Preliminary Studies On Hormones And Differentially Expressed Genes During The Induction And Development Of Bletilla Pseudobulbs

The development process of Bletilla striata(Thunb.)Reichb.f.Has an important influence on the yield and quality of medicinal materials.The development of pseudobulbs is the result of the combined action of external environmental factors and internal regulatory factors.In this study,Bletilla seeds were used as materials to study the cultivation conditions of tissue cultured seedlings.The tissue culture seedlings were used as materials to study the endogenous hormone content and differentially expressed genes during pseudobulb formation and development.Below are key research findings:1.Bletilla seed germination test research shows that with 3/4MS as the basic medium,the overall results of Bletilla seed germination are better than MS and 1/2MS.On the medium composed of 3/4MS+0.7mg/LNAA+0.2mg/L6-BA+potato70g/L+30g/Lsucrose+7g/L agar,the seeds germinated fastest,and the germination rate reached 74% at 20 d 3/4MS+0.5mg/L6-BA+0.7mg/LNAA+0.3mg/LIAA+0.4mg/LKT medium formula is more suitable for Bletilla tissue culture seedling proliferation;3/4MS+0.3mg/LNAA+0.1mg/LIAA+0.4mg/LKT The medium formula is suitable for Bletilla plant growth and development;the hormone concentration suitable for root growth is 1mg/L6-BA,0.3mg/LIAA and 1.2mg/LKT.2.Bletilla pseudobulb induction studies have shown that when 3/4MS is selected as the basic medium,the induction rate of pseudobulbs is better than MS and 1/2MS.On the basis of 3/4MS,adding 0.5mg/L6-BA,0.3mg/LNAA is more suitable for the growth of Bletilla tissue culture seedlings,and the induction rate of pseudobulb is high;IAA has the same effect on Bletilla pseudobulb formation,followed by 6-BA and KT,3/4MS as the basic medium,adding 0.5mg/LNAA,0.3mg/LIAA and 0.8mg/LKT can better induce the formation of pseudobulb And the time is relatively short,which is beneficial to the increase of the number of sprouts,the number of roots and the growth and development of the pseudobulb.3.The division of the quality grade of Bletilla tissue culture seedlings and the scientific and reasonable verification results show that the quality of Bletilla tissue culture seedlings is divided into 3 grades,the diameter of pseudobulb of class I seedlings ?7mm,plant height ?8cm;Diameter ?5.5mm,plant height ?4cm;Class III seedling pseudobulb diameter ?4mm,plant height ?4cm.The results of the survival rate of grade seedlings show that the larger the diameter and plant height of the pseudobulbs of Bletilla tissue culture seedlings,the higher the survival rate of Bletilla plants after transplanting,that is,the survival rate of grade I seedlings> the grade II seedlings> the survival rate of grade III seedlings> The average average growth value of straight bulbs of class I seedlings is greater than that of class II seedlings and smaller than that of class III seedlings,but for individual plants,their growth is much greater than that of class II and III seedlings,the germination rate and new shoots of pseudobulbs The number of germination is also relatively high,and the number of new shoots is generally 1-2 per plant.4.The results of the division of the formation and expansion of Bletilla pseudo bulbs show that the diameter and fresh weight of white and pseudobulbs show an "atypical" S-shaped curve during the growth and development.The formation of pseudobulb in tissue culture seedlings is divided into: the formation stage,the expansion stage,the middle expansion stage,the bifurcation stage and the later expansion stage.LC-MS determination of endogenous hormones(GA3,ABA,IAA,and ZR)in the five stages of pseudobulb in Bletilla tissue culture seedlings found that the diameter and fresh weight of the pseudobulb showed a very significant positive correlation between There is a certain correlation between traits,but the significance is different.Except that the correlation between endogenous ZR and pseudobulb diameter and fresh weight is not significant,the correlation between other hormones and pseudobulb diameter and fresh weight has reached a significant or extremely significant level.The weights were significantly positively correlated.GA3 had a very significant negative correlation with pseudobulb diameter and a significant negative correlation with fresh weight.ABA was extremely significantly negatively correlated with pseudobulb traits.GA3/ABA was significantly negatively correlated with pseudobulb diameter,but not negatively correlated with fresh weight.IAA/ABA was extremely significantly positively correlated with diameter and fresh weight,with correlation coefficients of 0.86**and 0.93**,respectively(GA3+IAA)/ABA has a very significant positive correlation with pseudobulb diameter and fresh weight,indicating that endogenous IAA plays a crucial role in the growth and development of pseudobulbs during the formation and expansion of pseudobulbs,and is a positive regulatory factor.5.The transcriptome sequencing results of the pseudobulb of Bletilla tissue culture seedlings found that 51,277 Unigenes were assembled and the average length was 941 nt.The GC content is 42.7985%,the number of N50 is 9277 nt,the length is 1605,the longest unigene length and the shortest unigene length are 13871 nt,201nt,and the total base number of Unigene is 48281634.Annotated 51,277 Unigenes to the databases NR,Swiss Prot,KEGG,and COG/KOG,and obtained 25964,16502,24969,14063 Unigenes,respectively.In addition,24,600(47.97%)Unigene failed to be annotated in the four major databases.The obtained unigenes were analyzed by GO,KEGG and KOG,and the biological functions(Molecular Function),biological processes and cellular components of GO function annotation were divided into 51 functional subcategories;a total of 5186 Unigene is assigned to 133 KEGG metabolic pathways,among which the metabolic pathways related to hormone synthesis include phytohormone signaling pathway(ko04075),zeatin biosynthesis(KO00908),MAPK signaling pathway-plant(KO04016),brassinolide Ester biosynthesis(KO00905),?-linolenic acid metabolism(KO00592),tryptophan biological metabolism(Ko00380);14,063 Unigenes were annotated in KOG,divided into 25 categories.The CDS prediction results show that a total of 27,560 CDS are obtained,with a prediction ratio of 53.74%.SSR analysis showed that a total of 36 types of 5955 SSR sites were searched.A total of 57 transcription factors were screened for the prediction of coding capacity of transcription factors,and a total of 1204 TF genes in gene families.The transcription factors with the largest number of genes were the b HLH and C2H2families.According to the threshold,the differentially expressed genes were selected.The results showed that XCQ-VS-PDQ had 206 differentially expressed Unigenes,including 115up-regulation and 91 down-regulation;PDQ-VS-PDZQ had 555,8 upregulation,and 547 Down-regulation;PDZQ-VS-FCQ has 591,including 62 upregulations and 529 down-regulations;FCQ-VS-PDHQ has 827,586 up-regulations and 241 down-regulations.For the enrichment analysis of GO and KO,Metapathic pathways(ko01100)are the most abundantly enriched genes in each comparison group,followed by Biosynthesis of secondary metabolites(ko01110).Hormone signal transduction pathways and synthetic pathways have significant differences in the expression of genes enriched to varying degrees,and related endogenous hormones are zeatin nucleoside,auxin,cytokinin,gibberellin,brassinolide,water Salicylic acid,abscisic acid,ethylene,jasmonic acid and indoleacetic acid.In summary,the results of this experiment will provide technical support for the grading and quality management of Bletilla tissue culture seedlings and the domestication and transplantation of tissue culture seedlings.It will lay the foundation for the research on the development mechanism of Bletilla pseudobulbs,and for the development of Bletilla pseudobulbs.The research of artificial control technology provides theoretical and technical reference,which is of great significance to promote the development of Bletilla basic research and cultivation.