Studies On Lipoxygenase And Its Associated Genes Related To Persimmon And Apple Fruit Ripening And Senescence

Softening and ripening speed are key factors affecting storage time and shelf life ofpostharvest fruit; therefore finding out internal mechanism is the basis to create new storagemethod. Lipoxygenase (LOX) is not only involved in fruit softening, maturation andsenescence process, but also takes part in jasmonic acid (JA) biosynthesis pathway which isrelated to ethylene and plays an important role in fruit maturation. In recent years, more andmore studies have been done to analyze LOX functions. Persimmon and apple belong toclimacteric fruit which is enjoyed by most consumers in consumer market. By now, nosequence information of LOX genes was reported on persimmon fruit, no systematicfunctional analysis of LOX family members was done on persimmon and apple fruit. In thisresearch, the relationships between LOX genes and the maturation and senescence process ofpersimmon (‘Fuping jianshi’) and apple (‘Golden Delicious’) fruit were preliminarily studied.Main findings were obtained as follows:1. Two LOX cDNA sequences, DkLOX1(GenBank accession No.JF436951) andDkLOX3(GenBank accession No.JF436950), were isolated from persimmon fruit usingRACE. Phylogenetic analysis indicated that they belonged to9-LOX. A recombinationprokaryotic expression vector pET-DkLOX1was constructed, which expressed a recombinantprotein of118kDa through SDS-PAGE electrophoresis.2. Persimmon fruit was treated with60mg L-1GA3and50mg L-1ABA after harvest.Physiological indicators were measured during fruit storage at room temperature (20~24℃)after treatment. The results showed that fruit treated with GA3had higher firmness and lowerMDA content, the peak of ethylene production and LOX activity were reduced, thereforeslowing down fruit softening and ripening process; ABA treatment promoted the fall of fruitfirmness and the rise of MDA content, ethylene production and LOX activity peaked3daysearlier than control, thus accelerating the process.3. Relative expression levels of DkLOX1and DkLOX3were analyzed by QPCR duringpersimmon fruit storage at room temperature (20~24℃) after GA3and ABA treatments,results showed that expression patterns of DkLOX1and DkLOX3were consistent with changes of LOX activity and ethylene production. Relative expression levels of the two LOXgenes were reduced by GA3treatment and peaked at15d in both treated and control fruit,while upregulated by ABA treatment and peaked3days earlier than control. DkLOX3wasmore sensitive to the two treatments.4. Physiological indicators of apple fruit were measured during12weeks (07/17to10/02)before commercial harvest of ‘Golden Delicious’. The steady increase in fruit size anddecrease in fruit firmness were observed, SPI increased obviously in the late stage, internalethylene were only obviously detected during last few weeks and had lower concentration.Gene family members of13-LOX and other genes related to JA biosynthesis pathway wereisolated from the12-week apple fruit by conventional PCR and QPCR, results showed thatthere were13genes expressed in both cortex and core tissue, including:4LOX (MdLOX22,MdLOX23, MdLOX28and MdLOX39),3AOS (MdAOS2, MdAOS3and MdAOS5),2AOC(MdAOC2and MdAOC5),2JMT (MdJMT2and MdJMT5) and2ERF (MdERF1andMdERF2).5. Expression patterns of the13genes above and MdACS3in cortex were detected byQPCR during12weeks before commercial harvest of ‘Golden Delicious’, results showed thattheir transcripts followed4changing modes: Ⅰ "early high", there were3genes, including:MdLOX28, MdAOC2and MdAOC5; Ⅱ "later high", a total of7genes, they were: MdLOX22,MdLOX23, MdAOS2, MdERF1, MdERF2, MdJMT2and MdACS3; Ⅲ:"no significantlychange", a total of2genes, they were MdAOS3and MdJMT5; Ⅳ "dropping all the time",there were2genes, including: MdLOX39and MdAOS5. Expression patterns of MdLOX23andMdAOS2were consistent in cortex and core tissue, their relative expression levelssignificantly increased at10/02.6. Fruit was sprayed with exgenous MeJA at SPI of1.2,1.9and3.5before commercialharvest of ‘Golden Delicious’. Physiological indicators were measured after each sampling.The results showed that, except extremely individual sampling points, no significantdifferences were found in firmness and SPI between treated and control fruit, while MeJA hadmore impact on endogenous ethylene production, when MeJA was applied at SPI of1.2,internal ethylene concentration increased obviously from09/18and peaked at the lastsampling point; when applied at SPI of1.9, internal ethylene concentration kept rising aftertreatment, peaking at09/18and then reduced; when applied at SPI of3.5, internal ethyleneconcentration increased at early sampling stage but decreased at late satge.7. Relative expression levels of LOX and AOS genes in cortex tissue of ‘GoldenDelicious’ were detected by QPCR after MeJA application at SPI of1.2,1.9and3.5. Theresults were: when MeJA was sprayed at SPI of1.2, except MdLOX39and MdAOS2, expression levels of the other genes rapidly increased on the first day after treatment; whenapplied at SPI of1.9, except MdAOS2, expression levels of the other genes were up-regulatedon the first day after treatment, MdLOX22, MdLOX23and MdLOX28had higher expressionlevels than control throughout all sampling points; when applied at SPI of3.5, exceptMdAOS2and MdAOS3, expression levels of the other genes increased on the first day aftertreatment and were higher than control during the entire sampling stage.