别藻蓝蛋白β亚基基因在莱茵衣藻中重组表达及其对光能传递的影响研究  

Recombinant Expression of theβ-subunit of Allophycocyanin in Chlamydomonas reinhardtii and Its Effect on Light Energy Transfer

作　　者：徐晓婷 臧晓南[1] 章峰 尚孟慧 李瑞[1] 毕莹 赵悦 马艺宁 Xu Xiaoting;Zang Xiaonan;Zhang Feng;Shang Menghui;Li Rui;Bi Ying;Zhao Yue;Ma Yining(Key Laboratory of Marine Genetics and Breeding,Ministry of Education,Ocean University of China,Qingdao 266003,China)

机构地区：[1]中国海洋大学海洋生物遗传学与育种教育部重点实验室,山东青岛266003

出　　处：《中国海洋大学学报（自然科学版）》2023年第9期77-88,共12页Periodical of Ocean University of China

基　　金：国家自然科学基金项目(31872555)资助。

摘　　要：为了探究别藻蓝蛋白与类囊体膜光合系统之间的光能传递规律,及其与异源类囊体膜光合系统之间光能转移的可能性,本研究构建了含有钝顶节旋藻(Arthrospira platensis)的别藻蓝蛋白β亚基基因(apcB),及合成藻蓝胆素的血红素氧化酶基因(ho)和铁氧还蛋白氧化还原酶基因(pcyA)的转化载体pHyg3-apcB-ho-pcyA,利用玻璃珠转化法将其导入莱茵衣藻中。聚合酶链式反应筛选验证阳性转化藻株,Southern Blot结果表明基因apcB、ho、pcyA成功转化入莱茵衣藻基因组中。而后将转化藻株与对照藻株均置于高光(50μmol·m^(-2)·s^(-1))和低光(25μmol·m^(-2)·s^(-1))条件、白光和绿光条件中培养,并检测别藻蓝蛋白β亚基的表达、总叶绿素含量、77 K低温荧光、光系统表观电子传递效率、生物量变化。结果显示:实时定量PCR分析和Western Blot检测表明外源基因apcB,pcyA和ho均在转基因藻株Cr-ApcBHP中得到了转录表达。高光和低光条件下光系统Ⅱ(PSⅡ)和光系统Ⅰ(PSⅠ)位置处的荧光峰均显著高于对照莱茵衣藻藻株CC-849,绿光中PSⅡ荧光峰(波长为690 nm)显著高于对照藻株;高光与低光和白光与绿光条件下转基因藻株的光系统Ⅱ的表观电子传递速率(ETRPSⅡ)值均高于对照藻株,但生物量并未见明显提高。这些结果表明转基因莱茵衣藻中可能存在自重组别藻蓝蛋白到光系统核心的光能传递,但是吸收的光能没有达到促进生物量积累的作用。本研究为利用重组表达蓝藻藻胆蛋白提高异源类囊体膜的光合作用进行了尝试。Allophycocyanin,a photosynthetic antenna protein in cyanobacteria and red algae,consists of an alpha subunit and a beta subunit.In order to investigate the light energy transfer between allochlorophyll cyanobacteria and cystoid membrane photosynthetic systems,and the possibility of light energy transfer between them and heterologous cystoid membrane photosynthetic systems,in this study,the transformation vector pHyg3-apcB-ho-pcyA was constructed with both theβ-subunit gene of allophycocyanin(apcB)of Arthrospira platensis and the heme oxidase gene(ho)and ferric oxide oxidoreductase gene(pcyA)for the synthesis of phycocyanobilin,and was introduced into Chlamydomonas reinhardtii using the glass bead transformation method.Positive clone screening PCR and Southern Blot showed that apcB was successfully transformed into the genome of Chlamydomonas reinhardtii.The transformed and control strains were then incubated in high light(50μmol·m^(-2)·s^(-1))and low light(25μmol·m^(-2)·s^(-1)),white and green light conditions,and examined the expression of theβsubunit of cyanobacteriaa,total chlorophyll content,77 K low temperature fluorescence,photosystem apparent electron transfer efficiency and biomass changes.The results showed that the exogenous genes apcB,pcyA and ho were transcribed and expressed in the transgenic strain Cr-ApcBHP by quantitative PCR and protein electrophoresis.The fluorescence peaks of photosystemⅡ(PSⅡ)and photosystemⅠ(PSⅠ)were significantly higher than those in the control strain CC-849 in both high light and low light conditions,and the PSⅡfluorescence peak(690 nm)was significantly higher in green light than that in the control strain.The apparent electron transfer rate(ETR PSⅡ)values of photosystemⅡwere higher than in the control strain in both high light and low light and white light and green light conditions,but no significant increase in biomass was observed.These results suggest that light energy transfer from recombinant allophycocyanin to the core of the photosystem may ex

关 键 词：别藻蓝蛋白β亚基 钝顶节旋藻 莱茵衣藻 重组表达 光能传递 

分 类 号：Q946[生物学—植物学]