叶和下胚轴,分别能达到90.10%的愈伤组织诱导率和77.67%的胚性愈伤组织诱导率;而子叶仅为66.73%和14.80%;下胚轴为52.80%和34.87%,均低于茎尖。所以,确定中国沙棘组培苗茎尖为最佳外植体。5.通过进行基本培养基和KT对中国沙棘体细胞胚植株再生的影响试验,结果发现:4种基本培养基WPM、MS、1/2MS、1/4MS中,WPM培养基促进效果最明显。在进一步通过降低糖含量及调整细胞分裂素KT浓度的试验之后,确定中国沙棘体细胞胚植株再生的最佳配方为:WPM+KT0.05mg/L+白砂糖20g/L+琼脂粉6.5g/L,植株再生率达到60.30%。6.通过Trizol法提取菠菜RNA,用DNAMAN软件设计引物BH4.24.1:5’-CTCGCCTTACCCTCTCAACTC-3’和BH4.24.2:5’-CTGTAAACTCGACCTTCTCTGCG-3’,用RT-PCR法克隆到BADH基因片段,经测序证实BADH基因被成功克隆。7.在己从菠菜中克隆到的BADH基因序列上、下游分别添加酶切位点BglⅡ（识别位点:agatct）和PmlⅠ（识别位点:cacgtg）,成功构建pCAMBIA1305.2-BADH表达载体,并进行了野生型拟南芥的侵染转化试验。8.pCAMBIA1305.2-BADH表达载体上既含有BADH基因,又含有潮霉素抗性标记基因,可以用甜菜碱醛和潮霉素两种筛选介质来筛选转基因植株,起到互相验证转基因植株的作用。试验结果表明:在20mM甜菜碱醛（BA’）筛选剂选择压力下,没有筛选到转基因株系;在含25mg/L潮霉素的筛选培养基上,获得4个转基因株系（HBT₀（1）、HBT₀（2）、HBT₀（3）、HBT₀（4））,4个对照株系（HKT₀（1）、HKT₀（2）、HKT₀（3）、HKT₀（4））。9.将通过25mg/L潮霉素筛选到的含BADH基因的拟南芥株系（HBT₀（1）、HBT₀（2））,通过PCR分子检测,证实BADH基因已转入拟南芥基因组中;将收获的T₁代种子在25mg/L潮霉素的培养基上筛选发现,转基因植株:非转基因植株的抗性筛选比率符合3:1的孟德尔遗传分离规律。10.含BADH基因的转基因株系（HBT₀（1）、HBT₀（2））的T₁代阳性植株及成熟种子在含不同浓度NaCl的培养基上,经过一段时间光培养和暗培养处理,根和下胚轴的生长都受到不同程度的影响,表现出一定抗盐能力;尤其在阳性植株表型分析中,含BADH基因的转基因株系在100mM NaCl培养基上,抗逆性明显优于对照株系,说明转基因株系能耐受100mM盐的逆境胁迫。11.含BADH基因的转基因株系（HBT₀（1）、HBT₀（2））的T₁代阳性植株及成熟种子在含20mM BA’的培养基上,经过一段时间光培养和暗培养处理,转基因株系的表型如根、下胚轴等形态指标比对照株系都有明显的伸长生长,表现出一定的抗逆性能;尤其在阳性植株表型分析中,含BADH基因的转基因株系与对照株系的表型差异仍表现在根的生长受到最明显的影响,与敏感性试验的结论一致,进一步验证了选用根的表型特征作为BADH基因筛选拟南芥转基因株系的筛选依据的可行性。另外,凭借根长表型为筛选判断依据进行试验时,发现以培养到15d左右为最合适判断时机,为BA’筛选转基因拟南芥提供了一定表型判断依据。更多还原

【Abstract】 As a kind of multi-function economic tree, seabuckthorn （Hippophae rhamnoides L.） is distributed widely in Asia-Europe, which has some important values in the field of ecology, medicine, food and so on. But now, there were some limiting factors in seabuckthorn propagation, that results in a low output about seabuckthorn choiceness seedlings and could not meet the great demands of markets. In the recent years, there is a very serious problem about seabuckthorn, so lots of experts gave a kind of unanimous analysis about the phenomenon, that was low anti- adversity ability effecting on inferior growth of seabuckthorn, and genetic transformation method of anti-adversity gene maybe the best choice to settle problem. The regeneration system by indirection somatic embryogenesis, not only is an effective technique means to get a number of choiceness seedlings, but also give a good base to have related to genetic transformation research in seabuckthorn. As an important economic fruit tree, seabuckthorn are applied widely in the food, medicine and feedstuff field. If BADH gene as aim gene and marker gene at the same time is transferred into plant, not only transformation tree can get high anti-adversity ability, but also the latency harm problem can be avoided from resistance marker gene. So, it is very significative to develop BADE gene as marker gene in genetic transformation research.During two years, on the base of reported research, the regeneration system of indirected somatic embryogenesis in seabuckthorn was achieved and there were some discussions and analyses about related factors of somatic embryogenesis. In genetic transformation research of Arabidopsis thaliana,some phenotypic difference were discussed about using BADH as marker gene to screen positive plants and those work also can offer strong supports for next researches. The results were as follows:1.During experiment of callus and embryogenic callus induction using shoot tips from tissue culture plant of seabuckthorn as explants, callus incidence rate was the highest on 1/4MS medium, which had key function to induce callus. Either using NAA or 2,4-D alone, embryogenic callus could not be induced, indicating that the combination of auxin and cytokinin is necessary in the process of inducing embryogenic callus. On the same time, a conclusion was summarized that the combination of NAA and KT was effective to embryogenic callus induction in seabuckthorn.2.On the base of experiment of callus and embryogenic callus induction in seabuckthorn, some factors were discussed related to embryogenic callus initiation. Finally, the achievements were as follows: the induction frequency of callus reached 90.10%, the induction frequency of embryogenic callus was 77.67% and the average number of somatic embryogenesis could up to 15.48. So the suitable medium to inducing embryogenic callus and somatic embryos was 1/4MS +KT1.0mg/L +NAA0.3mg/L +granulated sugar 30g/L +agar6.5g/L +CH0.5g/L.3.By the research about different inoculation manners of seabuckthorn cotyledon and hypocotyl on inducing medium, the result showed that different inoculation manners could affect inducing effect of callus and embryogenic callus, and the inducing rate from high to low in turn about cotyledon was cotyledon incision downside >abaxial side downside > adaxial side downside, and the under side inoculation of inclined hypocotyl was better than horizontal placement.4.The research results about shoot tip, cotyledon, hypocotyl in seabuckthorn were inoculated respectively on inducing medium, indicated that the effect of shoot tip was better than cotyledon and hypocotyl, which had 90.10% callus inducing rate and 77.67% embryogenic callus inducing rate. Otherwise, the callus inducing rate and embryogenic callus inducing rate of cotyledon were only 66.73% and 14.80%, respectively, and the callus inducing rate and embryogenic callus inducing rate of hypocotyls were 52.80% and 34.87%, respectively. The inducing rate of both cotyledon and hypocotyl were all very lower than shoot tip’s. As a result, shoot tip was the best explant during the inducing embryogenic callus experiment.5.By experiment of the plant regeneration from somatic embryo in seabuckthorn under the effect of KT and basal medium, WPM had the best effect on promoting few plants regeneration in seabuckthorn among MS, 1/2MS 1/4MS and WPM mediums. Meanwhile, reducing granulated sugar and design different concentrations KT were used to have the experiment of plant regeneration. Finally, the best regeneration medium was:WPM +KT 0.05mg/L +granulated sugar 20g/L +agar6.5g/L +CH0.5g/L. And regeneration frequency was 60.30% on the medium.6.Using Trizol method to extract RNA from spinach, using DNAMAN sofeware to design primer BH4.24 1:5’-CTCGCCTTACCCTCTCAACTC -3’ and BH4.24.2:5’-CTGT AAACTCGACCTTCTCTGCG -3’and using RT-PCR method to clone BADH gene, the sequence was cloned successfully by DNA Sequencing analysis.7.After restriction sites BglⅡ（agatct） and PmlⅠ（cacgtg） were added successfully to upstream and downstream of the cloned BADH sequence from spinach, the expression vector pCAMBIA1305.2-BADH was constructed and transformed in Arabidopsis thaliana subsequently.8.Because of both BADH gene and hygromycin resistance gene existing on the expression vector pCAMBIA1305.2-BADH,transformed plant could be selected using BA’ and hygromycin, which had a function to validate each other. The results showed that transformed plant was not selected on 1/2MS supplemented with 20mM BA’.On the other hand, four transformed plant lines （HBT₀（1）,HBT₀（2）,HBT₀（3）,HBT₀（4）） and four control plant lines （HKT₀（1）,HKT₀（2）, HKT₀（3）,HKT₀（4）） were got on 1/2MS supplemented with 25mg/L hygromycin, respectively.9.By PCR, transformed plant lines with BADH （HBT₀（1）,HBT₀（2）） was detection, the test result showed BADH gene had been transformed into Arabidopsis thaliana DNA genome. After T₁ generation seeds were selected on selective medium supplemented with 25mg/L hygromycin, there was a 3:1 Mendelian’s segregation law between transgene and non-transgene plants.10.T₁ generation positive plants and mature seeds from transgenic plant lines with BADH （HBT₀（1）,HBT₀（2）） were cultured on different concentrations NaCl mediums in the condition of light and night, root and hypocotyl were affected at different extent and exhibited some anti-NaCl ability, especially at the phenotypic analyse of positive plants, transformed plant lines with BADH could survived and grew strongly than control plant lines on 100mM NaCl medium, indicating transformed plant could suffer the stress treatment of 100mM NaCl.11.T₁ generation positive plants and mature seeds from transgenic plant lines with BADH （HBT₀（1）,HBT₀（2）） were cultured on 20mM BA’ medium on the condition of light and night, the phenotypic characters of transformed plant with BADH, such as root and hypocotyl were longer than control plants, which indicated some resistance function in transformed plant lines, especially at the phenotypic analyse of positive plants, the difference of root was the best obvious between transgenic plants with BADH and control plants, which was consistent with the conclusion of sensitivity test and further verified the feasibility of using the root phenotypic characters to screen positive plants with BADH in Arabidopsis thaliana. On the side, by right of the selective standard in root length,15 days was the suitable time to judge transgenic plant lines, which offered some phenotypic selectable characters to select transformed Arabidopsis thaliana by BA’ selective agent.更多还原