Studies On Characterization Of Carbohydrate Metabolism And Expression Of Relative Genes During Development Of ’White’ And ’Hongyang’ Kiwifruit

Kiwifruit is one of the most popular and important fruits worldwide, which has distinctive flavor and delicious taste, as well as numerous health benefits from high vitamin C content, dietary fiber and mineral nutrients. Actinidia eriantha ’White’ and Actinidia chinensis var.rufopulpa’Hongyang’have been successfully bred and are considered to be rare and novel among the germplasm resources. Up to now, studies on characteristics of carbohydrate metabolism, relative genes cloning and expression of ’White’ and’Hongyang’, their fruit development characteristics and correlation between the sugar metabolism and the occurrence of Actinidia bacterial canker, which significantly influence their further development and utilization on breeding cf high sugar and superior quality, remain largely unknown. Therefore, the changes in fruit carbohydrate content (starch, glucose, fructose and sucrose), enzyme activity in relation to carbohydrate metabolism throughout the growing season, as well as relative genes cloning and expression of sugar metabolism, the differences in starch accumulation and characteristics of sugar metabolism during fruit development of’White’and’Hongyang’were contrastively analysed. It is expected that this study will provide a theoretical guidance for increasing fruit sugar content and improving flavor and quality of kiwifruit. The main results were shown as follows:1. Investigation on the characteristics of fruit development of ’White’ based on BBCH scaleBy analysing characterization of’White’fruit morphology, growth and development and dynamics of carbohydrate and organic acid accumulation during fruit development from anthesis through to fruit senescence, a standard model for describing fruit development of ’White’ based on the BBCH scale was proposed. The findings showed that there were three main stages during the ’white’ fruit development:cell division (BBCH scale stage from 70 to 74); fruit expansion (starch accumulation, BBCH scale stage from 74 to 80); fruit maturation (BBCH scale stage from 80 to 90).’White’, which could be ripen on the vine, has a distinctive taste and flavor, as well as a long edible period, as its fruit possesses high fructose content, moderate sugar component distribution and high organic acid accumulation.2. Characteristics of carbohydrate metabolism during fruit development of ’White’and ’Hongyang’ kiwifruit(1) It was cleared that ’Hongyang’ was high-starch accumulation cultivar, while ’White’was low-starch accumulation cultivar. The higher rate of starch accumulation, which was also maintained for a longer period of time, was found in’Hongyang’, and the starch peak was much higher in’Hongyang’than in’White’. The rate of starch accumulation and the maximum starch content in ’Hongyang’ were 0.685 mg·g-1FW·d-1 and 70.78 mg·g-FW, and they were 1.34 and 1.69 fold higher than those in’White’, respectively. The period of starch accumulation of ’Hongyang’ was 21 days longer than that of ’White’. When starch content in ’White’ fruit rapidly dropped, starch accumulation of ’Hongyang’ fruit linearly increased. As a consequence, the higher level of starch concentration in ’Hongyang’ fruit maintained until harvest, while almost all starch in ’White’ had been converted into soluble sugars prior to harvest.(2) Correlations between metabolites and enzyme activities were measured and analysed. ADP-glucose pyrophosphorylase (AGPase) was identified as a key enzyme for starch accumulation in kiwifruit berries, in addition to this, the variation of neutral invertase (NI), acid invertase (AT) and sucrose phosphate synthase (SPS) activities may be the causes of high or low contents of starch, dry matter and soluble sugars in kiwifruit berries. Fruit starch content was positively and significantly correlated to AGPase and SPS activities, but was negatively and strongly correlated to NI and AI activities, and the positive correlation coefficient to starch for ’Hongyang’ was higher than for’White’. AGPase and SPS activities were always stronger in ’Hongyang’ than in ’White’. In contrast, NI and AI activities were consistently much lower in ’Hongyang’ than in’White’.3. Cloning of relative genes of carbohydrate metabolismSix cDNA fragments and two complete cDNA of genes encoding some key enzymes related to the sugar metabolism were cloned from total RNA isolated from’White’fruit by the methods of homologous cloning, combined by RT-PCR and RACE. Five genes have been submitted to the GenBank. The cDNA fragments of sucrose transporters (AeSUT), was 309 bp, encoding 103 amino acids, and shared 80% homology with that of Verbascum phoeniceum. The cDNA fragment of SPS (AeSPS) was 264 bp, encoding 88 amino acids, and shared 98% homology with that of Actinidia chinensis. The cDNA fragment of SuSy (AeSUSY) was 483 bp, encoding 146 amino acids, and shared 82% homology with that of Vitis vinifera. The cDNA fragment of AI (AeINA) was 744 bp, encoding 248 amino acids, and shared 81% homology with that of Solanum lycopersicum. The cDNA fragment of NI (AeINN) was 1725 bp, encoding 575 amino acids with a predicted molecular mass of 65.46 kDa, and shared 83% homology with that of Vitis vinifera and Manihot esculenta. The cDNA fragment of FK (AeFk1) had 990 bp open reading frame (ORF), encoding a deduce protein of 329 amino acids with a predicted molecular mass of 35.34 kDa, and shared 99% homology with that of Actinidia chinensis. Phylogenetic analysis revealed that AeFK1 was close with that of Actinidia chinensis. The complete cDNA of AGPase (AeAGP1) was 1758 bp in length with 1518 bp ORF, encoding a deduce protein of 505 amino acids with a predicted molecular mass of 55.54 kDa, and shared 99% homology with that of Actinidia chinensis. Phylogenetic analysis revealed that AeAGP1 was close with that of Actinidia chinensis. The cloning of full-length UGPase cDNA(AeUGP1) was 1732 bp in length with 1428 bp ORF, encoding a deduce protein of 475 amino acids with a predicted molecular mass of 51.84 kDa, and shared 92% homology with that of Populus tremula. Phylogenetic analysis revealed that Ae UGP1 was close with that of Ipomoea batatas.4. Selection for RT-qPCR of the most stable reference genes of kiwifruitThe expressions of four frequently-used reference genes (ACTB, TUB, 18Sr RNA, GAPDH) were analyzed in the different tissues and fruits at different developmental stages, respectively, at the mRNA level by using the real-time quantitative PCR. According to the analysis by geNorm program, the stabilities of the four candidate reference genes were different, TUB and ACTB were both stably expressed in six different tissues. 18SrRNA was most stably expressed in the different fruit development stages.5. Expression of relative genes of carbohydrate metabolism of kiwifruit(1) Real-time quantitative PCR analysis indicated that AeAGP1, AeUGP1, AeSPS, AeSUSY, AeINA, AeINN, AeFK1 and AeSUT genes could be expressed in fruits, roots, stems, leaves and flowers of ’White’ kiwifruit. The expression level of AeAGP1 was high in leaves and low in flowers, while AeUGP1 was high in stems and also low in flowers. The expression levels of AeINA, AeINN and AeFKl were high in roots and low in flowers, and the accumulation of AeINN transcripts in young fruits and leaves was low too. The expression levels of AeSPS, AeSUSY and AeSUT were low in roots and high in flowers, young fruits and stems, respectively.(2) The different expressions of relative genes of carbohydrate metabolism were analyzed during fruit development of ’White’ and ’Hongyang’. The expression levels of 7 genes such as AeUGPl, AeINA, AeINN AeSPS, AeSUSY, AeFkl and AeSUTin’Hongyang’ were lower than those in’White’, while the expression quantity of AeAGPl in’Hongyang’ in the different fruit growing stages far exceed that in’White’. The expression levels of AeAGP1, AeUGPl, AeINA, AeSUSY, AeFkl and AeSUT in ’White’ decreased with the fruit development, while the expression quantity of AeSPS increased at first, reaching its maximum at 133 DDA, then decreased gradually. The transcript abundance of AeINN in ’White’ decreased during cell division, then increased sharply, and decreased again, at last increased gradually. With the fruit development of’Hongyang’, the expression levels of AeUGP1, AeINA and AeINN decreased, while the transcript abundance of AeAGP1 increased at first, then decreased, and increased again, decreased at last, and the transcript abundance of AeSPS sharply increased at first, then rapidly declined, increased at last. The expression levels of AeSUSY, AeFkl and AeSUT in’Hongyang’increased at first, then decreased, reaching their maximum at 56 DAA,28 DAA and 84 DAA, respectively.(3) Significant up-regulation of expressions of AeAGP1,AeUGP1, AeINA and AeSUSY were observed in fruits treated with CPPU (N-(2-chloro-4-pyridyl)-N’-phenylurea)during the development of’Hongyang’ kiwifruit. The expression levels of AeAGPl, AeUGP1, AeINA and AeSUSY under the treatment of CPPU were 6.65-fold,3.46-fold,6.47-fold and 9.03-fold higher than those in control fruits, respectively. And the expression peak of AeSUSY gene in CPPU treatment fruits was 7 days earlier than that in control fruits. During the fruit development of’Hongyang’kiwifruit, expression level of AeINN was down regulated before 84 DAA, and thereafter was up regulated by CPPU treatment; expression level of AeSPS was up regulated during cell division, and then was down regulated by CPPU treatment; expression level of AeFKl was down regulated from 28 DAA to 63 DAA, while was up regulated from 70 DAA to 105 DAA in CPPU treatment fruits. The expression patterns of AeSUT in CPPU treatment fruits showed negative regulation, which was 4.88-fold lower than that in untreated fruits.6. Molecular identification of Actinidia bacterial canker and the occurrence of canker in correlation with sugar contentsDNA fragments from six representative strains amplified by PCR primers based on the 16S-23S rDNA intertranscribed spacer region, were the same as the GenBank accession number of D86357 and AY342165 of Pseudomonas syringae pv. Actinidae.18 germplasm materials were identified for resistance by three methods of in vitro leaves injection inoculation, in vitro shoots needle inoculation and live branches needle inoculation. It was concluded that the isolates from the canker infected kiwifruit branches were Pseudomonas syringae pv. Actinidae. A. chinensis ’Hongyang’ was found to be the most susceptible, A. eriantha ’White’ and ’Miniwhite’ were high resistant, and A. deliciosa ’Hayward’ was between them. And the different kiwifruit cultivars were inoculated by Psa. It was observed that the soluble sugar and starch in shoots and leaves had positive and significant correlation with the occurrence of Actinidia bacterial canker, with the coeffcients of 0.946, 0.896,0.971,0.996, respectively. The soluble protein contents in shoots and leaves were negatively and strongly correlated with the occurrence of Actinidia bacterial canker, with the coeffcients of -0.962,-0.972, respectively.