| Bletilla striata is a perennial herbaceous plant of the family Orchid.It is rich in polysaccharides,benzyls,terpenoids and other chemical components.It has the functions of scavenging free radicals,antibacterial,antioxidant,preventing cardiovascular disease,hemostasis and moisturizing the lungs.It has anti-tumor effects and is also a safer pharmaceutical raw material and pharmaceutical excipients.The most important active substance in Bletilla striata is Bletilla striata polysaccharide,which can be used not only in the food industry,but also in medicine,such as wound dressing and drug delivery.Bletilla striata has good medicinal effects and broad market prospects,wild Bletilla striata has been exploited in large quantities and its resources have been seriously lost.However,artificially cultivated Bletilla striata still cannot meet people’s normal needs due to the long growth cycle and other reasons.Therefore,studying the cultivation technology of Bletilla striata and the biosynthesis of Bletilla striata polysaccharides,and increasing the yield and polysaccharide content of Bletilla striata have become the primary tasks of Bletilla striata researchers.This paper(1)conducted transcriptome analysis of Bletilla striata tuber from three different years,and revealed the physiological and biochemical molecular mechanisms of Bletilla striata polysaccharide metabolism and tuber enlargement and development.Based on transcriptome data,the β-fructofuranosidase gene was screened out.Carry out cloning and functional identification;(2)Screen Bletilla striata symbiotic bacteria,observe its growth-promoting effect on Bletilla striata,construct a red fluorescent prokaryotic expression vector,transfer it into Bletilla striata symbiota to construct engineering bacteria,and observe the symbiotic bacteria pair through the symbiosis of engineered bacteria and Bletilla striata The colonization process of Bletilla striata young roots provides a theoretical basis for cultivation techniques to improve the yield and quality of Bletilla striata.The main research results are as follows:A total of 76 552 Unigenes sequences were obtained through sequencing analysis of the transcriptomes of 1-year-old,2-year-old and 3-year-old Bletilla striata tuber.After comparing with the seven big data,we got 52 219 annotated Unigenes,accounting for 68.12% of all Unigens.Comparing the transcriptome data of1-year-old,2-year-old and 3-year-old Bletilla striata tuber,37,825 differential genes were found,involving 47 biological functions.These 47 biological functions can be divided into molecular functions,cellular components,and biological processes.Three major categories.KEGG enrichment analysis found 134 signal pathways,metabolic pathways and secondary metabolite synthesis pathways are the most important signal pathways,59 key enzyme genes are involved in the synthesis of mannose and many pathways are involved in the synthesis of glucose;there are 12 pathways Differential genes related to tuber enlargement participate in the regulation of Bletilla striata tuber enlargement growth.q RT-PCR analysis verified that the expression of 6 representative genes related to root enlargement and sugar metabolism tended to be consistent with the transcriptome results.Using the transcriptome analysis data of Bletilla striata tuber growth and development,the differentially expressed β-fructofuranosidase gene(FFase)related to Bletilla striata polysaccharide synthesis was screened out,and its full length was cloned for functional identification.The gene was cloned by nested-PCR technology to obtain a full length of 1743 bp,and bioinformatics analysis was performed on it.Construct its prokaryotic expression vector,induce expression and purification to obtain the protein.The study found that when the concentration of IPTG is 1.0m M,the induction time is 6h,and the induction temperature is 37℃,the protein induction effect is best.The p H2GW7.0-35S-FFase plant overexpression vector was constructed to prepare for the next step of preparing transgenic plants and functional identification.A symbiotic bacterium was isolated from the roots of Bletilla striata and was identified as Pseudomonas fluorescens by morphology and molecular identification.Co-cultivation with Bletilla striata seedlings promotes the growth of Bletilla striata.The p BBR1MCS-2-m RFP1 red fluorescent protein prokaryotic expression vector was constructed,and it was successfully transferred into the isolated Pseudomonas fluorescens and expressed.The colonization of Bletilla striata larvae was successfully observed using a laser confocal microscope. process. |
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