| Pogostemon cablin(Balnco)Benth.is one of the Labiatae Stamidae plants,also is the "top ten of Guangdong Traditional Chinese Medicine",with the efficiency of arresting vomiting and eliminating dampness.In order to solve the problem of the decline of resistance and resource shortage,and to avoid the chimeric defects caused by chemical induced polyploidy,the polyploid breeding method with P.cablin protoplast symmetry fusion was considered.In addition,on the basis of the previous studies,we carried out the transcriptome analysis of P.cablin suspension cells added MeJA elicitor and blank control.The original data has been established and related transcription factors has been mined.It is a way to further explore mechanism of MeJA signaling pathway involved in the regulation of patchouli alcohol biosynthesis,and it provides a new thought and method in order to improve the quality of the medicine and sustainable development of P.cablin source.The main results are as follows:(1)Protoplast separation of P.cablin suspension cellsThe effects of pretreatment method,enzyme pH and enzymolysis temperature on the separation of protoplasts were investigated on the basis of the original experiment.The optimum conditions were as follows: Patchouli suspension cells in logarithmic growth phase which had growthed 9-15 d was selected as the raw materials.The mixed enzyme liquid was 1.5% cellulase,0.5% hemicellulase,0.8% pectinase(m/v),with 0.4 moL/L mannitol osmotic adjustment;the temperature was 25 ? and pH was set to 5.8;suspension cells were oscillated by enzymatic hydrolysis at 50 rpm for 12 h in a dark environment.The final yield was 13.2×105 protoplasts/g,and the activity was 81.8%.(2)Protoplast Culture of P.cablin suspension cellsThe culture methods,the culture density,the hormone ratio,the ammonium content,the carbon source and the acid hydrolysis casein content were studied.Finally,the MS1 medium with half ammonium salt was used to embed sodium alginate.The hormone was selected by 0.2 mg/L NAA and 2.0 mg/L 6-BA;the culture density was about 2.0×105 protoplasts /m L;the sucrose content was 1%;and the acid hydrolyzed casein was 500 mg/L.(3)Protoplast fusion of P.cablin suspension cellsIn this experiment,the product selection was carried out by the physical selection method to determine the range of fusion product diameter.The range of screening products was 69.33-87.35 ?m by measurement and statistics.The effects of PEG concentration,fusion time,fusion density and the amount of fusion solution on the fusion were investigated by using high pH and Ca2+ method.Finally,40% PEG 6000 was selected to promote the chemical fusion for 30 min;the fusion solution was added to the volume of protoplast suspension by 0.5 times;the fusion density was 2.0 × 105 Protoplasts/mL;the polymerization rate was 57.19%.The fusion products were incubated with sodium alginate for 2 months.And then the regenerated callus was transferred to the induced cluster bud culture medium.The regenerated plants were not observed yet.The cultivation method was to be further optimized.(4)Transcriptome sequencing of P.cablin suspension cells induced by MeJAA total of 662270 Unigenes were obtained in P.cablin suspension cells supplemented with MeJA elicitor and blank control group by Illumina sequencing technology.The rate of reference was 74.04%.328994 Unigenes were obtained in the KOG database,covering 25 functional categories.There were 607785 Unigene annotated in GO database.Compared with three functional categories biological process,cellular component,and molecular function in the GO database,the annotated Unigenes respectively matched 339437,129556,138792 of the mentioned functional categories.163979 Unigenes were annotated in KEGG database,belonging to the 345 metabolic pathways,and 8 of them were terpene related involving 943 Unigenes.There were 5 synthase genes annontated in mevalonate(MVA)pathway of terpenoid backbone biosynthesis involving 44 Unigenes,and 7 synthase genes annontated in 1-deoxy-D-xylulose-5-phosphate(DXP)pathway of terpenoid backbone biosynthesis involving 20 Unigenes.83 Unigenes were annotated in Sesquiterpenoid biosynthesis pathways,including 6 synthase genes: farnesyl-diphosphate farnesyltransferase,squalene monooxygenase,NAD+-dependent farnesol dehydrogenase,(E,E)-alpha-farnesene synthase,(-)-germacrene D synthase and premnaspirodiene oxygenase.In addition,185221 SSRs were identified,and the most of which were monomers,accounting for 64.04%,followed by dimers,accounting for 23.21%.The above content is a kind of reference information for the further study on mechanism of MeJA signaling pathway involved in the regulation of patchouli alcohol biosynthesis and genetic improvement of P.cablin. |
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