Cloning, Expression Characteristics Of Ascorbate Peroxidase Gene In Tea Plant (Camellia Sinensis) And Physiological Response To Abiotic Stress

Ascorate peroxidase(APX), a kind of heme proteins existing in higher plants and eukaryotic algaes, was firstly found in1970s. In recent years, the researches about APX updated repidly, especially that APX, the key enzyme of AsA-GSH cycle scavenging H2O2produced in chloroplast, cytoplasm and microbody, had attracted more and more researchers’ attention. APX has higher affinity for H2O2than other antioxidant enzymes and in chloroplasts, CAT, a H2O2scavenger, is not yet to be found, so H2O2is mainly detoxified by APX in chloroplasts, hence APX plays an important role in maintaining normal photosynthetic ability of plant under abiotic stresses.Tea plant (Camellia sinensis), an economic crop, originates in tropic and subtropic regions and likes warm and moist climate whilst cold and drought stresses are always serious problems limiting tea distribution and production, so the mechanism of cold-resistance and drought-resistance of tea plant has been paid more and more attention. In this works, three ascorbate peroxidase genes, microbody ascorbate peroxidase (mAPX), chloroplastic stromal ascorbate peroxidase (sAPX) and thylakoid-bound ascorbate peroxidase (tAPX), respectively, were amplified via RACE technology with EST fragments screened from cDNA library prepared from the whole organs transcriptome and cold-induced leaves of Camellia sinensis, respectively and subsequently biological information of amino acid encoded by these APX genes was predicted and analysed. Differential expression of APX genes in different tissues of tea cultivars under overwintering and drought stress was studied by RT-PCR and qRT-PCR. Moreover, APX activity and chlorophyll fluorescence parameters were also determined. The main results are listed as follows:1.mAPX was named Cs mAPX and its cDNA full-length was1158bp, with867bp ORF encoding288amino acid, which contained a transmembrane domain at C-terminal located on peroxisome’s membrane tAPX was named Cs tAPX and its cDNA full-length was1424bp, including1296bp ORF encoding431amino acid, which had a transmenbrane domain at C-terminal located on chloroplastic thylakoid-bound membrane sAPX was named Cs sAPX, cDNA full-length of which was1579bp, containing1149bp encoding382amino acid and no transmembrane domain. These genes have been submitted to the GenBank and the accession number were JQ011380, JQ740734and JQ011381, respectively.2.Multiple sequence alignment showed that the three APX genes all contained two conserved functional domains: active site and heme-bouding site. In addition, sAPX and tAPX also contained two conserved structure domain:(K-N-I-E-E-W-P) and (E-T-K-Y-T-K-D-G-P-G-[A|N]-P-G-G-Q-S), which were the conserved domains of chloroplastic APX. The phylogenetic tree indicated that Cs mAPX, Cs sAPX, Cs tAPX and Cs cAPX (Accession No: EU547804) belonged to two families.3. Expresion characteristics of four APX genes in different tissues of tea plant were studied via RT-PCR. The results showed that Cs cAPX distributed equally in roots, stems and leaves, which did not exhibite tissue-specificity. However, Cs mAPX, Cs sAPX and Cs tAPX exhibited tissue-specificity and mainly distributed in leaves.4. The change of APX activity and chlorophyll fluorescence as well as expression level of the four APX genes during overwintering were studied with Camellia sinensis cv.“Chanong98”, cv.“Yingshuang” and cv.“Shuchazao” one-year old tea seedlings as materials. The results showed that the change trend of APX activities firstly increased and then decreased among tea varieties with maximum in January The trend of chlorophyll fluorescence parameters was the same among varieties with Fv/Fm, Fm and Fv/Fo firstly decreasing then increasing as well as Fo increasing at the beginning then decreasing RT-PCR and qRT-PCR showed that the expression level of cAPX and sAPX increased dramatically in December and January while mAPX and tAPX increased slightly.5. The changes of APX activtiy among three varieties was from descend to ascend and then to descend with the increase of drought stress Fv/Fm, Fm and Fv/Fo decreased and Fo increased during drought stress The expression level of mAPX and tAPX were not evident under drought stress, however, the level of sAPX increased dramatically and the cAPX was inhibited under drought stress.