Analyses Of Photosynthetic Characteristics And Chlorophyll Biosynthesis Of Allotetraploid In Cucumis

Cucumis hytivus(Cucumis × hytivus J.F.Chen&J.H.Kirkbr,2n = 4x = 38)is a newly synthesized allotetraploid between a wild cucumber(C.hystrix,2n = 2x = 24)and a cultivated cucumber ’Beijingjietou’(C.sativus,2n = 2x = 14).In contrast to green leaves of C.sativus and dark green leaves of C.hystrix,the color of C.hytivus leaves was yellow-green in seedling stage but can recover to nearly ’normal’ green in mature stage.Based on the leaf color mutation,this study comparatively analyzed the plant growth,pigments content,photosynthetic characteristics and chloroplast ultrastructure of leaves,as well as the chlorophyll synthetic metabolism and key enzyme genes,preliminary discussed on the causes of leaf chlorosis and explored the impact of allopolypoidization on the new species and laid foundation elucidating the mechaims of leaf chlorosis.1.Characterization of photosynthesis and plant growthThe pigments analysis showed that Chl a,Chl b and Chl content in the young leaves of C.hytivus were significantly lower than its parents,and slowly increased to normal level in the mature leaves.This demonstrated that lower chlorophyll content is the direct reason to leaf chlorosis.The result of plant growth,biomass allocation and seed weight and size indicated that the growth of C.hytivus was not adversely affected by the Chl deficiency,the data of measurement were intermediate and paternal-biased inherited in C.hytivus.The same trend was also observed in photosynthetic parameters.However,the chloroplast ultrastructure obviously showed that the chloroplasts of young leaves in C.hytivus contained narrower grana stacks and fewer thylakoids,and the thylakoid lamellar structures appeared visibly looser,and were irregularly arranged,compared with those of its parents.Thus we speculated that the lower chlorophyll content may caused by the disturbed development of chroloplast.2.The biosynthesis of chlorophyllThe measurement of the intermediates of chlorophyll synthesis metabolism showed that the content of 8-aminolevulinic acid(ALA),porphobilinogen(PBG)and uroporphyrinogen(Urogen Ⅲ)in young leaves of C.hytivus accumulated distinctly,yet corporporphyrinogen Ⅲ(Coprogen Ⅲ)content reduced remarkably,and then protopor-phyrin Ⅸ(Proto Ⅸ),Mg-protoporphyrin IX(Mg-Proto IX)and protochlorophyllide(Pchlide)were all decreased accordingly.Whereas the content of Coprogen Ⅲ increased in mature leaves of C.hytivus.It suggested that the biosynthesis of Chl in C.hytivus may be blocked at the step of converting Urogen Ⅲ to Coprogen Ⅲ.The qRT-PCR analysis of 19 key genes involved in chlorophyll synthesis showed that POR,HEMA1 and HEME2 were significantly down-regulated in C.hytivus compared to its parents.Besides,HEME2 which encoded uroporphyrinogen decarboxylase can catalyze the synthesis of Coprogen Ⅲ.The results indicated that the expression of POR,HEMA1 and HEME2 may contribute to the reduced chlorophyll level in the young leaves of C.hytivus.3.Gene cloning of POR,HEMAl and HEME2Three important genes,POR,HEMA1 and HEME2,involved in the chlorophyll biosynthesis from C.hytivus and its parents were cloned.POR gene from C.hystrix contained a 1199 bp ORF encoding 399 amino acids protein,and there were 14 different nucleotide sites and 1 different amino acid compared with C.sativus.The obtained cDNA of HEMA1 gene from C.hystrix was 1659 bp in length which encoding 552 amino acids.Compared with C.sativus,there were 25 different nucleotide sites and 3 different amino acids.HEME2 gene from C.hystrix contained 1179 bp ORF encoding 392 amino acids and resulting in 6 different nucleotide sites blast to C.sativus.Through the analysis of sequence alignment,base mutation was unfounded in the process of polyploidization,however there was an obvious phenomenon of genetic recombination.Therefore we assumed that there was no base mutation in the key enzymes genes of chlorophyll synthesis,however the recombination of different gene sequence may affect the gene expression.Besides,the interaction between homologous genes may cause changes of methylation which not involved in DNA sequence changes in the process of allopolyploidization.