| Cymbidium is a kind of traditional and precious flower with important ornamental,cultural,and economical value.Different Cymbidiums have diverse tissue culture characteristics and micropropagation efficiencies.Therefore,it is of great significance for further studying in tissue culture characteristics,clarifying the molecular mechanism of shoot regeneration in vitro and genetic mechanism underlying the different shoot regeneration capacity in Cymbidium in order to promote the research progress and industry development of Cymbidium in our country.In this paper,investigations were carried out on micropropagation characteristics of different genotype Cymbidiums,screen of reference genes for q RT-PCR analysis in tissue culture process,comparative analysis in endogenous hormone and transcriptome during shoot regeneration between Cymbidium ‘Xiaofeng' and Cymbidium sinense ‘Qijianbaimo',and identification and cloning of candidate genes related to shoot regeneration in Cymbidium.The main results are as follows:1.When cultured on MS medium without exogenous hormone on darkness,rhizomes or PLBs of Cymbidium aloifolium,C.Maureen Cater ‘Dafeng',Cymbidium ‘Xiaofeng',Cymbidium ‘Guofeng' and C.sinense ‘Qijianbaimo' did not differentiate but proliferate,however,shoots and roots were regenerated from those cultured under light.The frequency of shoot regeneration was 100%,100%,99.17% and 95.29%,respectively,and the frequency of root regeneration was 98.67%,10.59%,63.33% and 87.50%,respectively.Under the same circumstance,no shoot and root were regenerated from the rhizomes of C.sinense ‘Qijianbaimo'.In the dark,rhizomes of C.‘Xiaofeng',cultured on MS medium with addition of 6-BA or/and NAA,had no shoot regeneration,proved that the role of light in inducing shoot regeneration could not be replaced by 6-BA and NAA.When cultured on MS medium supplemented with 1 mg/L NAA,roots V were differentiated from the rhizomes of C.‘Xiaofeng',indicating that light was not essential for root regeneration.2.Ge Norm,Norm Finder and Bestkeeper software tools were applied to test the expression stability of reference genes of 18 S ribosomal RNA(18S),26 S ribosomal RNA(26S),Actin(ACT),Elongation factor 1 alpha(EF1?),Glyceraldehyde-3-phosphate dehydrogenase(GAPDH),Photosystem I P700 apoprotein B(Psa B),RNA polymerase B(Rpo B),Ribosomal protein S3(Rps3),Ribosomal protein S19(Rps19)and beta-tubulin(TUB).The results indicated that Rps3,GAPDH and ACT were suitable to be used as reference genes in q RT-PCR analysis during micropropagation of Cymbidium aloifolium,C.Maureen Cater ‘Dafeng',C.‘Xiaofeng',C.‘Guofeng' and C.sinense ‘Qijianbaimo'.3.Rhizomes of C.‘Xiaofeng' and C.sinense ‘Qijianbaimo',cultured on medium without exogenous hormone on darkness,were exposed to light.Shoot regeneration occurred after 10 d,root regeneration occurred after 20 d,the leaves were unfolded after 25 d,and the elongated roots were formed after 30 d in C.‘Xiaofeng',in contrast,rhizomes from C.sinense ‘Qijianbaimo' proliferated slowly and did not differentiate.There was significant difference in endogenous hormone content of indole-3-acetic acid(IAA),zeatin(Z),N6-isopentenyladenine(i P),dihydrozeatin(DHZ),brassinosteroids(BR),methyl jasmonate(Me JA),abscisic acid(ABA)and gibberellic acid 3(GA3)in the rhizomes exposed to light for 0,1,5,10,and 20 d between C.‘Xiaofeng' and C.sinense ‘Qijianbaimo'.Except for Z,other endogenous hormone contents in C.‘Xiaofeng' were higher than that in C.sinense ‘Qijianbaimo'.The content of IAA,i P and Z reached a maximum value at ten days after exposure to light in C.‘Xiaofeng'.4.Rhizomes of C.‘Xiaofeng' and C.sinense ‘Qijianbaimo' exposed to light for 0,1,5,10,and 20 d were subjected for RNA-seq analysis on Illumina Hiseq X platform.158,352 unigenes were obtained.KEGG enrichment analysis showed that most gene were enriched to carbon metabolism,amino acid biosynthesis,ribosome,starch and sucrose metabolism,spliceosome,protein processing in endoplasmic reticulum,purine metabolism,RNA transport,plant hormone signal transduction pathway and so on.DEGs analysis between C.‘Xiaofeng' and C.sinense ‘Qijianbaimo' demonstrated that there were 8718,8940,10435,8967,12598 and 9890 DEGs in the rhizomes exposed to light for 0,1,5,10,and 20 d,respectively,and these DEGs were mainly enriched to spliceosome,m RNA surveillance and aminoacyl-t RNA biosynthesis pathways.There were 969,541,151,150 and 212 DEGs at different shoot regeneration stages in C.‘Xiaofeng',in contrast,there were 1091,1207,111,98 and 44 DEGs in C.sinense ‘Qijianbaimo'.These DEGs were mainly enriched to photosynthesis,photosynthesisantenna proteins,circadian rhythm-plant,porhyrin and chlorophyll metabolism,carbon fixation in photosynthetic organisms and flavonoid biosynthesis pathway.5.Through quantitative real-time PCR(q RT-PCR)analysis,HY5,YUC8,TIR1,ARF11,GH3.1,GH3.5,LOG8 and AHK candidate genes related to shoot regeneration were screened out.6.Full length of YUCCA6 c DNA from C.‘Xiaofeng' cloned with SMARTer RACE is 2058 bp,which contains a 5' untranslated region(5' UTR)of 259 bp,a coding sequence(CDS)of 1221 bp,and a 3' untranslated region(3'UTR)of 575 bp.Full length of GH3.8M c DNA is 2028 bp,which includes a 5'UTR of 57 bp,a CDS of 1803 bp,and a 3'UTR of 165 bp.Full length of AHK4 c DNA is 4419 bp with an untranslated region of 996 bp at 5' end,a CDS of 2865 bp,and an untranslated region of 558 bp at 3' end.Full length of CKX9 c DNA is 2248 bp.It is consisted of a 5'UTR of 77 bp,a CDS of 1560 bp,and 3' UTR of 608 bp.7.The open reading frame and genome sequence of AHK4,YUCCA6,GH3.8M and CKX9 from C.‘Xiaofeng' and C.sinense ‘Qijianbaimo' were amplified by PCR and homologous cloning.According to sequence analysis,ORFs of four genes between C.‘Xiaofeng' and C.sinense ‘Qijianbaimo' are completely consistent in sequence,and so do the genome sequences.The length of YUCCA6 is 3351 bp,with 4 exons and 3 introns.The length of GH3.8M is 2597 bp,with 3 exons and 4 introns.The length of AHK4 is 8067 bp,which contains 11 exons and 10 introns.The length of CKX9 is 1831 bp with 4 exons and 3 introns,there is an alternative splicing site in the third intron,therefore,two types of m RNA can be generated after transcription.The splicing site of intron conforms to ‘GT-AG' rule.8.Overexpression and RNAi vectors of AHK4,YUCCA6,GH3.8M and CKX9 were constructed,and transformation was carried out via Agrobacterium-mediated transformation method using rhizomes of C.‘Xiaofeng' and C.sinense ‘Qijianbaimo' as receptor material.No transgenic plants were obtained.In conclusion,our study revealed that light plays an essential role in shoot regeneration,and dynamic changes of endogenous hormone content and transcriptome were clarified during shoot regeneration.Genes related to shoot regeneration were preliminarily identified.DNA sequences of AHK4,YUCCA6,GH3.8M,and CKX9 were obtained.These results lay a solid foundation of further research on function analysis of shoot regeneration gene,genetic basis underlying different micropropagation characteristics and molecular breeding of micropropagation character in Cymbidium. |
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