Establishment Of Tissue Culture And Regeneration System Of Sea Buckthorn (Hippophae Rhamnoides) And Cloning And Expression Analysis Of Its HrFAD2 Gene

Sea buckthorn(Hippophae rhamnoides)is an excellent tree species with important ecological functions and great economic development value.The establishment of tissue culture regeneration system for this species is an essential prerequisite for its molecular genetic improvement.In recent decades,there have been many reports on tissue culture of sea buckthorn.However,the ratios of the regenerated plants are currently not high and the transplant survival rates are also low and unstable.Therefore,the tissue culture regeneration techniques need to be further improved and optimized.FAD2(Fatty acid desaturase 2),a key enzyme for the production of linoleic acid from oleic acid,is closely related to both plant stress resistance and the quality of vegetable oil.So far,cloning of the full-length FAD2 gene from sea buckthorn and its characterization have not been reported.In this thesis,study on the tissue culture of Chinese sea buckthorn(H.rhamnoides.subsp.sinensis Rousi)was carried out.Moreover,the full-length Hr FAD2 gene was cloned and its expression analysis and RNAi vector construction were performed.The main results are as follows:1.The sterile cultures of wild branches were obtained,but their survival rate was low(13.8%)and their growth was poor.2.The medium for efficient callus induction from cotyledons and hypocotyls of aseptic seedlings are composed of WPM salts+0.8 mg/L 6-BA+0.3 mg/L NAA+100mg/L Vc and WPM salts +0.5 mg/L 6-BA+0.3 mg/L NAA+100 mg/L Vc,respectively,and the callus induction rates were 90.9% and 44.9%.3.The medium suitable for the differentiation of regenerated buds and their growth is composed of WPM salts+0.5 mg/L 6-BA+0.2 mg/L NAA+100 mg/L Vc.The differentiation rates of regenerated buds from cotyledon-derived and hypocotyl-derived calli were 80.6% and 56.0%,and the average regenerated buds per callus were 13.6 and16.1,respectively.4.When the regenerated shoots were rooted in the bottles with growth medium,the rooting rate was low(36.4%)and the roots grew slowly.However,when the regenerated shoots were directly transplanted into nutritious soil for rooting(rooting outside the bottle),the rooting rate increased(42.9%)and the roots grew rapidly.5.The full-length c DNA and g DNA of Hr FAD2 gene were cloned,and the coding region of the latter does not contain intron.The coding protein of Hr FAD2 consists of383 amino acid residues and the conserved histidine clusters necessary for the enzyme activity of FAD family exist in its amino acid sequence.The protein also has a signal sequence for endoplasmic reticulum localization.In molecular evolution,Hr FAD2 is closely related to soybean Gm FAD2-1 and tung oil tree Vf FAD2.6.RT-q PCR analysis showed that the transcription level of Hr FAD2 gene was highest in young fruits,followed by lateral roots,semi-ripe fruits and ripe fruits,and lowest in twigs and leaves.7.Based on plant expression vector p CAMBIA3301,the RNAi vector of Hr FAD2 gene was successfully constructed.This study lays a foundation for further establishment of a high-efficiency tissue culture and plantlet regeneration system of Chinese sea buckthorn and functional analysis of the Hr FAD2 gene.