Construction Of Normalized Full-Length CDNA Library From Nai And Cloning And Expression Profile Analysis Of Related Genes From Nai Browning Fruit

"Nai"(Prunus salicina) belongs to the prunus family, Rosaceae, and believed tobe originated from FuJian province, southern of China. Nai is one of the fomous,special and excellent fruits which has the shape of peach and the pulp of plum, themediate sweet and sour taste. Fruit browning always occur at the stage of fruit harvestand storage. The browning fruit has normal appearance, but brown pulp, especiallythe pulp near the fruit cavity. Fruit browning not only deteriorate fruit edible qualityand storage property, but also deprive of the value of commodities, seriouslyinfluenced the market reputation of Nai fruit and economic benifit. So, the issue of thefruit browning has become the bottleneck of industry development of Nai fruit.This research took Oil Nai fruit at early stage of fruit browning as material,combined the SMART library-construction technology and DSN homogenizationmethod, constructed a full-length normalized cDNA library of Oil Nai fruit withbrowning. After massive5'EST sequencing and function annotation analysis, theselection of six genes (numbered NFH-102, NFH-C23, NFH-F21, NFH-17,NFH-633and NFH-99, respectively) possibly related to Oil Nai fruit browning havebeen undergone bioinformatics analysis, promoter cloning analysis, Real-time PCRanalysis and the various gene expression of these genes under different biotic stressand abiotic stress, according to the result of EST gene function anticipation andprevious research related to fruit browning. The objectives were to understand themolecular mechanisms of Oil Nai fruit browning.1Total RNA was extracted from the fruit of Oil Nai with beginning of browning,then reverse transcribed into cDNA by two-step amended SMARTTMcDNAsynthesized method which has higher proportion of full-length cDNA. The full-lengthnormalized cDNA library was constructed by the combination of long distance PCR,DSN (Duplex-Specific Nuclease) treatment and directed cloning. The titer of primarylibrary was about2.0×106pfu/ml and99%clones were recombinant, and the lengthof insert cDNAs were about1.5kb. The cDNA library was well normalized with8.4%redundancy. A total of720random clones in this cDNA library were thensequenced by5'EST. A total of valid684ESTs were attained by fragment assembly,removing the contaminated sequences and frameshift mutation. Then647unigenes were attained, which included21contigs and626singlets. Annotation by the onlineBlast2go search against GenBank database on NCBI web server revealed that mostof genes with predicted function were related to energy metabolism, protein synthesis,degradation of secondary metabolites, cell wall metabolism and transcription factors.These results will lead to further research of mechanism of browning fruit inmolecular level and develop good foundation of cloning related important genes ofbrowning from Oil Nai.2According to the function prediction of the six selected genes and the result ofprevious research related to fruit browning, the analysis of the six selected genes hasconducted from bioinformatics, promoter cloning and the various gene expression ofthese genes under different biotic stress and abiotic stress, the main result asfollowed:(1) Phenylpropanoid metabolism pathway is one of the important pathways ofsecondary metabolism. Phenylalanine ammonialyase (PAL) which catalyze the firststep of phenylpropanoid metabolism pathway, is the key enzyme and rate–limitingenzyme of phenylalanine metabolism pathway. This research has separated PAL genewhich has a full sequence of2497bps containing2154bps open read frame, a119bps5′UTR and a224bps3′UTR, respectively, encoding718amino acids withmolecular weight of78kDa and the isoelectric point of6.6. Systematic cladogramanalysis revealed that PAL gene cloned and the counterpart from Prunuspseudocerasus belong to the same cluster, which had conserved regions ofPhenylalanine ammonialyase–Histidine ammonialyase (PAL-HAL). Compared withcontrol, PsPAL showed a differential expression mode in varying degrees attranscriptional level under various abiotic stress and ethylene treatment. After woundand low temperature treatment, PsPAL showed a obvious up-regulated expressionmode. After high temperature and oxygen free treatment, PsPAL gene wasup-regulated first and then down-regulated. The gene expression showed up-down-up mode under ethylene treatment.(2) Three expansin genes have been separated from the full-length normalizedcDNA library, named PsEXP1, PsEXP2and PsEXP3with Genbank accession numberJN675711, JN675712and JN675713, respectively. The full length of these genes were 1303bp,1392bp and1384bp with open read frame of759bp,780bp and759bp,respectively. Systematic cladogram analysis revealed that PsEXP1, PsEXP2andPsEXP3belonged to different sub-family with various biological function ofα-expansin. Genome DNA sequence amplification revealed that all of these threegenes contained three exons and two introns. At the upstream of PsEXP1, PsEXP2and PsEXP3, three regulatory sequence with length of899bp,744bps and596bpswere acquired respectively. All of these regulatory sequences had the specialstructural character, such as TATA-box, CAAT-box, anaerobic induction element,endosperm expression element and photoreaction element et al. The result ofexperiment showed that all of the three genes have transcribed through the entiregrowth and developmental period except PsEXP1was absent during the early stage offruit development. The expression of these three genes were induced by ethylene andwound, but not by high and low temperature and oxgen free.(3) It revealed that the sequence of NFH-633shared high homogeneity with thesequence of MdNAC from Malus domestica by5′EST sequencing of the full-lengthnormalized cDNA library. This clone named PsNAC and submitted to Genbank withaccession number of JN67510has a1077bp length of ORF with359aa and39kDamolecular weight. The homogeneity between PsNAC and the counterpart from Malusdomestica, popar and Navel orange was more than60%. Bioinformatics analysisshowed that the expression of PsNAC might be relevant to decrepit and environmentalstress etc. Real-time PCR revealed that PsNAC transcript was up-regulated by wound,ethylene and oxygen free treatment, especially by ethylene, whereas it wasdown-regulated by high temperature and unchanged under low temperature treatment.(4) WRKY transcription factor was another transcription factor separated from thefull-length normalized cDNA library. So far as, this WRKY transcription factorseparated here, named PsWRKY, was reported the first one from Nai fruit. It has beensubmitted to Genbank with accession number of JN675708, with full length of1836bp and encoding533amino acids. Semi-quantitative analysis demonstrated that it hadhigh expression level in browning fruit than normal fruit. Bioinformatics analysis ofthe sequence of amino acids deduced by this gene showed that it has the character ofWRKY family with two WRKY conservative domain, two zinc domain of C2-HC style and a putative nucleus-located signal PKRR. PsWRKY protein could bind to Wbox to activate the GUS gene expression. Transient expression analysis in onionepidermal cells suggested35S:: PsWRKY-GFP fusion protein was localized in thenucleus,Real-time PCR revealed that PsNAC transcript was down-regulated by woundand high-temperature treatment, whereas it was up-regulated by ethylene, oxygen freeand low temperature treatment.3Polyphenol oxidase (PPO) has been regarded to be a critical enzyme in foodtechnology. A new clone, named PsPPO2, was separated from cDNA library preparedfrom mature leaf of the Prunus salicina. The deduced amino acid sequences shared81%and80%identity with the PPO from Hebei Yali pear and Fuji apple and shared55%and56%identity with the PPO gene from P.salicina leaf and fruit whichprevious isolated, respectively. RT-PCR showed that three PPO gene display uniquepatterns of expression in developmental specific manner in leaf and fruit tissue. In theprocess of leaf development, the PsPPO2mRNA was detectable at all stages. Incontrast, PsPPO1and PsPPO3were expressed in young tissue. PsPPO2and PsPPO3expression are higher at the early stages of fruit development, then dramaticallyreduced as the fruit ripened. The PsPPO1mRNA was detectable only at the browningfruit. Upon wounding, PsPPO1was significantly induced in mature fruit, whereas thelevel of PsPPO2and PsPPO3were not affected by mechanical damage.