The Role Of Auxin Signaling Pathway Related Genes In The Development Of Bulbil For Two Species Of Plant

In this thesis,Pinellia ternata(Araceae)and Lilium sulphureum(Liliaceae as main materials)and Pinellia cordata(Araceae,as supplemental materal)were selected to explore roles of auxin in bulbil development.Sequencing of transcriptomes and expression profiles were carred out based on various tissues at differential bulbil developmental stage for these three species.Five candidate genes were screened: PteAUX1,LsuAUX1 and PcoLAX2 that were involved in auxin polar transportation,PteARF11 and PcoARF11 that were involved in auxin responses.Full-length CDS sequence of these candidate genes were carred out by RACE method.The AthAUX1 gene sequence of Arabidopsis thaliana was used as a reference to predict the transmembrane region of PteAUX1,LsuAUX1 and PcoLAX2,to show mutation sites,and so on.Phylogenetic tree were constructed and homology analysis were carried out based on sequences of cloned and family members from NCBI to affirm identity of cloned sequences.Fuethermore,PteAUX1,PteARF11 and LsuAUX1 were quantitified by qPCR to show its relationship with bulbil development.Results of the study are listed as follows:1.Based on results of transcriptome sequencing of P.ternata,P.cordata and L.sulphureum,793,656 and 487 Unigenes that were related to auxin metabolism and signal transduction were screened,respectively.For these Unigenes,87,66 and 36 Unigene are involved in auxin biosynthetic pathways,respectively;The inactive metabolic pathway-related Unigene were 39,74 and 71,respectively;Unigenes associated with the auxin transport pathway were 58,81 and 102,respectively.609,435 and 278 Unigenes were screened in the signal transduction pathway,respectively.The transcriptome sequencing of P.ternata has identified the YUCCA gene and the AAO gene;the transcriptome sequencening of P.cordata has identified the nitrilase(NIT),YUCCA gene and indole aldehyde oxidase(IAO)genes;the transcriptome sequenceing of L.sulphureum has identified NIT,TAA1/SAV3(Tryptophan Aminotransferase of Arabidopsis 1/Shade Avoidance 3),YUCCA,and IAO genes.These results showed that the pyruvate pathway was the common auxin biosynthesis pathway for these three species.Howerever,the acetonitrile pathway was only not identified in P.ternata among these species.The results of expression profiling showed that the petiole of was compared with that of bulbil,and between bulbil and tuber.DEGs involved in auxin biosynthesis,inactivation metabolism,transport and signal transduction in petiole/bulbil and bulbil/tuber of P.cordata were 280 and 184 Unigenes,respectively;in early bulbil/late bulbil and early bulbil/stem of L.sulphureum were 197 and 189,respectively;in petiole/bulbil and early bulbil/late bulbil of P.ternata 173 and 50,respectively.2.Regulational candidate genes related auxin metabism and signal transduction were screened based on DEGs of bulbil development and comaprision among these species.Five genes were selected as candidates: Unigene4372_All(PteAUX1),Unigene12362_pcoor(PcoLAX2),Unigene20438_lisul(LsuAUX1),CL8576.Contig1_All(PteAEF11)and Unigene1025_pcoor(PcoARF11).These candidate genes were targets for more researching works.3.The LsuAUX1 gene was cloned by RACE,posses 2037 bp nucleotieds,and encoded495 amino acids.The PcoLAX2 gene was cloned by RACE method,encoding 530 amino acids by 2313 bp nucleotides.The full-length cDNA sequence of PteAUX1 gene was 2327 bp,encoding 492 amino acids.The PteARF11 gene was obtained by sequencing the full-length cDNA,PteARF11 gene with 4097 bp,encoding 1022 amino acids.PcoARF11 gene colned by RACE obtained two fragments: 4127 bp and 3549 bp fragments,encoding 715 amino acids and 975 amino acids,respectively.4.The construction of phylogenetic tree and homologous comparison for cloned sequences and other members of AUX1/LAX protein family from Arabidopsis thaliana were carried out to affirm identity of cloned sequences.Cloned seuqnces in P.ternata and L.sulphureum are more close to AthLAX1,so they were nameds as PteAUX1 and LsuAUX1,respectively.The cloned sequences in P.cordata was more close to AthLAX2,so they were named as PcoLAX2.The predicted transmembrane regions of these AUX1/LAX protein inthree plants and Arabidopsis were the same.It were proposed that LsuAUX1,PteAUX1 and PcoLAX2 protein were located on the cell membrane and contained 11 transmembrane regions just like to AthLax1/2.Comaprision to AthAUX1 protein,the mutation sites were found mainly at the C-terminal and N-terminal region.There was no mutation at the functional important sites.So,LsuAUX1,PteAUX1 and PcoLAX2 were functioned in each species.It should play an important role in the transportation of auxin during the development of bulbil.5.Results of quantifying for PteAUX1,LsuAUX1,and PteARF11 in different tissues of P.ternata and L.sulphureum showed that auxin is transported from the apical leaves to the bulbil primodia or early bulbil.However,auxin transportation from leaves to early bulbil were decreased when differential bulbil growth continuously.Auxin reponses factor(such as PteARF11)possessed consequently various expression.