【作者】 刘红全；

【导师】 戴继勋； 于文功；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2004， 博士

【摘要】 紫菜是我国海藻养殖业的支柱产业之一也是主要的出口创汇海产品。近年来，随着紫菜栽培业规模的逐年扩大，对紫菜优良品种的供应要求越来越高。紫菜易于养殖，藻体形态结构简单，细胞发育分化易于调控，酶解紫菜产生原生质体及原生质体的再生技术已经成熟，易于进行分子水平上的研究。因此，开展紫菜基因工程研究，利用现代分子生物学的手段培育紫菜新品种是十分必要的，也具备现实可行性。同时，也有望将紫菜作为生物反应器来生产有用的外源蛋白。为此，我们以条斑紫菜的原生质体为受体，进行转基因研究，初步建立其转基因体系。 纯系培育是传统紫菜育种及进行紫菜生物工程研究的先决条件。本研究选取青岛地区野生条斑紫菜(Porphyra yezoensis)，利用酶解制备原生质体进行再培养的方法培育了营养体纯系PY-qingdaol。对PY-qingdaol的18S rRNA基因进行了克隆和序列测定。将该基因与从GenBank获得的22个紫菜进行了分析、比较，并以相邻连接法构建了紫菜较为一致的系统发生树。结果表明利用紫菜18S rDNA基因序列差异可较精确的进行紫菜种间乃至品系的划分，这为进行紫菜的种质鉴定提供了一个新的思路。 为确立电击法转化条斑紫菜原生质体的条件、确定有效的启动子并验证以18S rDNA为外源基因整合位点的可行性，构建了以条斑紫菜18S rDNA片段为同源臂、CaMV35S为启动子、GUS基因为报告基因的同源重组型表达载体，并利用电击法对外源GUS基因在条斑紫菜原生质体瞬间表达进行了初步探索。实验结果表明CaMV35S启动子能够在条斑紫菜原生质体中启动GUS基因的瞬间表达；电击法是条斑紫菜基因转移的有效方法；当电场强度为2KV／cm，脉冲时间为300ms，脉冲次数为1次时，外源基因的转化效率较高；以18S rDNA片段为同源臂的同源重组型载体可以提高GUS基因在条斑紫菜原生质体中的瞬时表达。 为确定合适的选择性标记基因，以条斑紫菜18S rDNA片段为同源臂，构建了分别利用SV40启动子和CaMV35S启动子的两种cat基因的同源重组型表达载体pQD一CAT一control和pQD一CAT一Enhancer。利用电击法将所构建的表达载体转化条斑紫菜原生质体。结果表明，SV40启动子和C咖V35S启动子均可以驱动cat基因在条斑紫菜原生质体中有效表达;所构建的以185 rDNA片段为同源臂的两种同源重组型载体均可实现cat基因在条斑紫菜原生质体中的稳定表达;当以cat基因作为选择性标记基因时，氯霉素可以作为紫菜原生质体基因转化的选择压力。 对小鼠的7天为IL cDNA，进行了克隆和序列测定，并构建了以条斑紫菜185rDNA片段为同源臂、cat基因为标记基因、了万才IL基因为外源基因、分别利用SV4O启动子和CaMV35S启动子的同源重组型表达载体pQD一TRAIL一CAT。利用电击法将其转化条斑紫菜原生质体，经氯霉素初步筛选，检测到外源基因己整合到紫菜基因组中。 进行了条斑紫菜原生质体的玻璃化冷冻保存研究，发现玻璃化保护剂VS6 (10%DMSO，30%甘油，10%蔗糖)的保存效果较好，其最佳冻存程序是:25%VS6过渡处理5 min后，用ooC预冷的VS6处理3 min，直接液氮保存，化冻时采用40oC水浴快速化冻。按此方法，冻存后的原生质体的存活率可达66.5%，并且能再生成叶状体。 总之，本研究初步建立了条斑紫菜原生质体的转基因体系，优化了电击法转化条斑紫菜原生质体的条件，确定了Sv40启动子和CaMV35S启动子为有效的启动子，cat基因可以作为选择性标记基因，以1 85 rDNA片段为同源臂的同源重组型表达载体可以有效的表达外源基因。此外还首次成功地进行了条斑紫菜原生质体的玻璃化冷冻保存，不仅为藻类的种质保存研究积累了资料，也可以为以后紫菜原生质体的遗传转化操作随时提供理想的实验材料。这些研究为进一步通过转基因的手段改良紫菜品种，实现以紫菜为生物反应器来大量生产外源蛋白奠定了重要的基础。更多还原

【Abstract】 Porphyra is the leading cultural species of the seaweed cultivation and the main export sea product in China. Recently, there is an increasing demand for good Porphyra cultivars with the enlargement of Porphyra cultivation scale. Porphyra is feasible for cultivation and its development is easy for regulation. The technique for preparation and regeneration of Porphyra protoplasts is perfect and the protoplast is a good material for research in molecular level. Therefore, it is necessary and viable to develop Porphyra genetic engineering research and breed new Porphyra cultivars via transgenic technique. Porphyra is also expected to be a bioreactor for foreign protein production because of the feasibility of cultivating of this alga. In this study, protoplast was used as receptor for transgenic research in order to construct an effective transgenic system for Porphyra yezoensis.The prerequisite of the traditional breeding and bioengineering research of Porphyra is the construction of pure lines. The wild P. yezoensis was collected from Qingdao and used to prepare protoplasts by enzyme digestion. The pure line PY-qingdaol was constructed by cultivating the protoplasts. The 18S rDNA of the PY-qingdaol was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed based on these sequences using neighbor-joining method. The results indicated the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.In order to establish the eclectroporation condition for P. yezoensis protoplasts transformation, confirm the effective promoter and test the feasibility of 18S rDNA used as foreign gene integration site, a targeting vector pQD-GUS was constructed containing a portion of the 18S rDNA of P. yezoensis and transformed it into the same strain protoplasts by electroporation. The results showed that CaMV35S promotor iseffective for transient expression of GUS gene in P. yezoensis. Electroporation is a useful method for foreign genes enter into P. yezoensis. The good condition for electroporation is that field strength was set at 2KV/cm and pulse length was set at 0.3 ms. GUS gene expression was greatly increased when 18S rDNA was used as homologous sequences to direct homologous recombination.In order to establish the suitable genetic selectable marker gene, two targeting vectors pQD-CAT-control and pQD-CAT-Enhancer were constructed which contained a portion of the 18S rDNA of P. yezoensis and cat gene promoted by SV40 and CaMVSSS respectively. The vectors were transformed into P. yezoensis by electroporation. The results showed that SV40 and CaMVSSS were effective promoters for cat gene expression in P. yezoensis. The targeting vectors containing the portion of the 18S rDNA of P. yezoensis could fulfill the stable expression of cat gene in P. yezoensis. Chloramphenicol could be used as selection pressure for positive transformant when cat gene was used as selectable marker gene.The mouse TRAIL cDNA was cloned and sequenced. A targeting vector pQD-TRAIL-CAT containing the portion of the 18S rDNA of P. yezoensis was constructed with TRAIL as foreign gene and cat gene as selectable marker gene. The vector was transformed into P. yezoensis protoplasts by electroporation 0 It was proved that foreign genes have incorporated into genome of P. yezoensis after preliminary selection with chloramphenicol.Cryopreservation of protoplasts of P. yezoensis by vitrification was studied. The results showed that VS6 (10% DMSO, 30% glycerol, 10% sucrose ) is seemed to be a preferable vitrification solution for P. yezoensis protoplasts. The best vitrification protocol using VS6 involves: loading with 25% VS6 for 5 minutes at 0 癈, dehydrating with ice cold concentrated VS6 for 3 minutes, immediately immersing in LN, and then warming in 40癈 water bath. Using this protocol, P. yezoensis protoplasts showed higher viability (66.5%) after Cryopreservation and could regenerate thallus.hi summary, the tra更多还原