| Fruit color is one of the important agronomic traits and appearance quality of goods for pear, and is also consumers’first sense before their purchasing decisions. Breeding pear cultivars bearing fruits with different fruit color to meet consumers’diversified needs is a long-term goal for pear breeding, and now works on pear fruit color is becoming a research focus. Study on fruit skin development of russet and green pears, and differentially expressed genes during fruit color formation in pear could help deeply understand the formation mechanism of pear russet fruit and green fruit, promote russet and green pear molecular breeding theory formation and breeding work. Russet pear and its green bud sports supply the ideas materials for pear fruit color research.In this article, biological characteristics was firstly studied on Huanghua pear (russet fruit) green skin bud sport——Green Huanghua. Secondly, studies on structure of fruit peel of ripe fruits and epidermal structure for Huanghua and Green Huanghua pear, fruit dot development, fruit skin development and epidermis development, the characters of Green Huanghua ripe fruit peels with different level of fruit rust and Huanghua pear russet peel were performed. Thirdly, high molecular weight RNA was isolated from the two kinds of pear peels sampled at the key stage of fruit skin development, and differentially expressed genes——the full length of pear defensin gene PpyDFNl and fragment of phenylalanine ammonia-lyase gene PpyPAL1in Huanghua and Green Huanghua pear peels were cloned by differential display reverse transcription polymerase chain reaction (DDRT-PCR) and rapid amplification of cDNA5’end (5’RACE). The sequence of PpyDFN1was further analy sized and subcellular location of PpyDFN1was studied. Next, the expression level of genes involved in phenylpropanoid secondary metabolites including phenylalanine ammonia-lyase gene PpyPAL1, cinnamic acid-4-hydroxylase gene PpyC4H and cinnamoyl-CoA oxidoreductase gene PpyCCR was determined in peels of Huanghua and Green Huanghua pear at different developmental stages to evaluate the level of secondary metabolism. Finally, the expression analysis of PpyDFNl in Huanghua and Green Huanghua pear, functional analysis in transgenic tobacco plants and bacterial inhibition in vitro were studied. The results were showed as follows.1. Green Huanghua is a stable green skin bud sport of Huanghua pear. Its fruit skin is green, maturiy is7to10days ahead of Huanghua pear, and the total soluble sugar, total soluble solids and titratable acidity is higher than that of Huanghua pear fruit, but the fruit hardness is lower than Huanghua pear. Besides that, other botanical characteristics and fruiting habit are close to Huanghua pear.2. Huanghua and Green Huanghua pear shared different fruit dot development pattern, in which the fruit dot on Huanghua fruit formed earlier and more intensively, developed faster and had the larger size than Green Huanghua fruit dot at the same developmental stage, whereas fruit dot on Green Huanghua fruit formed later, developed slower; Huanghua pear fruit surface wax before10weeks after flowering (WAF) was more, but continually decreased from10WAF, whereas Green Huanghua pear fruit surface wax was constantly present on the fruit from the observed fruit of4WAF to ripe fruit; the cuticle of Huanghua fruit was basically intact before10WAF, but the cuticle at the edge of fruit dot began to crack at10WAF, and the whole fruit cuticle completely cracked at the maturity, whereas Green Huanghua fruit cuticle from the young fruit to fruit ripening remained intact and rarely rupture; Before10WAF, the fruit epidermal membrane of Huanghua and Green Huanghua pear were both intact, colorless and transparent, but after10WAF, the fruit cuticle of Huanghua pear gradually cracked and finally completely cracked into pieces, exposed epidermal cells suberized and the epidermal membrane showed russet, whereas the epidermal membrane of Green Huanghua pear was almost intact, colorless, transparent and suberization seldom occurred for epidermal cells; Ripe pear peel structure followed by the cuticle, the epidermal cell layer and sub-epidermal cell layer from outside to inside. For ripe Huanghua pear, its fruit surface was rough, cuticle completely cracked, the epidermal cells exposed or underneath the cuticle suberized, showing russet, and could not be stained by phloroglucinol solution, whereas for ripe Green Huanghua pear, its fruit surface was smooth, cuticle was intact and epidermal cells underneath it was green or colorless, without suberization. The epidermal membrane of ripe Huanghua pear fruit was russet, and determined the fruit color, whereas the epidermal membrane of ripe Green Huanghua pear fruit was coloeless and transparent, did not determine the fruit color.3. Green Huanghua pear peel with heavy rust was the same as Huanghua pear peel in substance, and the differences of fruit peel with light rust and heavy rust was the degree of cuticle injury, the suberized degree of epidermal cells, and the integrality of the suberized epidermal cell layer.4. A method to isolate high molecular weight RNA (HMW RNA) and low molecular weight RNA (LMW RNA) from pear peels was developed using the Huanghua and Green Huanghua pear peel as materials. Using the cDNA synthesized from the HMW RNA of fruit peels as template, DDRT-PCR was carried out to clone differential expressed gene fragments. Pear defensin gene PpyDFN1(accession number HM044853) and fragment of phenylalanine ammonia-lyase gene PpyPAL1, as differentially expressed genes were cloned by5’RACE and5’homologous degenerate primers amplification based on the differentially expressed fragments. The two genes’differential expression in Huanghua and Green Huanghua pear peels of8WAF by quantitative real-time PCR (qRT-PCR). The expression level of PpyDFN1and PpyPAL1in Huanaghua pear peel was6.7times and2.2times higher than that in Green Huanghua pear, respectively.The full length cDNA of Huanghua pear defensin gene PpyDFN1was567bp, containing5’untranslated region (UTR) with a length of72bp,3’UTR with a length of258bp, and coding region with a length of237bp. The5’UTR contained a (TC)7simple repeat sequence. The coding region encoded a polypeptide composed of78amino acid residues with molecular weight (MW)8.65kDa and isoelectric point (PI)9.36. BLASTP analysis indicated the polypeptide was precursor of defensin PpyDFN1. Signal prediction indicated that the first31amino acid residues was the signal peptide of PpyDFN1, and the left47amino acid residues was the mature defensin. Comparative homology modeling of PpyDFN1indicated that, PpyDFN1had a typical defensin structure consisting of a triple-stranded antiparallel (3-sheet and an α-helix, which was organized in a βαββ Clone and analysis of the genome sequence of PpyDFN1indicated that the genome sequence of PpyDFN1in Huanghua and Green Huanghua showed no difference and they both contained one intron and two exons. Results of bombardment on onion epidermal cells using the constructed vector for PpyDFN1subcellular location showed that defensin PpyDFN1located on the cell wall.5. PpyPAL1, PpyC4H and PpyCCR showed higher expression level in peels of Huanghua and Green Huanghua pear from6WAF to10WAF, and the expression level of PpyPALl and PpyCCR in Huanghua pear peel was apparently higher than that in Green Huanghua pear. The period of above genes’higher expression according with the stage of fruit dot forming and enlargement, to some extent reflected the strong secondary metabolic activity in the peel at fruit dot forming and enlargement stage. Whereas the higher expression level of PpyPALl and PpyCCR in Huanghua pear peels, reflected the stronger phenylpropanoid secondary metabolic activity in Huanghua pear peel than that in Green Huanghua pear peel.6. PpyDFNl gene was constitutively expressed in roots, leaf, stalk, sepal, petal, stigma and style, stamen, seed, flesh, and fruit peel. The expression level of PpyDFNl was depended on fruit dot developmental stages and PpyDFNl had a higher expression level during fruit dot forming and enlargement stage, whereas had a lower expression level in the later stage for fruit dot development; PpyDFNl showed a higher expression level in Huanghua pear peel than that in Green Huanghua pear. Wounding, ethylene, SA, MeJA, and H2O2strongly induced PpyDFNl expression, but ABA did not.7. Transgenic plants constitutively expressing the pear defensin PpyDFNl exhibited enhanced resistance to B. cinerea infection. Transgenic tobacco plants expressing PpyDFNl had shorter internodes, lower plant height, delayed flowering time, shorter flower organs, shorter stalks, thicker stalks and thicker flower tube. Root of T1generation seedlings harboring the PpyDFNl gene was significantly shorter than that of seedlings from wild type seeds. PpyDFN1was isolated from the transgenic tobacco leaves using the Nickel-ion resin and the eluate containing PpyDFNl could inhibit the growth of Agrobacterium tumefaciens. |
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