豆血红蛋白基因lba的克隆及其转化衣藻叶绿体

降低细胞内的氧气含量是提高莱茵衣藻产氢效率的重要手段之一。本研究首次尝试将豆血红蛋白基因lba转入衣藻叶绿体中表达,利用豆血红蛋白具有与氧可逆结合的特性,期望降低转基因衣藻细胞内的氧气含量,达到提高衣藻产氢效率的目的。实验结果证明,lba成功转入到衣藻叶绿体中,且对其生长没有产生显著影响,这为下一步调控Lba在衣藻叶绿体中表达活性和提高衣藻产氢效率奠定了实验基础。     

谷氨酸废液培养莱茵衣藻的产氢研究

通过在一般培养基中添加尿素、谷氨酸、葡萄糖等有机物,考察了菜茵衣藻混合营养培养的产氢效果。结果表明,含尿素混合营养培养菜茵衣藻产氢是含铵氮的培养基培养的6倍,无硫含尿素的混合营养培养具有最佳的产氢效果,是有硫含铵氮的培养基的10倍。谷氨酸及葡萄糖的添加对其生长和产氢也有促进作用,对菜茵衣藻生长和产氢促进作用的尿素的最佳添加量为0．25g/L,谷氨酸的最佳添加量为0．7g/L,葡萄糖的添加量为0．2g/L。     

同步化培养后莱茵衣藻生物量和总RNA含量的变化

为了探索莱茵衣藻经光/暗同步化培养后的细胞生长和总RNA的变化规律。本研究检测了16h光/8h暗同步化培养后莱茵衣藻的生物量和总RNA含量的变化规律。结果,在同步化培养结束后的前28h,莱茵衣藻的生物量呈现有节律的阶梯增长;在同步化培养结束后的28～48h,这种阶梯式增长方式逐步消失。在同步化培养结束后的前24h,总RNA含量呈现有节律的峰-谷-峰变化;在同步化培养结束后的24～48h,这种变化幅度逐步减小,节律周期也逐步缩短。对比同步化培养后莱茵衣藻生物量和总RNA含量的变化可以得出,同步化培养后莱茵衣藻的同步化节律仍然可以维持一定时间;但随着连续光培养时间的延长,这种节律逐步消失,通过测定生物量和总RNA含量的变化可以跟踪同步化培养后莱茵衣藻的同步化变化。     

衣藻的藻种保存`扩大培养和观察

衣藻属(Chlamydomonas)是常见的淡水绿藻。在生长季节里,衣藻常在营养特别是氮、磷较丰富的坑塘中有时长成纯群,但在池水或缓流中则常与其他藻类等混生,不易采集。在非生长季很难找到它们。这给教学和科研带来许多困难。几年来,我们对衣藻的藻种保存、扩大培养进行观察试验,总结出一些经验,现介绍如下:     

伊蚊Ⅴ-ATPA基因RNAi干涉载体构建及其转基因衣藻对伊蚊致死作用

蚊媒传病严重危害全世界人类的生命健康,其中,伊蚊在传播疾病方面扮演着十分重要的角色,是登革热、寨卡病毒病、黄热病、基孔肯雅病的媒介昆虫.因此,对伊蚊的控制在防治上述传染病中起着重要的作用.本项研究构建了伊蚊Ⅴ-ATPA基因的RNAi表达载体pMaa7IR/Ⅴ-ATPAIR,通过玻璃珠法转化莱茵衣藻(Chlamydomo...     

转基因莱茵衣藻hemHc-lbac 和根瘤菌共培养提高产氢培养条件的优化

转基因莱茵衣藻hemHc-lbac(transgenic Chlamydomonas reinhardtii hemHc-lbac)以不同比例与日本慢生大豆根瘤菌(Bradyrhizobium japonicum)混合, 在不同光照条件下进行产氢培养, 以确定产氢的最优条件和探索产氢提高的机理。结果表明藻菌共培养的最优产氢条件为25 ℃、光照30 μE⋅m–2⋅s–1、生长至饱和期的菌和藻体积比为1: 80, 产氢量达到最大, 约为278 μmol⋅mg–1Chl, 是对照组80 μmol⋅mg–1Chl 的3.5 倍。藻菌共培养提高产氢量的主要原因是体系中氧气浓度的降低而使氢化酶活性提高、以及衣藻生物量的增加。该研究为利用藻菌共培养及转基因的方法提高微藻光合生物制氢效率提供了重要实验基础。     

莱茵衣藻Nfr-4突变株中类胡萝卜素含量的变化及其对藻生长的影响

文章对相同条件下培养的莱茵衣藻野生型CC-137和八氢番茄红素脱氢酶(phytoene desaturase,PDS)基因突变株Nfr-4的生长进行分析;并用反相高效液相色谱分析总有色类胡萝卜素以及叶绿素含量变化,结果表明两者生长的差异明显;Nfr-4突变株的单细胞叶绿素和总有色类胡萝卜素含量高于野生型CC-137的。     

不同培养条件下莱茵衣藻细胞中SGAT酶活性的变化

利用模式单细胞植物莱茵衣藻,研究不同培养条件下细胞中丝氨酸：乙醛酸氨基转移酶活性的变化情况。结果表明：莱茵衣藻SGAT酶活性的最适pH介于5￣7之间,当pH高于7以后,酶活性逐渐下降;随着细胞密度增加,SGAT酶活性降低;光强可显著影响SGAT酶活性,在一定光强范围内,随着光照强度的增加,酶活性增强;乙酸作为莱茵衣藻的唯一异养碳源也会影响SGAT酶活性,两者间呈正相关;提高氧浓度,显著地提高了细胞内SGAT的酶活性;当二氧化碳浓度增加时,细胞内SGAT的酶活性也略有升高;40℃高温和15℃低温处理后,SGAT酶活性均降低。此外,提高氧浓度时细胞内Gly含量增加,Ser含量减少,Gly/Ser的比值从0.79提高到1.49。     

连续光照转暗培养联合药物处理实现衣藻细胞的高水平同步化

单细胞衣藻(Chlamydomonas)是光合作用和植物细胞周期等生物学过程研究的一个重要模式系统, 同步化培养是进行相关研究的必要手段。该研究探索了连续光照转暗培养联合细胞周期阻断剂实现莱茵衣藻(Chlamydomonas reinhardtii)细胞高水平同步化的新方法, 并利用流式细胞术对同步化程度进行了精确的分析。结果表明, 连续光照转暗培养或联合S期阻断剂可以使衣藻细胞同步化到G1期或G1/S期边界; 连续光照转暗培养联合M期阻断剂或者在“加入-释放”S期阻断剂后再加入M期阻断剂可以使衣藻细胞同步化到M期, 同步化水平可达80%。具体的同步化培养步骤要根据研究对象(特别是某些衣藻突变株系)的特性和研究目的确定。     

转基因稻及其非转基因亲本对照在间隔种植条件下的转基因漂移

随着转基因技术的迅速发展, 越来越多的转基因作物被培育出来。转基因作物的外源转基因通过花粉传播向非转基因作物的漂移, 会影响非转基因作物品种的种子纯度, 从而可能导致一系列生物安全问题。为了研究转基因栽培水稻(Oryza sativa)中的外源转基因通过花粉介导向非转基因水稻品种逃逸的可能性及其频率, 我们选用3个含双价抗虫基因(Bt/CpTI)的转基因水稻品系及其相对应的非转基因水稻亲本品种(近等基因系)进行了转基因漂移的实验。为了获取在近距离状况下转基因水稻与非转基因水稻品种之间的转基因漂移频率, 采用了转基因与非转基因水稻品种间隔种植的栽培方式, 分别在福建省福州市和海南省三亚市的转基因环境安全实验地进行实验, 并利用潮霉素抗性筛选标记基因来鉴定转基因和非转基因稻的杂种。共检测了从非转基因水稻品种随机收获的70,056颗种子, 以此计算转基因漂移频率。结果表明, 在相邻种植的情况下, 由这3个转基因水稻向对应的非转基因水稻品种的转基因漂移的频率比较低(0.275–0.832％)。如此近距离条件下获得的低转基因漂移频率表明, 对于严格自花授粉的水稻而言, 通过一定的隔离措施, 能有效地降低由花粉介导的转基因漂移导致的非转基因种子混杂。     

衣藻生物制氢的研究进展

综述了利用衣藻生产氢气作为再生能源的研究进展。分别介绍了衣藻产氢的代谢机理、培养条件、衣藻氢化酶的特性以及利用分子生物学手段、生物信息学手段和生物工程技术提高衣藻生物制氢效率的方法,包括氢化酶的氧耐受性的改造、外源氢化酶基因的表达、影响衣藻产氢的关键基因的筛选、利用缺硫培养基和固定化培养方法提高氢气产量等。最后,还对利用衣藻生物制氢的可行性和经济性进行了分析,对其发展方向提出自己的看法。     

外源基因在莱茵衣藻叶绿体中的表达

把莱茵衣藻(Chlamydomonas reinhardtii)叶绿体作为生物反应器来表达外源基因具有广阔的应用前景。人们利用莱茵衣藻叶绿体表达体系已成功表达多种重组蛋白,其中包括人类药用蛋白。综述了莱茵衣藻叶绿体转化的方法、影响外源基因表达的主要因素以及外源基因在莱茵衣藻叶绿体表达研究进展。     

Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene （apcA and apcB） into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.     

Recombination and Heterologous Expression of Aliophycocyanin Gene in the Chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires theintroduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins ormetabolic pathways.In order to accomplish the expression of multiple genes in a single transformationevent,we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonasreinhardtii chloroplast expression vector,resulting in papc-S.The constructed vector was then introducedinto the chloroplast of C.reinhardtii by micro-particle bombardment.Polymerase chain reaction and Southernblot analysis revealed that the two genes had integrated into the chloroplast genome.Western blot andenzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria couldbe correctly expressed in the chloroplasts of C.reinhardtii.The expressed foreign protein in transformantsaccounted for about 2%-3% of total soluble proteins.These findings pave the way to the reconstitution ofmulti-subunit proteins or metabolic pathways in transgenic C.reinhardtii chloroplasts in a single transformationevent.     

盐胁迫状态下莱茵衣藻849光合特性的初步研究

以莱茵衣藻（Chlamydomonas reinhardtii）为研究对象,对其在不同NaCl浓度胁迫条件下的光合特性进行了初步研究。结果表明0.04 mol·L^-1的NaCl对莱茵衣藻的生长无显著影响,而0.075 mol·L^-1的NaCl使莱茵衣藻生长速率下降了50%。在低光照强度下,NaCl胁迫下的衣藻光合速率和呼吸速率在最初7 h内比对照组高;而在较高光照强度下,NaCl胁迫下的衣藻的光合速率和呼吸速率均比对照组低;而且光合速率和呼吸速率升高和下降的幅度与NaCl浓度成正比。     

Hydrogen production by photoautotrophic sulfur-deprived Chlamydomonas reinhardtii pre-grown and incubated under high light

We have previously demonstrated that Chlamydomonas reinhardtii can produce hydrogen under strictly photoautotrophic conditions during sulfur deprivation [Tsygankov et al. (2006); Int J Hydrogen Energy 3:1574-1584]. The maximum hydrogen photoproduction was achieved by photoautotrophic cultures pre-grown under a low light regime (25 microE m(-2) s(-1)). We failed to establish sustained hydrogen production from cultures pre-grown under high light (100 microE m(-2) s(-1)). A new approach for sustained hydrogen production by these cultures is presented here. Assuming that stable and reproducible transition to anerobiosis as well as high starch accumulation are important for hydrogen production, the influence of light intensity and dissolved oxygen concentration during the oxygen evolving stage of sulfur deprivation were investigated in cultures pre-grown under high light. Results showed that light higher than 175 microE m(-2) s(-1) during sulfur deprivation induced reproducible transition to anerobiosis, although the total amount of starch accumulation and hydrogen production were insignificant. The potential PSII activity measured in the presence of an artificial electron acceptor (DCBQ) and an inhibitor of electron transport (DBMIB) did not change in cultures pre-grown under 20 microE m(-2) s(-1) and incubated under 150 microE m(-2) s(-1) during sulfur deprivation. In contrast, the potential PSII activity decreased in cultures pre-grown under 100 microE m(-2) s(-1) and incubated under 420 microE m(-2) s(-1). This indicates that cultures grown under higher light experience irreversible inhibition of PSII in addition to reversible down regulation. High dissolved O(2) content during the oxygen evolving stage of sulfur deprivation has a negative regulatory role on PSII activity. To increase hydrogen production by C. reinhardtii pre-grown under 100 microE m(-2) s(-1), cultures were incubated under elevated PFD and decreased oxygen pressure during the oxygen evolving stage. These cultures reproducibly reached anaerobic stage, accumulated significant quantities of starch and produced significant quantities of H(2). It was found that elevation of pH from 7.4 to 7.7 during the oxygen producing stage of sulfur deprivation led to a significant increase of accumulated starch. Thus, control of pH during sulfur deprivation is a possible way to further optimize hydrogen production by photoautotrophic cultures.     

丙肝病毒融合抗原基因NS3-C定点整合入衣藻叶绿体基因组的研究

用ＰＣＲ 方法从丙型肝炎病毒（ＨＣＶ） ｃＤＮＡ 文库中克隆了两段ＤＮＡ 片段,即ＨＣＶ 基因组非结构ＮＳ３区抗原基因（约０．７ ｋｂ）和核心抗原Ｃ区抗原基因（约０．６ ｋｂ）的ｃＤＮＡ 片段。在两段ｃＤＮＡ 间加入连接肽Ｓｅｒ－ Ｐｒｏ－ Ｇｌｙ－ Ｓｅｒ 的密码子序列,构建成融合抗原基因ＮＳ３－ Ｃ。将该融合基因与衣藻叶绿体基因ａｔｐＡ 的启动子和ｒｂｃＬ 基因的３′末端连接,得到丙肝病毒融合抗原基因ＮＳ３－ Ｃ表达盒,再将该表达盒与选择标记基因ａａｄＡ 表达盒和衣藻叶绿体基因组同源片段连接,构建成衣藻叶绿体转化载体ｐＳＳ６。基因枪法转化衣藻叶绿体,经壮观霉素筛选获得转化再生的单藻落,对转基因衣藻的ＰＣＲ 和Ｓｏｕｔｈｅｒｎ 杂交分析表明,融合抗原基因ＮＳ３－ Ｃ已整合到衣藻叶绿体基因组中。