Mining And Research Of Functional Genes In Response To Shading In Pinellia Ternata

Pinellia ternata（Thunb.）Briet.,with the dried tubers used as medicine,is one of the famous Traditional Chinese medicines that is endowed with cough and phlegm effects.Recent studies have revealed its more functions including anti-tumor,anti-fertility,hypolipidemic,and liver protection effects.However,it is usually suffered from environmental stress,which causes the withering of above ground parts,regarded as“sprout tumble”during growth process.Shading has been proved to delay the sprout tumble formation（STF）.However,due to the lack of genetic information,researches are mainly focused on the shading mode and effect on tuber yield,the molecular mechanism governing shading-regulated growth has not been reported.In this study,we investigated the physio-biochemical effect and yield of shading on P.ternata,identified key functional genes through comparative transcriptome analysis,preliminarily characterized two key genes.The main results are as follows:1.Shading affects P.ternata growthShading alleviated the sprout tumble and increased tuber yield.STF rate under shading was 40%lower than control.Compared to control,the tubers under shading are twice as larger as control.Chlorophyll fluorescence parameter（initial fluorescence,maximal fluorescence,and qP）has been promoted,chlorophyll a:b and NPQ value have been inhibited.Catalase（CAT）activity was largely increased.It suggests that shading decreased STF by enhancing chlorophyll fluorescence parameters and photosynthesis.2.Screening and determination of internal reference genes（IRGs）of P.ternataTo screen suitable internal reference genes for field shading experiment,8 IRGs Pt18S,PtEF1α,PtGAPDH,Ptsec3a,Pttubulin,PtL8,PtAPRT and Ptactin were selected for detection on materials from shading and high temperature conditions and different tissues.Based on RT-qPCR results,we calculated the Ct value and analyzed stability of IRGs by geNorm,NormFinder and BestKeeper.Under shading,Pt18S and PtL8 have a good expression stability,Pt18S and PtAPRT express relatively stable under high temperature,but Pt18S is stable among different tissues.Thence,Pt18S is the suitable IRG for P.ternata.3.Mining the key genesCombining next generation sequencing（NGS）and third generation single-molecule real-time（SMRT）sequencing,the whole plants of P.ternata from full sun-light and shading treatment were sequenced.Totally 136,163 non-redundant transcripts were obtained,with an average N50 length of 2578 bp.At day 20 of treatment,shading induced 6384 deferentially-expressed genes（DEGs）.Gene annotation and functional analyses revealed that these genes were related to auxin signal transduction,polysaccharide and sugar metabolism,phenylpropanoid biosynthesis and photosynthesis.The expressions of sixteen DEGs PtPIF3,PtSOD,PtCAT,PtPOD,PtARF1,PtXET,PtRGP,PtCAB151,PtRUB,PtPIN1,PtARF19,PtSUS,PtCHS,PtHY5,PtPIN6 and PtGR were verified by RT-qPCR.The RT-qPCR results are consistent with transcriptome data.4.Functional characterization of two key genesPtCAT and PtSUS,showing significant change in shading experiment,were selected for functional characterization.The overexpression vectors of the two genes were transformed into Arabidopsis thaliana for screening the T₃ homozygotes.When heat-treated at 43.5°C for 3.5 h and recovered at 24°C for 7 d,the survival two PtCAT and PtSUS-expressing Arabidopsis thaliana were 60%and 83%,higher than40%of wild-type Arabidopsis,respectively.The results indicate that PtCAT and PtSUS can positively regulate thermal tolerance of Arabidopsis,speculating they may take an important role in P.ternata growth during shading treatment.