Study On Glyoxalase-Based Breeding To Increase The Salt Tolerance Of Daylily(H.fulva)

Plants accumulate excessive amounts of toxic aldehyde compounds(such as methylglyoxal)under biotic-and abiotic stress conditions.Glyoxalase can effectively remove such compounds to maintain homeostasis.Previous studies have shown that overexpression of glyoxalase genes significantly increased plant salt tolerance.Improving plant resistance to stress conditions has always been one important target of plant breeding.The glyoxalase activities of daylily cultivars were measured,and the cultivars with highest and lowest glyoxalase activities were selected to establish glyoxalase based salt-tolerance screening system.The selected daylily cultivars with different glyoxalase activities were treated with different culture media with different salt concentrations,and the glyoxalase activity,methylglyoxal and malondialdehyde contents of the leaf samples at different treatment times were measured to verify the correlation between salt tolerance and glyoxalase activity.In addition,the functions of daylily glyoxalase genes were also studied.The experiments of this thesis are mainly divided into the following four parts.(1)Six putative reference genes of daylily were screened by analyzing the RNA-Seq data,and the real time fluorescence quantitative PCR.(2)Glyoxalase activities of 30 randomly selected daylily cultivars were determined.The daylily cultivars with the highest and lowest glyoxalase activity were selected for salt tolerance determination.The correlation between daylily salt tolerance and glyoxalase gene related transcripts were analyzed.(3)The selected daylily cultivars with different glyoxalase activities were cultivated culture media containing different concentrations of sodium chloride.And the expression levels of various daylily glyoxalase transcripts were detected by real-time fluorescence quantitative PCR.(4)Hf Gl XI-1,the main responsible transcript for glyoxalase activity,were cloned and an over-expression binary vector was constructed.And the pollen tube pathway transformation method was used to generate transgenic plants,which were identified by genomic DNA amplification.This is the first report for evaluating the stability of daylily reference gene of quantitative reverse transcription polymerase chain reaction(q RT-PCR),which provides the basis for the gene expression analysis of daylily.The full-length c DNA of Hf Gl XI-1 was cloned by analysis of RNA-seq data,and transgenic daylily plants over-expressing Hf Gl XI-1 were obtained by the pollen tube pathway transformation method to elucidate the biological function of the glyoxalase gene in daylily,to provide new methods for breeding daylily cultivars with salt resistance.Understanding the correlation between salt stress tolerance and the activity of daylily glyoxalase I activity will provide a theoretical basis for the rapid and accurate selection of salttolerant ornamental plants.The main conclusions of this thesis are as follows.1.Six putative daylily candidate reference genes for q RT-PCR [actin-depolymerizing factor 7-like protein(Hf ADF),glyceraldehyde 3-phosphate dehydrogenase(Hf GAPDH),TIP41-like family protein(TIP41),alpha tubulin(Hf TUB),elongation factor-1a(Hf EF-1-?)and polyubiquitin gene(Hf UBQ)] were obtained by analysis unigenes of the third generation RNA-seq.There are many transcripts identified from daylily RNA-seq data for aforementioned putative reference genes.The number of transcripts of Hf UBQ is up to 40.The best reference genes for studying six different organs of daylily were Hf TIP41 and Hf GAPDH.Six putative candidate reference genes were selected for daylily with high salt stress treatment: Hf ADF,Hf TUB,Hf GAPDH,Hf TIP41,F-box protein(Hf F-box)and Hf UBQ for q RT-PCR.The results here showed that the optimal reference genes of daylily with salt stress treatment were Hf F-box and Hf UBQ,and Hf TUB was not recommended.2.Two daylily cultivars with relatively higher glyoxalase activity were selected by measuring the activity of glyoxalase I.They were H.fulva �Stella de Oro'and H.fulva�Serenity Morgan'.Two cultivars with relatively lower glyoxalase activity were also selected.They were H.fulva �Double Talk',H.fulva �Big City Eye'.The fore selected daylily cultivars were treated with culture media containing different concentration of Na Cl.And it was found that the leaves of H.fulva �Double Talk',H.fulva �Big City Eye' began to turn yellow on day 1,while that of H.fulva �Stella de Oro'and H.fulva�Serenity Morgan' began to have obvious yellowing on the second day.Thes data indicated that H.fulva �Stella de Oro' and H.fulva�Serenity Morgan'were tolerance to salinity,and H.fulva �Double Talk',H.fulva �Big City Eye' were sensitive to salinity?3.Correlation analysis between methylglyoxal(MG)content,glyoxalase activity and salt tolerance showed that there was a significant positive correlation between the salt tolerance of daylily and glyoxalase activity.The higher the glyoxalase activity,the stronger the salt tolerance of daylily.4.q RT-PCR of daylily cultivars under salt stress condition was performed to compare the expression level of daylily glyoxalase related transcripts.Correlation analysis was performed on the glyoxalase I activity and expression levels of glyoxalase related transcripts at 1 day under high salt stress.The results here showed that there was a significant positive correlation between glyoxalase enzyme activity and transcript 24211(named Hf Gl XI-1),a weak positive correlation with 20549.Therefore,it is speculated that the transcript 24211 plays a major role in the activity of glyoxalase,providing a basis for salt resistance gene cloning and genetic transformation of daylily.5.The coding sequence(CDS)of daylily glyoxalase(Hf Gl XI-1)was cloned by RT-PCR according to the RNA-seq data.6.The CDS of Hf Gl XI-1 is 885 bp in length and encodes 295 amino acids.Molecular evolutionary tree analysis shows that this protein encoded by the gene belongs to Clade I with glyoxalase activity.The protein structure prediction shows that Hf GLXI-1 is mainly composed of random coils,alpha helices and extended chains.Its ligands are divalent cobalt ions and glutathione,which are consistent with the properties of glyoxalase.The result of PCR amplification showed that the size of the amplified genomic DNA band was close to the molecular weight of the gene CDS.The sequencing results showed that the above two sequences were completely consistent speculating that there was no intron in the Hf GLXI-1 gene.7.The 35S::Hf GLXI1 fusion gene was constructed,and 5 positive daylily transgenic plants were obtained by pollen tube pathway transformation,and verified by genomic DNA amplification.