Selection Of Reference Genes And Expression Analysis Of HSFA1 In Amorphophallus

Real-time quantitative PCR(RT-qPCR)is a preferred method for analyzing gene expression currently.While the stable reference genes are a prerequisite for accurate quantification of gene expression levels.Amorphophallus is a kind of high value vegetable crop in southwest China,and some important functional genes in Amorphophallus are helpful to the improvement of Amorphophallus genetic improvement.In addition,Amorphophallus,a kind of poor heat tolerance crop,is currently only suitable for cultivating in moderate high altitude areas.Studying the expression levels of the Heat shock transcription factor A1(HSFA1)gene which is the main transcriptional regulator under heat stress will help to understand the thermoregulation mechanism of Amorphophallus,and it has important theoretical help to improve the heat resistance of Amorphophallus.We used the homologous cloning method and cloned the partical sequences of 9 candidate reference genes including Glyceraldehyde-3-phosphate dehydrogenase(GAPDH),Elongation factor 1-α(EF1-a),Eukaryotic initiation factor 4A(EIF4A),H3.2 histones(H3),Cyclophilin(CYP),β-Actin(ACTB),β-Tubulin(TUB)、Ubiquitin(UBQ)、 Ribosomal protein L16(RP)and HSFA1 in conserved areas by using Amorphophallus albus and Amorphophallus konjac as experimental materials,and identified the expression stability of the candidate reference genes under heat stress,waterlogging stress and across different tissues.The full length of HSFA1 was obtained by the method of Rapid-amplification of cDNA ends(RACE),and we predicted the open reading frame.And the subcellular localization patterns of HSFA1 genes in Amorphophallus were identified by onion epidermal subcellular localization experiment.At the same time we analyzed the relative expression levels of these genes at 41 ℃ across 0-8h in two species of Amorphophallus by using the screened combination of the stable reference genes as the internal control.The relative results were as follows: 1.cloning 9 reference genes of A.albus and A.konjacBased on the homologous conserved sequence of relative neighbor species in NCBI,we cloned the conserved coding region of nine reference genes(GADPH,ACTB,RP,UBQ,H3,TUB,CYP,EIF4 A,EF1-α)by using homologous cloning method in two species of Amorphophallus.And we predicted the coding amino acid residues based on the results of sequencing.We compared the genetic relationship between species through the construction of evolutionary tree.The results showed that the species which retain the closest kinship relationship with Amorphophallus are Anthurium andraeanum and other plants,and these plants belong to a same family with Amorphophallus.In addition,there are many species whose kinship relationships also close to Amorphophallus are somemonocot such as Elaeis guineensis,Phoenix dactylifera and Musa acuminata subsp et al.,which provides a reference for the homologous cloning of some related genes in Amorphophallus.2.Comparison of the stable reference genes under three conditions in two species of AmorphophallusBy applying RT-qPCR method and designing quantitative primers,we calculated Cq values of candidate reference genes expression across different tissues and under heat and waterlogging stress.The Cq values were transformed based on the principles of geNorm,NormFinder,and Bestkeeper.By calculating the stability values of different candidate reference genes and comparing the results of the stability values,we identified the stable reference genes in two species of Amorphophallus under different experimental conditions(heat stress,waterlogging stress and different tissues).The results revealed that EF1-α and EIF4 A were the two most stable reference genes in all sets.EF1-α,EIF4 A,H3,and UBQ were the most stable reference genes under heat stress.EF1-α,EIF4 A,and TUB were the stable reference genes under waterlogging;EF1-α,EIF4 A,and CYP were the stable reference genes across different tissues.3.Isolating HSFA1 gene in two species of AmorphophallusBased on the homologous conserved amino acid sequence of relative neighbor species and codon preference,we designed degenate primers and cloned the sequences with 1221 bps by using homologous cloning method.Using RACE methods,the downstream and upstream fragments of these genes was isolated.The stitching results showed that the total length of these genes were 1909 bp and had reached downstream the Ploy A tail region.ORF Finder pridected that these genes had the open reading frames with the length of 1569 bps which encoded 522 amino acid residues.The accuracy of RACE amplification was verified by designing full length primers.Domain analysis showed that this gene has a similar domain of HSFA1 gene in other species,C terminal containing AHA motif can activate downstream gene expression.The construction of neighbor tree showed that they had the closer relationship with monocot,but the similarity was all under 81%.Transmembrane structure prediction showed that they have no transmembrane structure.4.Analyzing the subcellular localization of these two proteins in onion epidermis cellsThrough constructing pCHF3-AkHSFA1-YFP and pCHF3-AaHSFA1-YFP fusion expression vector,we determined its location in onion epidermal cells.We found that these two proteins were mainly localized in the nucleus and cytoplasm.5.Obtaining the relative expression pattern of HSFA1 gene across different tissues in two species of AmorphophallusWe treated experimental materials of Amorphophallus under the high temperature of 41℃ and analyzed the expression of HSFA1 in the leaves and roots across 0-8 h in A.albus and A.konjac by using RT-qPCR and selecting EF1-α+H3+UBQ as reference genes.It was found that the expression level of HSFA1 in two species showed an overall increasing trendency and reached a peak at 2h after heat treatment.and had high expression abundance in leaves,which demonstrated that Amorphophallus is a thermosensitive crop.In comparison with the data of different tissues,we found that the expression of these genes in leaves were much higher than that in roots,which indicated that HSFA1 had high abundance of expression in leaves.By comparing with the expressions in two species,the expression of AaHSFA1 was higher than that of AkHSFA1 at different time points,which explained the reason why the heat tolerance of A.albus was stronger than that of A.konjac from genetic.In summary,the results of this study could provide an important theoretical reference and technical support for the analysis of gene expression and the heat-resistant regulatory network in Amorphophallus.