| Larch is one of the most important timber and ecological tree species in North China.A number of germplasm resources with good growth quality have been obtained through selection breeding and cross breeding.Due to the frequent occurrence of extreme climate conditions,the spring drought in Northeast China is serious,which affects the survival and growth of larch.It is necessary to select drought resistant varieties,but the conventional breeding improvement cycle is longer.Genetic improvement from the molecular aspect can shorten the breeding cycle,but the research on the molecular mechanism of drought response in larch is far behind other tree species.miRNA is a class of non coding single stranded sRNA molecules involved in the regulation of post transcriptional gene expression in plants.It mainly regulates gene expression by degrading target mRNA or inhibiting translation of target mRNA,thus changing plant growth traits or stress resistance.In order to improve the fine characters of Larix and propagate fine germplasm resources rapidly,immature zygotic embryos of Larix kaempferi 3×Larix gmelinii 9 were used as explants to induce embryogenic calli,and the genetic transformation system of deciduous embryogenic calli was constructed and optimized.Meanwhile,the primary function analysis of larch sRNA under drought stress was carried out,and the corresponding miRNAs were screened out and Lol-miR11467 was transformed,and the downstream target genes of transgenic cell lines were predicted.The physiological changes under abiotic stress of drought,salt and alkali and the gene function of transgenic cell lines were analyzed.The main results are as follows:(1)Construction and optimization of genetic transformation system of hybrid larch.The induction rate of embryogenic callus was related to cone collection time.The highest induction rate was obtained on July 1,which was the best collection time of immature zygotic embryos.After disinfection with 75%alcohol for 1 min and 3%NaClO for 10 min,and inoculated in BM medium containing 1.0 mg/L 2,4-D+0.2 mg/L KT,the induction rate of embryogenic callus was the highest(10.33%).The best subculture period was 12 d in BM medium containing 0.5 mg/L 2,4-D+0.2 mg/L KT.The highest number of somatic embryogenesis was obtained on 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose.45 mg/L ABA and 75 g/L PEG4000 could promote the number of somatic embryos,and the number of somatic embryos reached the maximum,which was 210/g.Agrobacterium tumefaciens mediated transformation of Larix was carried out.Being infected with OD600=0.5 infection solution for 20 minutes and co-culture in dark for two days.It was found that the resistant calli were screened in the screening medium containing 4 mg/L Hyg.The genetic transformation efficiency of pCAMBIA1301 was the highest,which was 45.56%.(2)MiRNAs mining of larch under drought stress.A total of 190 miRNAs were predicted by miRDeep2 software,and the total number of target genes was 6284,a total of 4964 miRNAs were annotated.A total of 59 miRNAs were differentially expressed,accounting for 31.05%of all predicted miRNAs(190),of which 33 were up-regulated and 26 were down-regulated.The enrichment analysis of differentially expressed miRNAs target genes found ABC transport,carotenoid biosynthesis,plant hormone signal transduction,N-glycan biosynthesis were significant enrichment,which showed that the regulation of miRNAs is jointly participated by a variety of metabolic pathways,and miRNAs may play an important role in the growth and development of larch,stress response and other different life processes.(3)The expression of miRNAs was analyzed in larch.Under different stress treatments,most miRNAs can respond to drought and saline alkali stress.Among them,novelmiR63 responded rapidly to PEG6000 and NaCl stress.After treated with different hormones,novelmiR63(Lol-miR11467)was found to be able to respond to hormones,novelmiR63 was compared with miR11467 of Picea,whose target gene is annotated as a heat shock protein,one of the biological protective macromolecular proteins,which has a certain regulatory effect in drought stress,and selected as a candidate gene for genetic transformation,and named Lol-miR11467.(4)Genetic transformation of Lol-miR11467 in larch.By agrobacterium tumefaciens mediated transformation,27 resistant calli were obtained.The transgenic cell line and wild type cell line were placed in 20%PEG6000,50 mM NaHCO3 and 250 mM NaCl medium respectively.Under different stresses,the POD activity of transgenic cell lines was lower than that of wild type,MDA content was higher than that of wild type,and soluble protein content was lower than that of wild type.It is speculated that Lol-miR11467 has the ability of negative regulation of drought and saline alkali stress in Larix.(5)Target gene prediction of Lol-miR11467.The results of GO and KEGG enrichment analysis of the three groups of different samples showed that all of them had the most significant enrichment in secondary metabolism biosynthesis,hormone response,flavonoid biosynthesis and phenylpropane biosynthesis,indicating that Lol-miR11467 may play an important role in the response to stress and other external stimuli and the metabolic pathway of phenylpropane biosynthesis in Larch.Further excavation of the differential genes shared by the three transgenic cell lines revealed the down-regulation of transcription factor genes,such as MYB,bHLH,NAC,and WRKY,bioprotective macromolecules such as LEA and heat shock protein,as well as glycosyltransferase and galactosidase,oxidoreductase and miRNA related to AGO protein genes,indicating that overexpression of Lol-miR11467 can negatively regulate the expression of these genes to make larch resist drought stresses.Using the differential gene sequence of the resistant callus transcriptome as the reference sequence,the target gene prediction of Lol-miR11467 was performed,and a total of 4 target genes related to drought stress and down-regulated were predicted.In summary,this study obtained the Lol-miR11467 resistant cell lines through the construction of hybrid larch genetic transformation system and the analysis of differential miRNAs under drought stress.The physiological index test showed that the drought resistance ability of the transgenic cell line was weakened.Finally,four Lol-miR11467 target genes that may be related to the weakened drought resistance were found.This research lays the foundation for the genetic transformation of larch or conifers and the molecular mechanism of miRNAs. |
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