Research On The Molecular Mechanism Of Panax Ginseng Seed After-ripening

Panax ginseng(Araliaceae,Panax),a perennial herb,is a valuable medicinal herb which has potent effects on regulating central nervous system,building immunity,relieving fatigue,anti-tumor,anti-diabetes and so on.According to research,P.ginseng has considerable economic value and broad application in production of drugs,cosmetics and health care products.Panax ginseng is mainly propagated by seeds,which have the characteristics of after-ripening and need to undergo a long period of morphological and physiological after-ripening.At present,the molecular mechanism of Panax ginseng seed after-ripening is not clear.Gibberellin and abscisic acid are key hormones to regulate seed dormancy and germination.In the process of seed after-ripening of Panax ginseng,seed embryos need to complete morphological and physiological after-ripening in order to germinate.WRKY transcription factor can regulate plant growth and development by regulating the expression of target genes.In this study,ginseng seeds are naturally stratified to select key genes of gibberellin and abscessic acid metabolism(GA2ox,GA20 ox,NCED,CYP707A),genes related to plant embryo development(SERK,BBM,WUS,FUS3,AGL15,LEC1)and WRKY transcription factors(WRKY3,WRKY6,WRKY40).Semi-quantitative PCR and real-time quantitative PCR are used to detect the expression levels of the above genes in the after-ripening process of Panax ginseng seeds.The results are as follows:(1)The newly collected Panax ginseng seeds are stratified and sampled every 45 days.According to the embryo morphology and embryo rate,the after-ripening process of ginseng seeds are divided into six stages,which are S1-early stage of morphological after-ripening.S2-middle stage of morphological after-ripening;S3-end stage of morphological after-ripening;S4-early stage of physiological after-ripping;S5-middle stage of physiological after-ripping;S6-end stage of physiological after-ripening.(2)The key genes of GA and ABA metabolism(GA2ox,GA20 ox,NCED,CYP707A),plant embryo development related genes(SERK,BBM,WUS,FUS3,AGL15,LEC1)and WRKY transcription factors(WRKY3,WRKY6,WRKY40)are successfully cloned and seque NCED.The results showed that the primer design and clone gene length were reasonable and the product specificity is good.(3)Panax ginseng 5Sr RNA is used as internal reference gene to carry out semi-quantitative PCR on the target gene.According to the results of electrophoresis,the expression of the target gene in Panax ginseng seed ripening process is preliminarily determined.Real-time quantitative PCR is used to detect the gene expression and analyze the results.During the after-ripening process of Panax ginseng seeds,key genes of GA and ABA metabolism: GA2 ox gene expression decreases continuously,and GA20 ox gene expression increases continuously.The expression of CYP707 A gene decreases first and then increased,and the expression of NCED gene decreases continuously.During the after-ripening process of Panax ginseng seeds,the expression of key genes of GA and ABA metabolism: GA2 ox gene decreases continuously,while the expression of GA20 ox gene increases continuously.The expression of CYP707 A gene decreases first and then increased,while the expression of NCED gene decreases continuously.Plant embryo development related genes: the gene expression levels all show the trend of increasing,but the amplitude of the increase is different.WRKY transcription factor: the expression of WRKY3 gene decreases continuously,the expression of WRKY6 gene increases continuously,and the expression of WRKY40 gene decreases continuously.(4)The CDS sequence of ginseng GA2 ox gene is cloned,named Pg GA2 ox,and submitted to Gen Bank.The login number is KT692958.1.According to the sequencing results,the length of CDS region of Pg GA2 ox gene is 987 bp,encoding 328 amino acids,which is 98.78% similar to the amino acid sequence alignment of Panax quinquefolius GA2 ox gene.(5)The CDS sequence of ginseng GA20 ox gene is cloned,named Pg GA20 ox,and submitted to Gen Bank.The login number is KX790244.1.According to the sequencing results,the length of CDS region of Pg GA20 ox gene is 1092 bp,encoding 363 amino acids,which is 98.90% similar to the amino acid sequence alignment of Panax quinquefolius GA20 ox gene.(6)The CDS sequence of ginseng WRKY3 gene is cloned,named Pg WRKY3,and submitted to Gen Bank for the landing number KX790243.1.According to the sequencing results,the CDS length of Pg WRKY3 gene is 1356 bp,encoding 451 amino acids,and the similarity of the amino acid sequence alignment with the American ginseng WRKY3 gene is 99.33%.This experiment analyzes the expression of several key genes of plant hormone GA and ABA metabolism,genes related to plant embryo development and WRKY transcription factor,which laid a foundation for studying the molecular mechanism of ginseng seed maturation.