Expression Analysis Of Genes In Embryo Development Of Cypripedium Japonicum

Cypripedium Japonicum Thunb.is a perennial herb of Cypripedium in Orchidaceae.It has a long and thin,transverse rhizome.The leaves are fan-shaped,the inflorescence is terminal.The flowers are big and colorful,and the flowering period is from April to May.The capsule is nearly spindle shaped,sparsely puberulent.It is found in the humid and humus rich soil under the forest,shrub forest,forest edge,beside the valley and shaded slope with an altitude of 1000-2000 meters,in the south of Shanxi,the south of Gansu,Anhui,Zhejiang,Jiangxi,and also in Japan.It has a certain medicinal value.Rhizome can be used as medicine for regulating meridians,dispelling wind and relieving pain,pruritus of skin,innominate swelling poison,bruise,rheumatism and arthralgia,snake bite,etc.The results showed that the seeds of C.Japonicum were seriously abortion and the germination rate was low under natural condition,which might be related to the formation of embryo.In order to reveal the molecular mechanism of low germination rate,we used transcriptome sequencing,micro RNA sequencing and degradation group sequencing to study the development process of embryo.At the same time,according to transcriptome sequencing results,we screened the internal reference genes of C.Japonicum.The main results are as follows:(1)High throughput sequencing was used to analyze the changes of gene expression during the five stages of embryo development.13761,3940,4556 and 6349 differentially expressed genes were screened from four adjacent groups(PTvs NP、PFvs PT、PFvs PS、PSvs PE).We analyzed the GO enrichment and KEGG pathway enrichment of these differential genes,and found that the differential genes mainly involved in multicellular biological development,hormone response and the biological process of embryo development ending in seed dormancy,and mainly involved in plant hormone signal transduction,starch and sucrose metabolism.We found that some genes related to embryo development and endogenous hormones play an important role in the process of embryo development.Among them,EMB506、MSI1、BOB1、ARP7、NFYA1、ARF4、HCC1 are important genes regulating the development of seed embryo,Mutation in EMB506 may make the embryo stay in the globular stage;The low expression of ARP will lead to fertility reduction or complete sterility of fruit.Some of the genes related to auxin,cytokinin and signal transduction are differentially expressed and play a regulatory role in five developmental stages.(2)Twelve housekeeping genes(ACT3、EF1、RPL26B、TIF3I1、SAMDC、CYP22、PP2A3、TUBB3 、 RPS4 、 UBC2 、 UPL1 、 RAN1)were selected from the transcriptome data of C.Japonicum.The stability of the 12 candidate genes was studied.The 12 candidate genes were expressed in different tissues and seeds at different developmental stages,but the expression amount was different.CYP22、UBC2、TUBB3 and RPS4 were stable in all samples;PP2A3 and TIF3I1 were the most stable in different tissues;TUBB3 and UBC2 were the most stable in seeds at different developmental stages.UBC2 and TUBB3 genes and their combinations were used as internal reference genes.The relative expression of ASK1 and SERK2 genes was detected to verify the reliability of UBC2 and TUBB3 genes and their combinations as internal reference genes.These stable internal reference genes have important reference value for quantitative analysis of gene expression of C.Japonicum.(3)In this study,1463543,5084343,8692446,2127279 and 1631381 valid reads were obtained from 5 samples.Most miRNAs were distributed in 20-24 nt,which accorded with the typical characteristics of Dicer enzyme cutting.When we analyzed these data further,320 miRNAs were selected,282 of them are known miRNAs belonging to 58 conservative miRNA families,including mir166,mir156,mir159 and other families.Among the 58 miRNA families,mir159 family has most family members,containing 13 mir159 genes.The results showed that the target genes were mainly enriched in biological processes,transcription regulation,protein phosphorylation,embryonic development,signal transduction and plant hormone signal transduction.Further trend analysis against differentially expressed miRNA was carried out,and the data obtained were combined with transcriptome and degradation group analysis.It was found that functions of target genes of mir156,mir171,mir160,mir166,mir168,mir390 and mir5179 families are mainly enriched in auxin response,protein coding and other biological processes.,playing an important role in the development of embryo of C.japonicum.In general,this study has mainly researched the gene expression,miRNA expression and target genes of five different developmental stages of C.Japonicum by high-throughput sequencing technology and preliminarily constructed the gene expression regulatory network during the development of C.Japonicum seed embryos,laying a foundation for revealing the gene expression regulatory mechanism during the development of C.Japonicum seed embryos.