Construction Of Antisense RNA Expression Vector Of Chlamydomonas Reinhardtii Containing Sulfate Transparent Gene

With the world wide shortage of energy, looking for renewable energy has become important. One of an important renewable energy is hydrogen. Recent studies have found that hydrogen production of Chlamydomonas reinhardtii will be greatly improved in sulfur deficiency state. However, this method can cause great harm to Chlamydomonas reinhardtii cells. In this study, Chlamydomonas reinhardtii anti-S’ulP2（sulfate transparent gene2） RNA expression vector was constructed. The results show that it could cause Chlamydomonas reinhardtii SulP2gene silence when the vector is introduced into Chlamydomonas reinhardtii cells. We hope that the hydrogen production effect in the transformed Chlamydomonas reinhardtii cells cultured in normal medium may be similar to the Chlamydomonas reinhardtii cultured in sulfur deficiency medium. The main contents and results are as follows:（1） Chlamydomonas SulP2partial fragment （about700bp） was cloned using the method of PCR. The sequencing results showed that the sequence of the gene is correct.（2） To construction of antisense expression vector pCh-sulp2CDSR, the partial SulP2gene was cloned into Chlamydomonas reinhardtii expression vector pChlamy₃in reverse orientation, controlled by Hsp70A-RbcS2promoter and RbcS23’ UTR terminator. The sequencing result showed that the open reading frame in the vector is correct.（3） The pCh-sulp2CDSR vector was transformed into Chlamydomonas reinhardtii cells by the method of electroporation. After screening by hygromycin, the transformation was detected using PCR method. The result showed that the sulp2gene had been transformed into Chlamydomonas cells.（4） The kinetic curves of transgenic Chlamydomonas reinhardtii （cultured in normal medium and sulfate-deficient medium） and wild type Chlamydomonas reinhardtii （cultured in normal medium and sulfate-free medium） was determined. The results showed that the biomass of transgenic Chlamydomonas reinhardtii and wild type Chlamydomonas reinhardtii vizs nearly same in the same cultured medium.（5） The mRNA levels of Rbcs, Rbcl, Atsl, Apr, Cys and Sir were measured using QRT-PCR method. The results showed that the mRNA levels of Rbcs and Rbcl in transgenic Chlamydomonas reinhardtii decreased slightly compared to wild-type Chlamydomonas reinhardtii. The mRNA levels of Atsl, Apr, Cys and Sir in transgenic Chlamydomonas reinhardtii increased significantly compared to wild-type Chlamydomonas reinhardtii.（6） The amount of hydrogen in wild-type and transgenic Chlamydomonas reinhardtii in TAP-S100, TAP-S70, TAP-S medium were measured. The results showed that comparing to wild-type algal, the hydrogen production in transgenic algal was improved significantly.