分枝相关基因CUC3保守区的克隆及其沉默研究
文献类型：学位论文
| 作者 | 贾海燕 |
| 学位类别 | 硕士 |
| 答辩日期 | 2008-05-29 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 过程工程研究所 |
| 导师 | 赵兵 |
| 关键词 | 罗布麻 烟草 CUC3 (Cup-Shaped Cotyledon3) 基因 农杆菌 基因沉默 |
| 其他题名 | Clone of the Conservative Region of Shoot Branching Regulatory Gene CUC3 and its Silence in Plants |
| 学位专业 | 生物化工 |
| 中文摘要 | 本文针对罗布麻分枝多造成纤维短、产量低等难题，研究克隆了其分枝相关基因CUC3（Cup-Shaped Cotyledon3）的保守区，并转化了罗布麻和烟草，为通过转基因技术控制罗布麻分枝奠定了基础。主要研究结果如下： 研究了罗布麻愈伤组织的诱导和分化。在16h/d光周期，对真叶和茎愈伤诱导最合适的培养基是MS附加0.5mg/L 6-BA和1.0mg/L NAA；真叶和茎愈伤组织的生长周期均为30d，但茎愈伤组织细胞生长速度较快；再分化诱导发现，罗布麻愈伤组织对激素诱导不敏感，不容易分化产生再生芽。 发根农杆菌对罗布麻的根外植体的发根诱导率达到80%以上；R1000感染的根外植体适合黑暗环境培养。在无激素的1/2MS培养基上，LBA9402和R1601诱导的发根可生成再生芽，诱导率为10 %，在附加0.2mg/L IBA的1/2MS培养基上2周内成根。PCR对发根及再生植株鉴定，证明T-DNA插入了植物基因组。 通过RT-PCR分别克隆了罗布麻和烟草CUC3基因的保守区cDNA，二者序列同源性达到98%。PCR鉴定、酶切和测序分析表明，正确构建了CUC3基因的反义和RNA干扰载体。用冻融法将表达载体转化农杆菌，PCR检测得到阳性克隆。 农杆菌分别转化烟草和罗布麻。烟草叶片在分化培养基（MS + 0.1mg/L NAA + 1.0mg/L 6-BA + 50mg/L Hyg + 500mg/L Cb）上4周后分化出芽；在生根培养基（MS +0.2 mg/L IBA +500mg/L Cb）上，2-3周后形成根；叶片GUS（β-葡萄糖甘酸酶）组织化学染色，阳性率为75%；通过PCR 检测HPT 基因（潮霉素磷酸转移酶基因）的DNA序列，获得30个阳性株系。半定量RT-PCR检测发现CUC3基因的表达量明显降低。在烟草转化阳性株系中获得5种表型：叶片融合，叶片卷曲，叶片缺失，叶片背腹特征不明显及分枝减少。罗布麻转化后，尚未获得再生植株。 |
| 英文摘要 | Apocynum venetum has too many branches to harvest high-quality fiber. In order to solve this problem, we cloned the conservative region of shoot branching regulatory gene CUC3 (Cup-Shaped Cotyledon3) and transformed it to Apocynum venetum and Nicotiana tabacum respectively. It provided a foundation for application of transgenic technology to control shoot branching of A. venetum. This research mainly focused on the following aspects: When different explants of A. venetum were cultured on MS medium supplemented with 0.5mg/L 6-BA and 1.0mg/L NAA in photoperiod 16h/d, the both callus induction rates were 100% for leaf and stem explants. Their growth circles were 30d on the solid MS medium supplemented with 1.0 mg/L 6-BA and 0.7 mg/L NAA, but the callus of stem grew fast than that of the leaf. Furthermore, the callus of A. venetum was less sensitive to plant growth regulators and difficult to obtain regenerated shoots through tissue culture. The transformation frequencies of Agrobacterium rhizogenes to root explants were up to 80%. Root explants infected by R1000 were more adaptable to be cultured under dark condition. Adventitious shoots were obtained from hairy roots induced by LBA9402 and R601 on hormone-free 1/2 MS medium, and the regenerated shoot induction frequency was 10%. Regenerated shoots rooted easily on 1/2 MS medium supplemented with 0.2mg/L IBA in 2 weeks. Polymerase chain reaction (PCR) analysis confirmed the integration of T-DNA to the genome of hairy root lines and regenerated plants. The conservative region of CUC3 gene of A. venetum and N. tabacum were cloned through RT-PCR, and they shared high homology (98%). PCR amplification, restriction and sequencing analysis indicated that anti-sense and RNA interference expression vector of CUC3 gene were correctly constructed. They were transformed to Agrobacterium by freeze-thaw method respectively, and positive clones were screened out by PCR amplification. Agrobacterium (with expression vector) transformed N. tabacum and A. venetum respectively. Regenerated shoots were formed in 4 weeks when the leaves of N. tabacum cultured on shoot-inducing medium (MS medium supplemented with 0.1mg/L NAA, 1.0mg/L 6-BA, 50mg/L Hyg and 500mg/L Cb). Shoots rooted on the root induction medium (MS medium supplemented with 0.2mg/L IBA and 500mg/L Cb) in 2-3 weeks. GUS (β-glucuronidase) histochemical assay indicated that the GUS positive rate was 75%. PCR analysis of HPT gene (Hygromycin phosphotransferase gene) indicated that there were 30 positive lines. Semi- quantitative RT-PCR detected that expression of CUC3 gene had a marked decrease. There were 5 distinct phenotypes in the positive lines as fellows: leaf fusion, leaf curliness, leaf incompleteness, leaf of dorsiventrality loss and branch decrease. However, no regenerated plants were got from A. venetum yet. |
| 语种 | 中文 |
| 公开日期 | 2013-09-13 |
| 页码 | 89 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1231]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 贾海燕. 分枝相关基因CUC3保守区的克隆及其沉默研究[D]. 过程工程研究所. 中国科学院过程工程研究所. 2008. |
入库方式： OAI收割
来源：过程工程研究所
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