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Cloning And Analysis Of Genes Involved In Cucumber Ethylene Signal Transduction Pathway And Improvement Of The Cucumber Genetic Transformation System

Posted on:2015-06-24Degree:DoctorType:Dissertation
Country:ChinaCandidate:B B BieFull Text:PDF
GTID:1223330422488735Subject:Biomedical engineering
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Cucumber (Cucumis sativus L.), an annual trailing herbaceous plant,belongs to the Cucurbitaceae family and is an economically important crop aswell as a model system for sex determination studies, owing to its rich diversityof sex expression.Ethylene plays an important role in plant development and stress responseas an important plant hormone. Ethylene can regulate seed germination, seedlinggrowth, leaf and flower abscission, fruit ripening, organ senescence and sexdetermination and so on. In regard to cucumber, ethylene has been demonstratedto promote female flower development and is highly correlated with thefemaleness in cucumber. To date, M and F gene that determine the ratio ofmale-to-female flowers in cucumber have been cloned and they are all encode1-aminocyclopropane-1-carboxylic acid synthase (ACS), a key rate-limitingenzyme in ethylene biosynthesis. In contrast, still little is known about the roleof ethylene signaling in cucumber, mainly because the important components inthe cucumber ethylene signaling pathway have not yet been separated andidentified. To gain a better understanding of this particular pathway in cucumber,in this study,we isolated three key genes involved in cucumber ethylene signaltransduction pathway from the monoecious plant line S52and conducted thebioinformatics analysis, expression pattern analysis and ectopic expression inArabidopsis mutant, main achievements have been gained as follows:1. The gene encoding CONSTITUTIVE TRIPLE RESPONSE1-Like (CTR1-Like) was isolated from cucumber, designated as CsCTR1. The cDNAsequence contained an open reading frame of2559bp encoding852amino acids.CsCTR1located on chromosome6and were single copy in cucumber genome.Genomic DNA sequence contained15exons and14introns. Multiple aminoacid sequence alignments revealed that the molecular structure of CTR1-likeproteins appears to be highly conserved in different plant species. Phylogenetictree revealed that plant CTR1were derived from a common ancestor duringevolution and CsCTR1was grouped into a cluster along with melon andpumpkin, belonging to the Cucurbitaceae family. Ectopic expression of CsCTR1in Arabidopsis ctr1-1mutant could restore its ethylene signaling ability,suggesting CsCTR1indeed perform its function as negative regulator of ethylenesignaling pathway. CsCTR1is constitutively expressed in all cucumber organsexamined including roots, stems, leaves, shoot apices, mature male and femaleflowers, as well as young fruits, however it was highly expressed in mature maleflowers. CsCTR1mRNA level gradually declined during male flowerdevelopment and increased gradually during female flower development.However, the expression level of CsCTR1always higher in male flowers thanthat in female flowers during whole floral developmental stages. Additionally,our results indicated that CsCTR1mRNA level could be induced by externalethylene in roots, leaves, and shoot apices. Between S52line (ffMM) and B52line (FFMM), a pair of near-isogenic lines (NILs) at F loci, the expression levelof CsCTR1in shoot apices and mature flower in B52was higher than that inS52.2. The gene encoding ETHYLENE INSENSITIVE2(EIN2) was isolatedfrom cucumber by RACE, designated as CsEIN2. The full-length of cDNAsequence was4565bp and contained an open reading frame of3873bpencoding1290amino acids. CsEIN2located on chromosome6and were singlecopy in cucumber genome. Genomic DNA sequence contained7exons and6introns. Multiple amino acid sequence alignments and phylogenetic treerevealed that the CsEIN2had very high homology with its counterparts fromother plant species and plant EIN2were derived from a common ancestor during evolution and CsEIN2was grouped into a cluster along with melon. Ectopicexpression of CsEIN2in Arabidopsis ein2-1mutant could partially recover itsethylene signaling ability. CsEIN2was constitutively expressed in all cucumberorgans examined including roots, stems, leaves, shoot apices, mature male andfemale flowers, as well as young fruits,however its expression level exhibitedsome differences among different tissues. There were no remarkable changes inthe accumulation of CsEIN2mRNA during male flower development. Duringfemale flower development, CsEIN2mRNA level was gradually increased andthen gradually declined from stage of FF3, however, the changes in expressionlevel was unremarkable. In addition, our results indicated that CsEIN2mRNAlevel could not be induced by external ethylene in roots, leaves, and shoot apices,it is suggested that CsEIN2might be regulated at protein level. Between S52line(ffMM) and B52line (FFMM), a pair of near-isogenic lines (NILs) at F loci, theexpression level of CsEIN2in shoot apices and mature flower in S52wasdramatically higher than that in B52.3. The gene encoding ETHYLENE INSENSITIVE3(EIN3) was isolatedfrom cucumber by RACE, designated as CsEIN3. The full-length of cDNAsequence was2560bp and contained an open reading frame of1908bpencoding635amino acids. CsEIN3located on chromosome1and were singlecopy in cucumber genome. Genomic sequence of CsEIN3did not contain theintron. Sequence alignment and phylogenetic analyses revealed that CsEIN3hashigh homology with other plant EIN3/EIL proteins that were derived from acommon ancestor during evolution, and CsEIN3was grouped into a clusteralong with melon. We found that CsEIN3is constitutively expressed in allcucumber organs examined including roots, stems, leaves, shoot apices, maturemale and female flowers, as well as young fruits,however its expression levelshowed some differences among various different tissues. We also found thatCsEIN3mRNA level was gradually increased during both male and femaleflowers development, however it was always higher in male flower than that infemale flower at each development stages. Between S52line (ffMM) and B52line (FFMM), a pair of near-isogenic lines (NILs) at F loci, the expression level of CsEIN3in shoot apices and mature flower in S52was dramatically higherthan that in B52.4. We optimized the cucumber genetic transformation system mediated byAgrobacterium tumefaciens using cotyledonary node of cucumber inbred linesS52. The results showed that there was the highest rate of regeneration (100%)when cotyledons just separated from husk, and each explant had4.13ofregenerated shoots. The gus transient expression rate from this treatment washigher than others. Higher efficiency of shoots regeneration was obtained fromthose explants with mild wounds. But explants with deep wounds had the lowesttransformation efficiency. On that basis, we also have studied the effects ofdifferent concentrations of6-BA and ABA on the regeneration from cotyledonnode. The results suggested that MS+1.5mg/L6-BA+0.5mg/L ABA+2.0mg/LAgNO3was the optimal formula. We also confirmed the critical concentration ofKanamycin for shoot and root induction was50mg/L. In addition, sometransformation parameters were optimized including pre-culture period,co-culture period and immersion time. The result showed that we could obtainhigher transformation efficiency when pre-cultured for1-2days, immersed for20minutes and co-cultured for3days. We also demonstrated that renewingculture integrated the proper screening concentration was important for genetictransformation using cotyledonary node of cucumber.In conclusion, this study will provide the basis for further study on the roleof CsCTR1, CsEIN2and CsEIN3in the molecular mechanism of the cucumberethylene signaling pathway and relevant biological processes of cucumber.Additionally, optimization of cucumber genetic transformation system alsoprovides the theoretical and technical platform for genetic engineering ofcucumber.
Keywords/Search Tags:Cucumis sativus (cucumber), ethylene signaling, CONSTITUTIVETRIPLE RESPONSE1(CTR1), ETHYLENE INSENSITIVE2(EIN2), ETHYLENE INSENSITIVE3(EIN3), genetic transformation
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