雨生红球藻和小球藻间的原生质体融合与糖异养融合子筛选

Cell Fusion between Haematococcus pluvialis and Chlorella kessleri and Screening of Glucose-heterotrophic Fusants

为了增强雨生红球藻的异养生长能力,提高规模化培养效率,本研究基于PEG介导的原生质体融合技术,在雨生红球藻SCCAP K-0084和小球藻SAG211.11a间开展了细胞融合以及糖异养融合子筛选研究,筛选中通过限制性糖异养条件和添加潮霉素分别抑制野生型的雨生红球藻和小球藻的生长,提高筛选效率。结果表明,小球藻SAG211.11a具有极强的异养生长能力,能够利用葡萄糖、果糖、乙酸钠、甘油、苹果酸以及琥珀酸进行异养生长,而雨生红球藻则仅能够利用乙酸盐进行异养生长,此外雨生红球藻SCCAP K-0084还具有极强的潮霉素耐受能力,可以被应用于融合子筛选中。研究中,雨生红球藻和小球藻的原生质体制备效率分别达到72.11%±3.94%和42.07%±3.73%,满足细胞融合需要,融合过程中观察到雨生红球藻和小球藻间的典型融合现象,并且在限制性糖异养筛选下获得了雨生红球藻形态的融合子克隆,融合子的生长速率相比于野生型雨生红球藻有明显的增强,三个融合子在10 d培养后细胞密度分别达到6.7×10~4、8.7×10~4、6.5×10~4个/mL,明显高于野生型的2.45×10~4个/mL,但是相比于小球藻10 d后1.4×10~6个/mL的细胞密度而言,异养生长能力仍然有限,而且在连续传代过程中融合子经常会出现表型分化的现象。这一研究证明,通过细胞融合杂交的方式能够使雨生红球藻获得糖异养的生长能力,但是异源基因组间重组效率有待进一步提高。

To improve heterotrophic growth ability of Haematococcus pluvialis,improve the efficiency of large-scale cultivation,cell hybridization and fusant screening between H.pluvialis SCCAP K-0084 and heterotrophic Chlorella SAG211.11a were carried out through PEG-mediated protoplast fusion.A screening condition only permiting heterotrophic growth based on glucose along with addition of hygromycin was utilized to improve the screening efficiency,which could inhibite growth of wild-typle H.pluvialis and C.kessleri,respectively.Results showed that,Chlorella kessleri SAG211.11a had strong heterotrophic growth capacity,which could metabolite glucose,fructose,acetate,glycerol,malic acid and succinic acid in its heterotrophic growth,while three H.pluvialis strains could just utilize acetate for its growth.And H.pluvialis SCCAP K-0084 exhibited strong hygromycin-resistant ability.These characters could be applied in the process of fusant screening.Protoplast-releasing rates respectively reached 72.11%±3.94%and 42.07%±3.73%in H.pluvialis and C.kessleri.And fusants with Haematococcus form were obtained on the screening condition only permiting heterotrophic growth on glucose after effective cell fusion identified through microscopic check.These fusants had faster growth than wild type H.pluvialis in the condition with glucose as substrates.After 10 d cultivation,the cell densities respectively arrived at 6.7×10^4,8.7×10^4 and 6.5×10^4 mL-1 in three independent fusants,obviously higher than 2.45×104 in wild type H.pluvialis.However,they were obviousy lower than 1.4×10^6 mL-1 in C.kessleri,which meant a low-efficient heterotrophic growth on glucose.It was also found that,many fusants were easy to lose their capacity of glucose utilization with extended inoculation.That might be due to inefficient recombination between two genomes.