【作者】 柴玉荣；

【导师】 薛乐勋；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2005， 博士

【摘要】 杜氏盐藻(Dunaliella salina, D. salina)属于绿藻门绿藻纲团藻目,是一种生长在海水或盐湖的嗜盐性浮游单细胞真核绿藻。细胞形态呈梨形或椭圆形,无细胞壁,胞内有一个大的杯状叶绿体,具有双鞭毛,可以在水中游动。该藻的突出特点是耐盐能力强,抗逆性极佳,可在0.05M-5M的盐水中生长,是迄今所发现的最耐盐的生物之一。 杜氏盐藻作为宿主表达外源基因,生产具有药用价值的蛋白质如血管抑素等,具有繁殖速度快、培养简单、表达产物下游纯化过程简单等优越性。在利用杜氏盐藻作为生物反应器生产药用蛋白质、口服疫苗等时,为使目的基因得到高效表达,必须寻找高活性的杜氏盐藻内源性启动子,建立杜氏盐藻的高效表达载体,使外源基因得到高水平表达,从而获取更多的具有治疗作用的外源蛋白质。 1,5-二磷酸核酮糖羧化酶/加氧酶(Ribulose 1,5-bisphosphate carboxylase/oxygenase, E.C.4.1.1.39简称RuBisCO)是光合生物进行光合作用的关键酶,催化光合作用中的羧化反应和氧合反应,由8个大亚基(RbcL)和8个小亚基(RbcS)组成。该酶是植物可溶性蛋白中含量最高的蛋白质之一,约占50%。因此,对于RuBisCO的研究具有重要的理论意义和应用价值。在转基因研究中,RuBisCO的RbcS基因启动子作为高活性的光调控型启动子在高等植物和藻类的遗传转化中应用较多。 本研究首先以简并引物PCR和RACE(Rapid amplification of cDNA ends, cDNA末端快速扩增)方法从杜氏盐藻中克隆了RuBisCO的小亚基基因(RbcS)的全长cDNA序列。根据此cDNA序列信息,设计位于RbcS基因上游的特异引更多还原

【Abstract】 Dunaliella salina （D. salina）, an autotropic unicellular eukaryotic green alga, which belongs to Chlorophyta, Chlorophyceae, Volvocales, has a thin cellular membrane, but lacks of rigid cell wall, and a single, large cup-shaped chloroplast with photosynthetic thylakoid membranes, pyreniod and abundance β -carotene globules. It is one of the most extremely halotolerant eukaryotes and can live in a variety of salt concentrations ranging from 0.05 to 5M solution of sodium chloride.The ease and cheap culture of D. salina make it a desirable host for production of natural compounds by genetic engineering or for exploitation as biological factories for the synthesis of novel high-value therapeutic compounds. However, the lack of efficient genetic transformation system has been one of major bottlenecks in the genetic manipulation of this microalga.Ribulose-1, 5-bisphosphate carboxylase/oxygenase （RuBisCO） is a bi-functional enzyme that catalyzes the initial step of photosynthetic carbonic reduction and photo-respiratory carbon oxidation cycles by combining CO₂ and O₂, respectively. RuBisCO as a hecadecamer is composed of eight large subunits （RbcL） and eight small subunits （RbcS） that assemble into a L8S8 holoenzyme in many eubacteria, cyanobacteria, algae and higher plants. The small subunits are encoded in the nucleus as precursors, then imported into the chloroplast and processed there. The promoter of RbcS has been showed to be a strong cis-acting element to drive the heterogeneousgene expression in algae and higher plants. In the present study a full-length cDNA of RbcS from D. salina was cloned and characterized. Then the 5’ franking regulatory region of RbcS was further isolated and used for trans-genetic study in D. salina.I Cloning and characterization of the RbcS genes from D. salinaMethods and Results1.1 Cloning of RbcS cDNA from D. salinaThe small subunit gene of ribulose-l,5-bisphosphate carboxylase/ oxygenase （RuBisCO） was obtained from D. salina. A pair of degenerate primers were designed according to conserved motifs ofETFSYLPPandMWKLPMFGofthe known RbcS and were used to amplify a cDNA fragment of RbcS from the total RNAs of D. salina treated with TRIzol.The resulting PCR product was cloned into pMD18-T vector and screened to determine its sequence. Homologous analysis of the deduced amino acid sequences was performed by BLAST and subsequently compared with GenBank data. An obtained cDNA sequence was 209 bp in length, which encodes 69 amino acids. The amino acid sequence shared high identify with other RbcS: 85 % to Volvox carteri, 84 % to Chloromonas sp. ANT3, 82 % to Chlamydomonas reinhardtii, 76 % to Acetabularia cliftonii, 70 % to Spinacia oleracea, 69 % to Zea Mays, respectively.According to the known cDNA sequence of RbcS from D. salina, 5’RACE and 3’RACE were performed to obtain 5’ and 3’ ends sequences of RbcS cDNA. Nucleotide sequence analysis and amino acid sequence BLAST indicated that the full-length RbcS cDNA from D. salina was long 570 bp, encoding 190 amino acids.1.2 RbcS gene was subcloned into the prokaryotic expression vector and expressed in E. coli BL21The full-length cDNA encoding the small subunit （RbcS） of ribulose-l,5-bisphosphate carboxylase/ oxygenase （RuBisCO） from D. salina was cloned by the method of reverse transcription polymerase chain reaction （RT-PCR）. A prokaryotic expression vector pET-S was constructed by inserting the cDNA of RbcS into the vector pET-30a（+） and transformed into E. coli BL21. Through expressionanalysis of the RbcS fusion protein by SDS-PAGE, a new specific band withmolecular weight of about 26 kDa was found when the bacteria were induced withIPTG13 Analysis of RbcS geneThe expression pattern of RbcS from D. salina was also analyzed by real time quantitative PCR and Southern blot. After cultured in darkness for 12h and in light for 4 and 9h, respectively, total RNAs were extracted from the cells of wild type D. salina and reverse-transcripted into cDNAs for PCR, respectively. The house keeping gene P -actin gene from D. salina was used as the inner control. The results demostrated that the expression of RbcS from D. salina cultured in light for 9h was enhanced compared with what that cultured in darkness for 12h. This result was consistent with the previous reports that the expression of RbcS can be induced by light, suggesting that the 5’upstream regulatory region of RbcS from D. salina may be one of light regulatory elements.Southern bolt showed that the gene family of RbcS from D. salina consisted of at least two gene members of RbcS gene.II Cloning and identification of the 5’ flanking region of RbcS gene from D. salinaMethods and Results2.1 Cloning and analyzing the 5’ flanking region of RbcSAccording to cDNA sequences of RbcS, the 5’ upstream regulatory region of RbcS was further cloned using genomic walking technique and its structural feature was characterized as well.The genomic DNAs from D. salina were digested with Dra I, EcoR V, Pvu II and Stu I, respectively. Genomic walking adapter were ligated to the ends of the digested DNA fragments. Genomic walking libraries including GWL1, GWL2, GWL3 and GWL4 were respectively constructed. The 5’ flanking region of RbcS from Genomic walking libraries of D. salina was amplified by the method of nested PCR and sequenced. A single specific PCR product of about 1.2 kb from the GWL1and GWL4 was generated, respectively. The partial sequences of 3’-end in the 1.2kb fragment were completely identical with the partial sequences of 5’-end of the cDNA of RbcS gene. Several conserved promoter motifs, such as TATA-box, CAAT-box, etc, and the GT tandem repeats were also found in this fragment. Therefore, the 5’ flanking sequence isolated from the 5’upstream of RbcS gene might be a promoter of RbcS gene from D. salina.2.2 Activity analysis of the 5’ flanking region of RbcS by transienttransformation2.2.1 Construction of transgenic expression vectors for D. salinaThe cloning vectors pMD-SPl （containing the 0.8 kb 5’ flanking region of RbcS gene and GT tandem repeats）, pMD-SP2 （including the 0.5 kb 5’ flanking region of RbcS gene without GT tandem repeats） and the expression vector pMDC-B （containing the promoter of carbonic anhydrase from D. salina and the bar-nos polyA fragment ） were digested with EcoRI, respectively. The fragments of the vectors and bar-nos polyA from pMDC-B were recovered on the gels, respectively. The phosphoric acids of 5’-ends were removed from the fragments of the vectors pMD-SPl and pMD-SP2 with calf intestine alkaline phosphatase （CIAP）. The bar-nos polyA fragment was fused to the fragments of the vectors pMD-SPl and pMD-SP2 to creat the transgenic expression vectors pSP-Bl and pSP-B2 for D. salina.2.2.2 Transformation and screening of D. salinaThe expression vectors pSP-Bland pSP-B2 containing the 0.8 kb and 0.5 kb of 5’ flanking regions of RbcS and the vector pMDC-B consisting of the promoter of carbonic anhydrase from D. salina were respectively transformed into the cells of wild type D. salina by electroporation and used to direct expression of the bialaphos resistance gene （bar） from Streptomyces hygroscopicus, a dominant selective reporter gene in the transformed D. salina. Direct selection of bar resistant transformants was achieved by allowing recovery in dark culture for 12h, light culture for 24h and following 3 weeks under standard culture condition prior to harvesting and plated on the solid medium containing 3.0 u g/ml of PPT. Then the clones of resistant transformants were picked into lipid medium for further culture. The cells oftransformed D. salina can grow better in the medium containing about 3.0 u g /ml PPT but not the cells of wild type D. salina, suggesting that the promoter of RbcS could drive the bar gene to express in the transformed D. salina. 2.23 Analysis of transformed D. salina2.23.1 PCR analysisA pair of primers were designed according to the bar gene sequence. Existence of the bar gene in the genome of the transformed D. salina was verified by PCR. The results of PCR indicated that a specific band of bar gene with about 500 bp was amplified from the transformed D. salina. In contrast, there was no amplification product from the wild type D. salina, demonstrating that the bar gene has been integrated into the genome of D. salina.The mRNA accumulations of the bar gene from obtained different transformants were compared by RT-PCR when eletroporated with the expression vectors pSP-Bl, pSP-B2 and pMDC-B. The results revealed that the mRNAs of the bar gene driven by the 0.8 kb and 0.5 kb 5’upstream regulatory regions of RbcS gene accumulate much more than that by the promoter of carbonic anhydrase, which exhibiting that the promoter of RbcS gene had a relatively powerful transcription regulatory activity.2.23.2 Southern blotThe integration patterns of chimeric gene carrying bar were analyzed by Southern blot. The DNAs of bar gene labeled by random primers with Digoxigenin-11-dUTP were used as the probe for hybridization on the nylon membrane blotted the digested genomic DNAs from the transformed D. salina. The signal intense of hybridization was detected by immunological technique. Results detailed of hybridization demonstrated that the bar gene had been integrated into the genome of the transformed D. salina.Id Conclusion1A full-length cDNA of RbcS gene of 812 bp in length has first been obtained from D. salina, which containing a complete open reading frame encoding 190 amino acids residues. The putative amino acid sequence of RbcS shares high homology with that from other organisms.2 The RbcS gene from D. salina has a higher GC content and higher GC preference at the third codon position.3 A prokaryotic expression vector carrying the RbcS gene has been constructed and the RbcS fusion protein can be expressed successfully in Rcoli BL21.4 A promoter of the RbcS gene has been isolated and it might be a light regulatorypromoter with relatively higher activity and safety in the transgenic D. salina.5 Electroporation may be a suitable and efficient method for nuclear transformation of D. salina.更多还原