Elsevier

Process Biochemistry

Volume 40, Issue 9, September 2005, Pages 2968-2972
Process Biochemistry

Cloning and structure analysis of hydrogenase gene from Chlamydomonas reinhardtii SE

https://doi.org/10.1016/j.procbio.2005.01.017Get rights and content

Abstract

The cDNA of Chlamydomonas reinhardtii SE encoding hydrogenase (HydA2) was obtained from the total RNA of C. reinhardtii SE by RT-PCR. The DNA of hydrogenase was amplified by PCR from the genomic DNA of C. reinhardtii SE. The cDNA and DNA of hydrogenase were sequenced, respectively. The structure of hydrogenase gene was analyzed by biology software. The open reading frame predicts that the hydrogenase is composed of 3584 bp encoding 505 amino acids in length with a predicted M.W. of 53.69 kDa. Ten exons (including 1518 bp) and nine introns (including 2066 bp) have been found in the hydrogenase, and there were two potential N-glycosylate sites, eight protein kinase C phosphorylation site, eight casein kinase II phosphorylation site and one sulphorylation in the sequence. The theory pI was 6.15. Total number of negatively charged residues (Asp + Glu) and positively charged residues (Arg + Lys) were 55 and 61, respectively.

Introduction

Nowadays, with the rapid increasing of energy wastage, the problem of the decreasing reserves of oil energy sources and environmental pollution have become more and more serious. Therefore, clean and renewable hydrogen has become the new potential resource and translating solar energy to hydrogen energy has been focused on it using the photosynthesis of cyanobacteria and algae [1], [16]. Now the efficiency of synthesizing hydrogen is very low by the use of algae, hydrogenase has a key role in the production of hydrogen photosynthesis. Producing hydrogen from algae may be improved by gene engineering technology [2], [3]. The gene structure and expression of the hydrogenase has been studied. So far, hydrogenase genes of many algaes have been reported, including hydA of the Scenedesmus obliquus, hydA of the Chllorella fiscal, hydA1 of C. reinhardtii, and hydA2 of CC-425 [4], [5], [6], [7]. There were many reports about hydA2. The cDNA and DNA of hydA2 from C. reinhardtii CC-425 had been reported by Forestier et al. The cDNA of hydA2 from C. reinhardtii 21gr had been reported by Happe and Kaminski [6]. The hydA2 cDNA from C. reinhardtii 21gr hydA2 was compared with C. reinhardtii CC-425 hydA2 DNA by Forestier et al., as a result, the cDNA of SE by the methods of PCR and RT-PCR and the gene structure was analyzed in this paper. The exon and intron of hydrogenase from C. reinhardtii SE were compared to the hydrogenase from C. reinhardtii CC-425 and that from C. reinhardtii CC-425. There were some difference in introns.

Section snippets

Isolation of C. reinhardtii SE total RNA

C. reinhardtii SE was provided by Wuhan Institute of hydrobiology, Chinese Academy of Sciences. The Trizol Reagent Total RNA Isolation Reagent Kit was taken from Life technology Company. C. reinhardtii SE [9] was cultured for one week under 25 °C, 3000 lux (12 h/day) with TAP (Tris–Acetate–phosphate) medium[8]. C. reinhardtii SE was centrifuged and ground immediately into a fine powder in liquid nitrogen. The cold powder was again ground in a homogenizer with 1.5 ml Trizol Reagent per 100 mg sample.

cDNA structure of hydA2 gene

Total RNA was extracted from C. reinhardtii SE and detected by agarose gel electrophoresis (Fig. 1). There were three bands indicating molecular weights equivalent to that of 28 s rRNA, 18 s rRNA and 5 s rRNA, respectively.

The primers for RT-PCR were synthesized and based on the published sequence of C. reinhardtii 21gr [7]. The total RNA of C. reinhardtii SE was used as a template for RT-PCR amplification, performed with primers of F1/R1. PCR products with primers F1/R1 resulted in a fragment

Discussion

In this paper the cDNA and DNA of hydA2 from C. reinhardtii SE was extracted. The nucleotide sequence of hydA2 cDNA and DNA was logged on GenBank, the GenBank accession number: AY436607) and analyzed. The sequence of hydA2 DNA from C. reinhardtii SE was compared to that from C. reinhardtii CC-425, they all consisted of 10 exon and nine intron. There was 98.2% homology between the exons of the hydA2 from C. reinhardtii SE and that from C. reinhardtii CC-425, but the difference between intron of

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