Cloning And Functional Analysis Of PpCHL1 In Kentucky Bluegrass

The full-length cDNA of 1391 nucleotides was cloned by RACE.The open reading fragments of PpCHL1 gene was 948bp in length,encoding 316 amino acid residues.The amino acid sequence compared by Blast revealed there was homology with chlorophyllase protein of other pants and the similarity to Triticum aestivum chlorophyllase was the highest with 74%.And there were conveserd typical lipase motify with serine as the active site.The gene encoding Poa pratensis chlorophyllase(PpCHL1),and its Genbank accession number is KU145126.The PpCHL1 promoter sequence was 1247bp long and contain multiple-homone responsive cis-acting elements,which including an TATA-box(core promoter element),Box4(a light responsive element),IAA responsive element(AuxRR-core),MeJA responsive element(TGACG-motif),CCGTCC-box(Plant meristem activation site),circadian(physiological regulation element).To select the most stable reference genes of Kentucky bluegrass for real-time quantitative PCR,we assessed tne mRNA expression in different tissues of six traditional candidate reference gene at various leaf development stages,under abiotic stress,or following various hormone applications.As a result,in Kentucky bluegrass leaves,GAPDH is recommended as the reference gene for analyzing the mRNA expression in different tissues;β-tublin was most consistent with different homones;EF-1α was the most stable gene under abiotic stress;ACT was stably expressed in the different leaf development stages.Hormone induction assays showed that gibberellins mainly regulated PpCHL1 expression.The expression level of PpCHL1 was different among root,stem and leaf,which the expression level in leaf was the highest,especlially in old leaf.Dark stress stimulated PpCHL1 highest expression in the middle treatment.Taken together,the results suggested that PpCHLl was closely involved in envriomental stimulation signal perception and involved in chlorophyll degradation.The coding region of PpCHLl was inserted into the expression vector 3302Y,which has a yellow fusion protein,and the recombined vector was named as 3302Y-PpCHLl.First,the fusion was then transformed into tobacco leaf,subcellular localization of transiently expressed PpCHLl-YFP fusion was detected by a confocal laser scanning microscope.The result revealed that PpCHLl localized in the nucleus and cytoplasm.Secondly,the 3302Y-PpCHLl was transformed into Arabidopsis,expecting to gain PpCHLl overexpression plant.After selection by antibiotics,the regenerated plants were obtained,DNA and RNA of different plants were extracted,PCR analysis of them showed some of them were positive lines.Chlorophyll content and chlorophyllase activities were measured in WT and transgentic Arabidopsis paints,the results showed some plants were transformed successfully.As a result,PpCHL1 is likely to sense environmental changes at the early stage of chlorophyll degradation.