The Study On Histology, Cytology And Enzyme Chemistry Of Sepiella Maindroni Digestive System

Chlorella is a common unicellular green alga, which belongs to the class Trebouxiophyceae and theorder Chlorellales. Chlorella is not only a health food for human and excellent feed for aquaculture, butalso has applied widely in many fields such as medicine, food, chemical industry, environment and ecology.The species Chlorella distribute widely in the nature and grow rapidly. It can not only grow in autotrophy,but also can be cultured in heterotrophic condition, so it is one of the best materials for photosynthesisinvestigation.Ribulose-1,5-bisphosphate carboxylase/oxygenase(EC4.1.1.39, abbreviations as Rubisco) is a keyenzymes for photosynthetic carbon assimilation. Its activity is an important factor for the efficiency ofcarbon assimilation, but its activity is regulated by Rubisco activase (RCA). In this study, the gene of rcawhich encodes Rubisco activase in Chlorella pyrenoidosa820was cloned and analyzed. Moreover, thetranscription level relations between the rca and rbcS (small subunit of Rubisco) under differentenvironmental factors were studied by Real-time PCR. This study has an important meaning of Rubiscoactivase’s structure and function in Chlorella, and provides reference of the mechanism of the activity ofRubisco.Firstly, two rca sequences in C. pyrenoidosa820was acquired by degenerate primers. One was a482bp cDNA sequence (named rca-1) and another was a561bp DNA sequence (named rca-2). The fulllength of3656bp rca-1and3200bp rca-2genes were got by genomic walking technology. The gene ofrca-1included10exons,9intron, and the putative "-35" element,"-10" element,“TATA-box”,“GC-box”,“CAAT-box” were also found in the5’-upstream sequence. The gene of rca-2was just one copy in C.pyrenoidosa820through Southern blot method.Then, the full length of1703bp rca-1sequence were obtained by the RACE method, which containeda1242bp open reading frame, a66bp5′-untranslated region and a395bp3′-untranslated region. Sequencecomparison showed that its homology with other green algae reached to78-85%. Sequence analysisshowed that the rca gene preferred to use the codons ended with G or C or T, and the putative isoelectricpoint and molecular weight was8.44and45.71kDa, respectively.Finally, the expression profiles of rca and rbcS genes from C. pyrenoidosa820under different lighttime, different salt and salicylic acid concentration were detected through the relative standard curvesquantitative analysis (the18S rDNA as the internal reference) by the real-time quantitative PCR. Theresults showed that rca and rbcS transcription level decreased with the light duration. And the mRNAquantities of the two genes varied a little under different salinity, except that rca mRNA reached to2.69fold in the37.5‰salinity. The expression quantities of rca and rbcS were inhibited by the addition of1.0to3.0mmol/L salicylic acid.