Establishment Of Leaf Regeneration System Of Ophiorrhiza Japonica Blume And Molecular Cloning Of The G10H Gene 3' End

Camptothecin is a kind of specific anticancer drug, which originally identified in the extracts of Camptotheca acuminata Nyssaceae. Being a woody plant, C.acuminata grows very slowly and the supply of CPT sustained by isolation from the original plant source is very limited, while the demand for CPT is rapidly increasing in recent years. Excitedly, it has been reported that hairy roots of herbaceous Ophiorrhiza pumila could produce CPT by Agrobacterium rhizogenes. However, it is difficult for domestic scholar to study CPT because O.pumila is very scarce in China. O.japonica, one indigenous Chinese medicinal plant, is also an herbaceous plant which belongs to Ophiorrhiza, hence it is speculated that CPT analogues may also exist in the roots of O.japonica similarly. In this paper, it has been studied on regeneration system of leaf explants from O.japonica, clone of 3'end of key enzyme G10H gene in CPT biosynthesis pathway and pre-test of genetic transformation of leaf, which try to produce CPTs from O.japonica by gene engineering. Up to now, there is no report concerned. Main research results were as follows:1 Regeneration system of leaf explants from O.japonica1.1 Callus formation and shoot differentiation1) Using leaves from about one-year-old field plants as explants, calli could be induced by auxin 2,4-D and NAA. The effect of calli induced in light condition was better than that in darkness. The optimal basal medium of callus proliferation was B5 medium and 6-24d was the index increasing phase of callus proliferation.There was a distinct effect on callus differentiation by incubation time and subculture passages of callus. The ability of shoot differentiation was the strongest when calli inoculated on initial medium was at 7d. With the increase of subculture passages of callus, the ability of shoot differentiation was decline.This research may provide the foundation for genetic transformation by using calli of O.japonica as initial material in the future.2) Using leaves from about one-month-old tube seedlings as explants, the leaf explants could form calli firstly and then differentiate shoots by combinations of BA and NAA. The optimal basal medium and the best combination of plant growth factors for shoot differentiation of leaf-derived callus were MS and BA2.5mg/L+NAA0.2mg/L,respectively. 18-27d of seedlings age, 0.5%PVP and 3-5d of pre-culture in darkness all could promote shoot differentiation of leaf-derived callus.This research can offer an effective approach for genetic transformation by using leaves of O.japonica as initial material in the future.1.2 Shoot proliferation and strength culture1) Using tip shoots as explants for proliferation, B5 basal medium was better than others. The results of orthogonal experiment design of BA, KT and NAA showed that effect on shoot proliferation of BA was better than that of KT and NAA.2) When strength culture, the results of orthogonal test design of KT, LH and CH revealed that KT could obviously promote growth of stem and leaf, and that LH and CH were favorable for elongation of stem and differentiation of axil.1.3 Root cultureThe best rooting medium was 1/2MS and the effect on rooting culture using by IBA was better than that by NAA. When the concentration of IBA was 0.5mg/L, the effect on rooting induction was the best.1.4 Transplanting tube seedlingsRooted plantlets were removed from rooting medium followed by rinsing out media and then transferred to small pots containing soil and sand, which were covered with glass beakers and kept under high humidity to prevent wilting for acclimatization in two weeks before being exposed to greenhouse conditions by removing the cover.2 Cloning of 3'end of G10H gene and pre-test of genetic transformation of leaf from O.japonica2.1 Cloning 3'end of G10H genePrimers were designed based on conserved sequences of species concerned from cytochrome P450 monooxygenase. 3'end of G10H gene was isolated from O.japonica. The 3'cDNA of O.japonica was 532bp, containing a part open reading frame (321bp) encoding a part peptide of 107 amino acids, with its encoding protein showing 84% similarity to G10H of Catharanthus roseus.2.2 Pre-test of genetic transformationThe results showed that the critical Hygromycin concentration of leaf differentiation was 20mg/L and the Cefataxine concentration of 100-200mg/L could be used to choose shoots.