Genetic, Physiological And Biochemical, Gene Expression Analysis Of Bush Mutant Cga In Cucurbita Moschata

Plants in Cucurbita moschata(Duch.ex Lam.)Duch.ex Poiret have trailing vines,which requires wide planting distances.Bush plants in C.moschata have some features that make them valuable,i.e.they allow higher plant densities,facilitate cultivation,and have a more concentrated maturity than vine counterparts. To understand the systematic regulation and physiological/molecular mechanism of vine development in C. moschata,the present study did the inheritance analysis and microscopic observation on the bush mutant cga and vine plants(wild type)derived from 'Non-vine 1' in C.moschata.Then the hormone sensitivity and physiological or biochemical differences between bush and vine plants in the cga and its wild type were studied.What'more,the differentially expressed genes during internode development of bush mutant were also studied by using cDNA-AFLP(cDNA-amplified fragment length polymorphism).The expression pattern of some transcript derived fragments(TDFs)were analyzed by RT-PCR(reverse transcriptase polymerase chain reaction).Finally,one gene that was related to the vine development in C.moschata was cloned and characterized from vine plants.The results were as follows:(1)Comparisons between the bush mutant cga and its wild-type habit of C.moschata in terms of various vegetative and reproductive characteristics revealed that the bush plants were characterized by short vine,short internode,small number of internode,few male flowers and early days to anthesis.In addition, the bush and vine plants had no difference in leaf length,leaf width,and petiole length.The study on vegetative and reproductive characteristics of F₁ revealed that it was similar with bush plants.Moreover, genetic analysis indicated that the cga have a monogenic inheritance,in which the bush genotype is dominant(Bu)to the vine genotype(bu).The measurement of hypocotyl growth and microscopic observation revealed that this bushy appearance of cga is the result of cell elongation inhibition.(2)The hormone sensitivity of bush and vine plants in C.moschata was studied by spaying with different concentrations of BR,IAA,GA₃,and GA₄₊₇.The result showed that all of the 4 hormones could not restore the vine elongation of bush plants.BR and IAA could restrain the vine elongation of bush and vine plants.The vine elongation of bush and vine plants could respond to GA₃ and GA₄₊₇.GA₃ and GA₄₊₇ could stimulate the vine elongation of bush plants by 188.9%and 52.9%.So,the bush mutant cga was neither hormone defect nor hormone insensitive mutant.What's more,BR and IAA could restrain the petiole elongation while GA₃ and GA₄₊₇could stimulate the petiole elongation of plants in C.moschata.However, GA₃ and GA₄₊₇had no influence on the leaf development of plants in C.moschata. (3)During the development of the bush mutant cga and its wild-type plants in C.moschata,the metabolism of reactive oxygen species(ROS)(hydrogen peroxide,H₂O₂;superoxide anion radical,O₂^-·), the activities of some antioxidant enzymes(superoxide dismutase,SOD;catalase,CAT;ascorbate peroxidase,APX;and peroxidase,POD),and POD isozymes,as well as the protein profiles and the levels of some endogenous hormones(Gibberellin A₃,GA₃;Gibberellin A₄,GA₄;and indole-3-acetic acid,IAA)were compared.Bush plants were found to have a significantly higher O₂^-·level than vine plants 28 days after germination and onwards.Moreover,there was no significant difference of H₂O₂ level between the two genotypes until 32 d,in which the difference was significant.Bush plants had a higher CAT activity than vine plants.In both genotypes analyzed,no significant differences of SOD activity were observed,and this was opposite to APX activity in which significant differences were observed during almost the whole sampling stages.What's more,APX activity in bush plants was significantly higher than that of vine plants at 24,28,32,and 36 d.POD activities of internode and leaf tissues of the bush plants were higher than those of respective vine-tissues.Roots of bush plants,however,had a lower POD activity than vine plants.In both bush and vine plants,the POD activities were lower in leaf tissues than in root and internode tissues.Each organ had a characteristic isozyme pattern.Electrophoretic comparisons revealed qualitative differences in POD patterns in internodes between bush and vine plants.Moreover,qualitative differences between internode and root protein profiles were found between bush and vine plants in C.moschata.Quantitative analysis by enzyme-linked immunosorbent assay(ELISA)indicated that the interodes of bush plants contain lower levels of GA₃,IAA,and GA₄ than those of vine plants in C.moschata.In addition,GA₃ deficiency was also found in the leaves and roots of bush plants.(4)The differentially expressed genes during internode development of C.moschata bush mutant cga were identified by using cDNA-AFLP method.58 differentially expressed transcripts have been successfully sequenced and identified.Among them are genes involved in metabolism and energy,unknown function protein,unknown gene,stress and defense,cell wall biosynthesis or modification,as well as genes encoding transcription factor and signal transduction.4 of these sequences have been shown to be differentially expressed between bush and vine plants of C.mosehata as well as different tissues using RT-PCRanalysis.(5)A novel gene encoding NADH dehydrogenase was isolated from a wild type of cga in C.moschata. This gene,designated as(?)ucurbita(?)oschata(?)sh related(?)(CmBul),was 545 bp in length and was composed of a 477 bp open reading frame,which had 99%nnucleotide similarity to the chloroplast ndhJ gene for NADH dehydrogenase subunit J from Brassica oleraeea.The deduced amino acid sequence of CmBul had 99%similarity to NADH dehydrogenase subunit J from Arabidopsis.Analysis of the basic characteristics of the CmBul protein revealed that it has one Respiratory chain NADH dehydrogenase 30 Kd subunit signature,three N-myristoylation sites,one Casein kinaseâ…¡phosphorylation site,and one Protein kinase C phosphorylation site.RT-PCR analysis showed that CmBul was expressed at a high level in the internodes and hypocotyls and was expressed stronger in elongating than in fully expanded internodes.In conclusion,results obtained in the present study suggest that CmBul gene might play important roles in vine elongation of tropical pumpkin.